targeted-toxins

Sharma A, Sharma R, Mackey C, Sahota P, Thakkar M (2017) Basal forebrain cholinergic neurons are vital for cortical desynchronization and behavioral arousal observed after nicotine consumption. Neuroscience 2017 Abstracts 241.1 / LL2. Society for Neuroscience, Washington, DC.

Summary: Purpose: Nicotine is an addictive constituent of tobacco which severely affects behavior. Sleep disruptions including reducing total sleep time, increasing sleep fragmentation and reducing sleep efficiency are very common in nicotine users. However, the underlying neuronal mechanism of how nicotine promotes desynchronization and disrupts sleep is unknown. We have shown that the basal forebrain (BF) is a key brain region, mediating nicotine’s effects on sleep-wakefulness (SFN 2015; Poster#166). The BF contains multiple neuronal phenotypes including cholinergic, GABAergic and glutamatergic subtypes. Thus, this study was designed to examine the neuronal subtype responsible for nicotine effects on sleep-wakefulness. As a first step, we focused on BF cholinergic neurons because BF cholinergic neurons are wake-promoting, express nicotinic receptors and supply acetylcholine to the prefrontal cortex, hippocampus and amygdala. We hypothesized that lesions of BF cholinergic neurons will attenuate nicotine induced cortical arousal/desynchronization. Methods: To test our hypothesis, adult male Sprague-Dawley rats were implanted with sleep recording electrodes and were divided into two groups: Lesion: Selective lesion of the BF cholinergic neurons was performed by bilateral administration of immunotoxin, 192-IgG-Saporin (SAP; 0.28 µg/0.5µL/side) in the BF; Sham (controls): Rats were bilaterally infused with saline (0.5µL/side). After injections, animals were left undisturbed for 3 weeks. Day 1: saline was administered subcutaneously at light/sleep onset. Day 2: Nicotine (0.3 mg/Kg) was administered at the same time. Sleep- wakefulness was examined for next 6 hours. On completion, animals were euthanized and the brains were processed for choline acetyltransferase (ChAT) immunohistochemistry to verify BF cholinergic lesions. Results: Our preliminary results: As compared to controls, lesioned rats, with a 64% reduction in cholinergic neurons, displayed attenuated nicotine induced cortical desynchronization and behavioral arousal. Conclusions: Our results suggest that the BF cholinergic neurons mediate nicotine induced cortical arousal/desynchronization that may be the cause of sleep disruptions in nicotine users.

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

Bowrey HE, James MH, Mohammadkhani A, Omrani M, Kane G, Aston-Jones G (2017) Chemogenetic activation of a retinal circuit that activates locus coeruleus neurons prevents the development of light- deprivation induced depression-like behavior. Neuroscience 2017 Abstracts 244.02 / NN6. Society for Neuroscience, Washington, DC.

Summary: Introduction: Chronic light-deprivation induces a depressive-like phenotype via a locus coeruleus norepinephrine (LC-NE)- dependent mechanism (Gonzalez and Aston-Jones, 2008). Suprachiasmatic nucleus (SCN) provides indirect circadian input onto LC via dorsomedial hypothalamus (DMH) (Aston-Jones et al 2001). SCN is therefore in a key position to integrate light information with LC via the pathway: retina→SCN→DMH→LC. We refer to this pathway as the Photic Regulation of Arousal and Mood (PRAM) pathway. We tested the hypothesis that increasing PRAM pathway activity prevents darkness-induced depression-like behavior. Methods: Expt 1. Sprague Dawley rats received intraocular injections of excitatory hM3Dq DREADD (AAV2-hSyn-hM3D(Gq)- mCherry) control virus (AAV2-hSyn-EGFP) or no virus. Rats were placed in continuous darkness for 8 weeks, and those that received virus were concurrently subjected to daily intraperitoneal injections of clozapine-N-oxide (CNO; 2 mg/kg), the DREADD-activating ligand. Rats were then subjected to assays of mood (saccharin preference test, elevated plus maze and forced swim test) or vision (electroretinagram: ERG). LC tissue was stained for Poly ADP ribose polymerase (PARP, a marker of apoptosis) and tyrosine hydroxilase (TH). Expt 2. To determine the retinal cell-type responsible for depression-like behavior, intrinsically photosensitive retinal ganglion cells (ipRGCs) of animals raised in 12:12 light:dark conditions were ablated using a saporin (SAP) toxin that selectively eliminates melanopsin-expressing cells (Mel-SAP). Two control groups received intraocular injections of vehicle and were kept in either continuous darkness or in 12:12 light:dark conditions. Ten weeks later, rats were subjected to identical analyses as those in Expt 1. Results: Expt 1. ERG analysis showed that CNO-activation of retinal DREADDs increased RGC activity. Constant darkness induced a depression-like phenotype in control animals, which was prevented by daily activation of retinal DREADDs by CNO. Expt 2. Mel-SAP induced a depression-like phenotype in animals maintained in normal light-dark conditions. This was also associated with increased apoptosis in LC-NE cells as seen with PARP staining. Conclusion: Dysregulation of the PRAM pathway may induce neural damage in LC-NE neurons that is associated with a depressive behavioral phenotype. DREADD-induced activation of RGCs can prevent depression-like behaviors that normally occur in rats kept in chronic darkness. The PRAM pathway presents a novel circuit for the regulation of mood, and thus a possible new direction for the treatment of some forms of depression in humans.

Related Products: Melanopsin-SAP (Cat. #IT-44)

Larin AA, Karpova SA, McCarley RW, Basheer R, Kalinchuk AV (2017) Glutamate and adenosine, basal forebrain and cortex: Cross-talk during prolonged wakefulness. Neuroscience 2017 Abstracts 72.2 /KK24. Society for Neuroscience, Washington, DC.

Summary: Recently we described a biochemical cascade which is critical in promoting recovery sleep (RS) after sleep deprivation (SD). It is initially triggered in the basal forebrain (BF) and later in the prefrontal cortex (PFC). This cascade includes production of inducible nitric oxide synthase (iNOS)-dependent NO followed by an increase in adenosine (AD). We hypothesized that iNOS induction is triggered by an increase in extracellular glutamate (Glu), and that the increase in AD prevents further rise in Glu via its inhibitory action on AD A1 receptor (A1R). To test this hypothesis, during 8h of SD, we first examined the time course of Glu and AD in BF/PFC. Further, to investigate the role of BF Glu receptors (GluRs) in this cascade, we measured the changes in BF/PFC AD and NREMs/delta after: a) stimulating BF GluRs by NMDA or AMPA without SD; b) blocking BF GluRs during SD by NMDAR or AMPAR selective antagonists. Finally, we measured Glu in the BF/PFC after blocking A1R. Furthermore, to determine the cellular target of glutamate effects, we examined the effects BF AMPA infusion on BF/PFC AD and NREMs/delta after BF cholinergic (ChBF) lesions using 192 IgG-saporin. Male rats were implanted with EEG/EMG recording electrodes and microdialysis guide cannulae targeting the BF and PFC. Microdialysis samples were collected during 8h SD and/or drug infusion. AD and Glu were measured using high performance liquid chromatography (HPLC) and ultra HPLC. To block NMDAR/AMPAR/A1R we used dizoclipine (MK-801)/6,7- dinitroquinoxaline-2,3-dione (DNQX)/8 cyclopentyltheophylline (CPT), respectively. 1) In the BF, Glu dramatically increased at the beginning of SD, followed by increase in AD at 2 h of SD. When AD maximized at 4 h of SD, Glu concurrently decreased to baseline. High AD levels were maintained till the end of SD. In the PFC, Glu significantly increased within 2h of SD. When AD increased at 5 h of SD, Glu returned to the baseline. 2) BF AMPA mimicked the effects of SD by increasing AD in both BF and PFC. NREMs/delta increased post AMPA-infusion. NMDA was not effective. 3) BF DNQX prevented AD increase during SD in BF/PFC and attenuated RS. MK-801 did not show any effect. 4) CPT Infusion to the BF/PFC induced dramatic increase in Glu till the end of SD. 5) Lesion of ChBF prevented BF/PFC AD increase during AMPA infusion and attenuated NREMs/delta post-infusion. A rapid increase in Glu during SD may be a trigger for the induction of iNOS-NO-AD cascade in both the BF and PFC. AD via A1R exerts a negative feedback on Glu neurotransmission, preventing its further rise and potential toxicity during long-term SD. The effect of Glu on SDinduced changes is primarily mediated via AMPAR, located on ChBF cells.

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

Llorente-ovejero A, Manuel I, Giralt MT, Rodríguez-puertas R (2017) Increase in cortical endocannabinoid signaling in a rat model of basal forebrain cholinergic dysfunction. Neuroscience 362:206-218.. doi: 10.1016/j.neuroscience.2017.08.008

Objective: To evaluate the eCB signaling in relation to the memory impairment induced in adult rats following a specific cholinergic lesion of the basal forebrain.

Summary: CB1 receptors present in presynaptic GABAergic terminals in the hippocampus are down regulated, but not those in cortical glutamatergic synapses.

Usage: 192-IgG-SAP was dissolved in aCSF under aseptic conditions to a final concentration of 130 ng/ml. aCSF or 192-IgG-SAP was bilaterally injected (1 ml/hemisphere) at a constant rate of 0.2 ml/min.

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

Leduc-Pessah H, Weilinger N, Fan C, Burma N, Thompson R, Trang T (2017) Site-specific regulation of P2X7 receptor function in microglia gates morphine analgesic tolerance. J Neurosci 37:10154-10172.. doi: 10.1523/JNEUROSCI.0852-17.2017

Summary: By selectively ablating microglia in the spinal cord using a Mac-1-SAP the authors demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats.

Usage: Mac-1-SAP or unconjugated Saporin control (15 μg) was administered by intrathecal injection.

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06), Saporin (Cat. #PR-01)

Xiao H, Li M, Cai J, Li N, Zhou M, Wen P, Xie Z, Wang Q, Chang J, Zhang W (2017) Selective cholinergic depletion of pedunculopontine tegmental nucleus aggravates freezing of gait in parkinsonian rats. Neurosci Lett 659:92-98.. doi: 10.1016/j.neulet.2017.08.016

Summary: Many patients with advanced Parkinson’s disease suffer from gait and postural impairments. The authors used Anti-ChAT-SAP (Cat. #IT-42) to specifically lesion neurons in the Pedunculopontine Tegmental Nucleus (PPTg) to examine the impact on gait performance. Adult male rats received either unilateral PPTg cholinergic lesion or bilateral PPTg lesion, at a dose of 250 ng. The authors conclude that the cholinergic neurons of pedunculopontine tegmental nucleus play a vital role in the occurrence of gait freezing in Parkinson’s disease.

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

Rosen S, Ham B, Drouin S, Boachie N, Chabot-Dore A, Austin J, Diatchenko L, Mogil J (2017) T-cell mediation of pregnancy analgesia affecting chronic pain in mice. J Neurosci 37:9819-9827.. doi: 10.1523/JNEUROSCI.2053-17.2017

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06)

Esko JD, Stanley P (2017) Glycosylation mutants of cultured mammalian cells. (eds. Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, Seeberger PH). In: Essentials of Glycobiology Chapter 49. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press doi: 10.1101/glycobiology.3e.049

Related Products: FGF-SAP (Cat. #IT-38)

Diepenbroek C, Quinn D, Stephens R, Zollinger B, Anderson S, Pan A, de Lartigue G (2017) Validation and characterization of a novel method for selective vagal deafferentation of the gut. Am J Physiol Gastrointest Liver Physiol 313:G342-G352. doi: 10.1152/ajpgi.00095.2017

Objective: To develop a new method that allows targeted lesioning of vagal afferent neurons that innervate the upper GI tract while sparing vagal efferent neurons.

Summary: CCK-SAP ablates a subpopulation of VAN in culture. In vivo, CCK-SAP injection into the NG reduces VAN innervating the mucosal and muscular layers of the stomach and small intestine but not the colon, while leaving vagal efferent neurons intact.

Usage: In vitro: each well was treated with a different dose of saporin conjugates (0, 2.4, 24, or 240 ng) for 24 h. In vivo: An equal volume (rat: 1 µl; mouse: 0.5 µl) of CCK-SAP (250 ng/µl) or Saporin (250 ng/µl) was injected at two sites rostral and caudal to the laryngeal nerve branch.

Related Products: CCK-SAP (Cat. #IT-31)

Mu D, Sun Y-G (2017) Itch induces conditioned place aversion in mice. Neuroscience Letters 658:91-96.. doi: 10.1016/j.neulet.2017.08.046

Summary: Consistently, ablation of itch‐specific neurons that express gastrin‐releasing peptide receptor in the spinal cord also abolished itch‐induced Conditioned Place Aversion (CPA), confirming that itch‐induced CPA is dependent on the spinal itch circuit.

Usage: Mice were given a single intrathecal injection (400 ng/5 μl each) of Bombesin-SAP (Cat. #IT-40) or Control Blank-SAP (Cat. #IT-21).

Related Products: Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)