targeted-toxins

Anderson ML, Nokia MS, Shors TJ (2014) The role of acetylcholine in learning: Cholinergic MSDB lesions retard trace eyeblink conditioning and decrease adult neurogenesis. Neuroscience 2014 Abstracts 177.10. Society for Neuroscience, Washington, DC.

Summary: Decades ago, acetylcholine was considered intrinsic to processes related to attention and/or learning and memory. However, in the last decade or so, this relationship has been questioned and with good reason. That said, only a few studies have addressed the involvement of acetylcholine in tasks that require an animal to associate stimuli separated in time, such as trace eyeblink conditioning. Trace eyeblink conditioning is associated with hippocampal theta rhythmic activity and dependent on adult neurogenesis in the hippocampus, both of which are thought to be mediated by cholinergic activity. In the present study, 192 IgG-Saporin (SAP) was infused into the medial septum diagonal band (MSDB) complex of Sprague-Dawley rats to selectively kill cholinergic neurons bilaterally or unilaterally. Each side of the MSDB predominantly projects to the ipsilateral hippocampal formation. Animals were considered to have a bilateral lesion if the number of neurons that express choline acetyltransferase was reduced by at least 80% compared to sham animals or a unilateral lesion if the difference in the reduction between hemispheres was greater than 30%. Animals with bilateral, unilateral or sham lesions were trained with trace eyeblink conditioning at least 14 days after the SAP infusion. Animals were given 200 trials for four days for 800 trials in total. Animals with a sham lesion made more conditioned responses over all 800 trials compared to animals with bilateral and unilateral lesions. Conditioned responses increased over time in all groups. Taken together, bilateral and unilateral lesions both retard but do not drastically impair learning. In two separate experiments, the effect of bilateral and unilateral lesions on adult neurogenesis and theta rhythms was assessed. Animals were injected with 5-bromo-2’-deoxyuridine (BrdU) to label dividing cell at least 14 days after the SAP infusion. Seven days later, the number of BrdU-positive cells in the dentate gyrus of the hippocampus of animals with bilateral and unilateral lesions was reduced by ~40% in both hemispheres. Hippocampal local field potentials were recorded from another group of animals. Seven days following the SAP infusion, relative theta power was reduced in the bilateral but not unilateral group. However, by Day 14 relative theta power was similar in all three groups. This data suggests that a reduction in the number of new neurons in the hippocampus may be a contributing factor to a trace learning deficit as a result of a MSDB lesion. Moreover, disrupting septohippocampal cholinergic activity even in one hemisphere only is enough to reduce hippocampal adult neurogenesis and retard learning.

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

Yegla B, Kelbaugh A, Mookhtiar A, Parikh V (2014) Prefrontal cholinergic overload and attentional capacities in aging. Neuroscience 2014 Abstracts 211.25. Society for Neuroscience, Washington, DC.

Summary: The cognitive reserve hypothesis of aging posits that brain activity attempts to cope with functional age-related changes. Individuals with lower cognitive reserve are considered more susceptible to cognitive decline and age-related pathologies. However, what neuronal mechanisms underlie cognitive reserve, and how these mechanisms provide compensation for age-related decline in attentional capacities remains unknown. The basal forebrain cortical cholinergic input system is a critical component of the brain’s attentional system. Healthy older adults show attentional load-dependent posterior-anterior shift in aging (PASA) characterized by higher activation of the prefrontal regions. However, it is not known whether prefrontal cortex (PFC)-driven cholinergic mechanisms compensate for age-related decline in attentional capacities. Here, we investigated the impact of partial cholinergic deafferentation of the PFC on attentional capacities in young and aged rats. The impact of cholinergic depletion on neuronal activation in the PFC and posterior parietal cortex (PPC) was also investigated using a semi-quantitiative c-fos immunohistochemistry procedure. Young and aged rats were trained in an operant sustained attention task (SAT) that required the animals to distinguish between signal and non-signal events to attain a reward. After attaining criterion (70% correct responses on both trial types), animals either received bilateral infusions of 192-IgG saporin or sterile saline into the PFC and the performance was monitored for 4 weeks. Aged rats required more training sessions to acquire criterion than young rats. However, post-criterion performance prior to lesion surgeries remained similar between the two age groups. Saline-infused aged rats show a greater number of c-fos expressing cells in the PFC but not PPC as compared to the young animals. Restricted loss of prefrontal cholinergic inputs produced attentional impairments in aged rats (SAT scores: 0.43±0.08 vs. 0.63±0.05 in young lesioned rats). Moreover, lesioned aged rats show reduced c-fos positive counts in the PFC as compared to aged intact rats. Collectively, these data suggest that PASA shifts and prefrontal overload foster top-down processes to maintain attentional capacities in aging. Moreover, these compensatory processes are triggered by prefrontal cholinergic inputs.

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

Burk JA, Otoya D, Leong C, Ng A, Kozikowski CT (2014) Assessing removal of illumination as a signal: Effects of loss of basal forebrain corticopetal cholinergic neurons. Neuroscience 2014 Abstracts 263.06. Society for Neuroscience, Washington, DC.

Summary: In the vast majority of experiments assessing visual attention in animal models, tasks are employed that require the detection of a stimulus that was previously absent. Relatively few experiments have employed procedures where the signal is the removal of a previously presented stimulus. In the present experiment, we modified a previously validated measure of a visual attention that required detection of a signal (illumination of a central panel light for 500, 100 or 25ms) from “blank” trials when the light was not illuminated. Loss of basal forebrain corticopetal cholinergic neurons has been shown to decrease signal detection in this task. We modified the task so that the central panel light was illuminated throughout the intertrial interval and a signal occurred when the light was turned off (4-s) whereas a blank trial occurred when the central panel light remained illuminated. Male FBNF1 hybrid rats were trained in this revised attention task and then assigned to receive infusions of 192IgG-saporin or saline into the basal forebrain. Rats were retrained in the task after surgery and then received one session with a houselight flashing in the back of the chamber throughout testing and a second session with the signal decreased from 4-to 2-s. During presurgical training, we observed that animals required a longer signal to maintain stable task performance when the signal involved turning off the central panel light. Surprisingly, loss of basal forebrain corticopetal cholinergic inputs was associated with higher rates of signal detection compared with sham-lesioned animals, although this effect was attenuated with subsequent training. Flashing the houselight decreased accuracy on blank trials, but did not differentially affect lesioned and sham-lesioned animals. Signal detection accuracy significantly declined in both lesioned and sham-lesioned animals when the signal duration was decreased. Collectively, these results suggest that task manipulations appear to have similar effects whether the signal involves turning the central panel light on or off. However, the neural mechanisms that are engaged during these two types of tasks appear to be different. Future work in our laboratory will explore the role of basal forebrain noncholinergic neurons in performance of a task with turning the central panel light off serves as a signal.

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

Kozikowski CT, Wolfe EL, Yanev PG, Burk JA (2014) Interactions between noncholinergic basal forebrain neurons and muscarinic receptors in attentional processing. Neuroscience 2014 Abstracts 263.16. Society for Neuroscience, Washington, DC.

Summary: Numerous studies have provided evidence that basal forebrain corticopetal cholinergic neurons are critical for normal attentional performance. However, the role of noncholinergic basal forebrain neurons in attention has not been well-characterized. Moreover, evidence regarding interactions between cholinergic receptor activity and noncholinergic basal forebrain neurons remains scarce. In the present experiment, rats (n=15) were trained in a two-lever sustained attention task that required to discriminate between brief illumination of a centrally located panel light (500, 100, 25 ms) from trials when the light was not illuminated. After reaching criterion performance, rats received infusions into the basal forebrain of saline (n=7) or the immunotoxin, GAT1-saporin (n=8), to lesion noncholinergic neurons. After re-establishing performance after surgery, all rats received systemic administration of the muscarinic receptor antagonist, scopolamine (0, 0.05, 0.20 mg/kg, ip). When attentional testing resumed after surgical recovery, lesioned animals’ task performance did not significantly differ from sham-lesioned animals. However, following the highest dose of scopolamine, lesioned animals exhibited a larger decline in signal detection accuracy compared to sham-lesioned animals. Additionally, lesioned animals’ omission rate was higher during injection sessions compared to sham-lesioned animals. These results suggest that noncholinergic basal forebrain neurons are not necessary for performance in a well-trained attention task. However, loss of these neurons renders animals’ attentional performance more vulnerable to decreased cholinergic system stimulation. Finally, the lesion-induced increase in omissions may reflect a role for noncholinergic basal forebrain neurons in processes beyond attention.

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

Dinh TT, Huston N, Ritter S (2014) Lesions of hindbrain catecholaminergic projections to nucleus accumbens, bed nucleus of the stria terminalis, lateral parabrachial nucleus or locus coeruleus do not impair glucoprivic feeding. Neuroscience 2014 Abstracts 256.05. Society for Neuroscience, Washington, DC.

Summary: We have shown previously that injection of the retrogradely transported immunotoxin, anti-dopamine beta-hydroxylase (DBH) saporin (DSAP), into the paraventricular nucleus of the hypothalamus (PVH) or arcuate nucleus, abolishes feeding in response to central or systemic glucoprivation. Since DSAP injection destroys DBH-expressing neurons with projections to the injection site, these results strongly implicate hindbrain catecholamine neurons as major mediators of glucoprivic feeding. In order to further define the essential circuitry underlying glucoprivic feeding, we injected DSAP into these additional sites: locus coeruleus (LC), accumbens shell (AcbSh), ventrolatersal bed nucleus of the stria terminalis (vlBNST) and lateral parabrachial nucleus (LPBN). These sites are innervated by hindbrain catecholamine neurons and some sites receive collateral innervation from PVH-projecting catecholamine neurons. Appropriate placement and volume for DSAP administration was determined by co-labeling of DBH-ir neurons with retrograde tracer injected into target sites. Lesions were confirmed by postmortem evaluation of DSAP injection site and by hindbrain catecholamine cell and terminal loss. We found that the feeding response to systemic glucoprivation was not significantly or permanently impaired by injection of DSAP into any of these sites. Based on our results to date, we tentatively conclude that direct projections from hindbrain catecholamine neurons to the LC, AcbSh, vlBNST and LPBN are not required for glucoprivic feeding. The hypothalamus appears to be the major recipient of direct innervation from catecholamine neurons required for glucoprivic feeding

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

Fu R, Chen X, Zho W, Li J, Ye J-H (2014) Selective ablation of mu opioid receptor expressing gaba neurons in the rostromedial tegmental nucleus promotes ethanol intake. Neuroscience 2014 Abstracts 267.30. Society for Neuroscience, Washington, DC.

Summary: BACKGROUND AND PURPOSE The cellular mechanisms underlying the aversive effect of ethanol that limits its intake are not well understood, although recent evidence has linked aversion with synaptic inhibition of dopamine neurons in the ventral tegmental area. Emerging evidence indicates that the rostromedial tegmental nucleus (RMTg), a newly defined midbrain structure exerts a major GABAergic inhibitory control over midbrain dopamine neurons and encodes aversive stimuli. The RMTg contains mostly GABAergic neurons and with dense μ-opioid receptor (MOR) immunoreactivity. However, the role of RMTg in the regulation of ethanol intake has not been well investigated. EXPERIMENTAL APPROACH We compared voluntary ethanol intake and locomotion in rats with intra-RMTg infusion of dermorphin-saporin or blank saporin. Dermorphin-saporin is a neurotoxin, which could selectively lesion MOR-expressing neurons. We measured ethanol intake in rats given intermittent access to ethanol (20% vol/vol) using a two bottle choice paradigm. We euthanized the rats, dissected their brains and analyzed the glutamic acid decarboxylase67 (GAD67) and MOR protein expression and immunoreactivity immediately following the behavioral test. KEY RESULTS In rats that received intra-RMTg injection of dermorphin-saporin, we observed a robust increase in the intake of and the preference to ethanol, and in the locomotor activity; but a significantly reduced GAD67 and MOR protein expression, as well as a massive loss of neurons with GAD67 and MOR immunoreactivity within the RMTg. We observed no such changes in rats that received injection of blank saporin or saline. Together, These findings indicate that MOR-expressing GABA neurons in the RMTg play a crucial role in the regulation of ethanol consumption, implicating the dysfunction of these neurons likely play a critical role in the pathogenesis of alcoholism, and that these neurons should represent an appropriate target for the development of therapeutic strategies against alcohol use disorders.

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

Kalinchuk A, Porkka-Heiskanen T, McCarley R, Basheer R (2015) Cholinergic neurons of the basal forebrain mediate biochemical and electrophysiological mechanisms underlying sleep homeostasis. Eur J Neurosci 41:182-195. doi: 10.1111/ejn.12766

Summary: Previous work has indicated that non-rapid eye movement during recovery sleep after sleep deprivation requires cholinergic neurons in the BF. The authors examined how BF cholinergic neurons affect the levels of HSP markers during sleep deprivation. Rats received 230-ng injections of 192-IgG-SAP (Cat. #IT-01) into the horizontal limb of the diagonal band/substantia innominata/ magnocellular preoptic area. The results indicate that cholinergic neurons in the BF are important for regulating the biochemical and EEG mechanisms that contribute to HSP.

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

Frankel AE, Nymeyer H, Lappi DA, Higgins D, Ahn C, Noe C (2014) Preliminary results from a phase I study of substance P-saporin in terminal cancer patients with intractable pain. Journal of Clinical Oncology 32:191. doi: 10.1200/jco.2014.32.31_suppl.191

Summary: Existing pain therapies are insufficient to control cancer pain in 10-15% of patients. Substance P (SP) and its receptor, neurokinin-1 (NK-1r) have been determined to play a major role in spinal transmission of chronic pain. Animal studies have demonstrated that disruption of the NK-1r pathway alleviates chronic pain caused by a variety of stimuli. The authors are conducting a Phase I clinical trial in humans (NCT02036281) assessing the ability of SP-SAP (Cat. #IT-07) to treat intractable chronic pain due to cancer. Patients have received intrathecal injections of 1, 2, or 4 µg of SP-SAP with no evidence of toxicity or neurological or cardiac abnormalities. Doses will escalate up to 90 µg.

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

Akiyama T, Tominaga M, Takamori K, Carstens M, Carstens E (2014) Role of spinal bombesin-responsive neurons in nonhistaminergic itch. J Neurophysiol 112:2283-2289. doi: 10.1152/jn.00409.2014

Summary: Recent papers have demonstrated that pruritogen-evoked scratching behavior is reduced or eliminated by intrathecal injection of Bombesin-SAP (Cat. #IT-40). In this work the authors build on those data by investigating if spinal neurons that are responsive to pruritogens administered intradermally are also responsive to a spinal infusion of bombesin. Through the use of intradermal chloroquine injections, spinal superfusion of bombesin, and noxious pinch, the overlap of neurons processing itch and nociception was examined. The results demonstrate that chloroquine- and bombesin-sensitive neurons are involved in the transmission of itch, and that these are a separate neuronal population from those involved in nociception.

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

Q: I ordered a control conjugate to use alongside my targeted conjugate, but the two products are at different concentrations. How much control conjugate should I use?

A: Conjugate products are often of differing protein concentrations, meaning dilution of one is usually necessary to ensure comparable amounts of control conjugate and targeted conjugate are used. This adjustment can be done on a molar basis or a protein concentration basis. The data sheet shipped with each Advanced Targeting System conjugate specifies the molecular weight of the product. There are various calculators available on the ATS website.

By using these tools, calculations can be done that will ensure the same number of molecules of both control and targeted conjugate are used in your experiment. Alternatively, if the molecular weights of the two products are similar, calculations can be done to use the same amount of control protein as targeted conjugate protein in your experiment.