targeted-toxins

Manuel I, Llorente A, Gonzalez de San Roman E, Merino L, Giralt M, Rodriguez-Puertas R (2014) Memory and cholinergic impairment using a new approach of bilateral lesion of rat cholinergic basal forebrain. Neuroscience 2014 Abstracts 134.02. Society for Neuroscience, Washington, DC.

Summary: The cholinergic basal forebrain neurons (CBFN), which innervate cortical, hippocampal and amygdaloid areas control learning and memory processes and are damaged in Alzheimer´s disease (AD). The aim of the present study was to characterize the model of selective induced CBFN death in the nucleus basalis of Meynert (nbM) of adult Sprague-Dawley rats by intraparenquimal injection of the specific CBFN immunotoxin 192IgG-saporin (SAP) (n=11; 1μl/side [135ng/μl]). Learning and memory behavior was evaluated with the passive avoidance (PA) test. The CBFN density and the presence of glial cells were evaluated by immunofluorescence (P75NTR, Neu-N, GFAP, Iba-1). The AChE activity and AChE+ neuron density were analyzed by staining reaction. A significant decrease in CBFN (P75NTR -ir) density was observed in SAP treated rats (-82,7% vs aCSF, p<0,001). We found that cognitive impairment in the PA test and the reduction in the CBFN density in nbM correlated with each other (P75NTR+-ir vs PA, r2=0,51, p<0,05). Similar results were obtained for the reduction in AChE staining in cortical areas (entorhinal cortex: r2=0,55, p<0,01), hippocampus (CA3 pyramidal: r2=0,49, p<0,01) and amygdala (anterior: r2=0,43, p<0,01). Immunofluorescence studies showed a high density of activated microglia (Iba-1-ir) and an abscence of astrocytes (GFAP-ir) in the SAP administration area. Moreover, using MALDI-IMS assay, some lipid species were modified around the lesion area in SAP treated rats. The obtained data on the above described model of CBFN death, mimics the cognitive and cholinergic system impairment described in AD patients.

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

Akiyama T, Tominaga M, Davoodi A, Nagamine M, Takamori K, Carstens MI, Carstens E (2014) Spinal antinociceptive effect of gastrin releasing peptide (GRP) via GABAergic inhibitory interneurons expressing the GRP receptor (GRPR). Neuroscience 2014 Abstracts 158.02. Society for Neuroscience, Washington, DC.

Summary: GRPR-expressing dorsal horn neurons signal itch. We investigated a role for such neurons in modulating the spinal neurotransmission of mechanical and heat pain in mice. In behavioral studies, we measured heat and mechanical paw withdrawal thresholds using Hargreaves and von Frey assays, respectively. Mice received intrathecal (it) administration of one of following (5 µL volume); bombesin (6.2 pmol), GRP (0.1 nmol), and GRPR antagonists RC-3095 (0.03 nmol) and BW2258U89 (1.5 nmol). It administration of bombesin or GRP significantly reduced both heat and mechanical withdrawal thresholds with a maximal effect 10 min post-administration. In contrast, it administration of RC-3095 and BW2258U89 significantly increased both heat and mechanical withdrawal thresholds with a maximal effect 10 min post-administration. Mice treated with it bombesin-saporin to ablate GRPR-expressing spinal neurons exhibited reduced heat and mechanical withdrawal thresholds. It GRP failed to elicit heat and mechanical hyperalgesia in these mice. In electrophysiological recordings from superficial lumbar dorsal horn neurons, either bombesin or RC-3095 was spinally applied during responses elicited by noxious mechanical or heat stimulation of the cutaneous receptive field on the hindpaw. Bombesin increased both noxious mechanical- and heat-evoked activity in bombesin-sensitive neurons, while RC-3095 decreased noxious heat-evoked activity. In bombesin-insensitive neurons, bombesin decreased both noxious mechanical- and heat-evoked activity, while RC-3095 increased both. We additionally employed a double-label strategy to investigate if GRPR-expressing dorsal horn neurons coexpressed GABA, a molecular marker of inhibitory interneurons. Approximately 10% of GRPR-positive neurons were immunopositive for GABA. These results indicate that a subset of GRPR-expressing neurons function as interneurons in a circuit that suppresses nociceptive transmission in the dorsal horn. Noxious mechanical and heat stimuli activate GRPR-expressing dorsal horn neurons. A GABAergic subset of these may serve as inhibitory interneurons that contribute to inhibition of spinal neurons signaling heat and mechanical pain. Alternatively, GRPR-expressing neurons may drive other subsets of inhibitory interneurons. The antinociceptive circuit described here can be activated by pruritogens. We propose that the relative activity in antinociceptive and antipruritic circuits within the dorsal horn modulates itch- and pain-signaling ascending neurons to result in the perception of itch or pain.

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

Borella TL, Takakura AC, Moreira TS (2014) C1 neurons excite A5 noradrenergic neurons during hypoxia condition. Neuroscience 2014 Abstracts 168.07. Society for Neuroscience, Washington, DC.

Summary: C1 neurons activate sympathetic tone and stimulate the hypothalamic-pituitary-adrenal axis in circumstances such as pain, hypoxia or hypotension. They also innervate pontine noradrenergic cells group, including the locus coeruleus (LC) and the ventrolateral pontine catecholaminergic region (A5). Activation of C1 neurons reportedly inhibits pontine neurons; however, because these neurons are glutamatergic and have excitatory effects elsewhere, we re-examined the effect of C1 activation on pontine noradrenergic neurons (A5) using a more selective method. We examined the neuronal effects of destroying C1 catecholaminergic neurons with unilateral injection of the immunotoxin anti-dopamine beta-hydroxylase-saporin (anti-DβH-Sap) into the A5 region during hypoxic condition. Bilateral injections of anti-DβH-Sap into A5 destroyed tyrosine hydroxylase (TH) neurons but spared facial motoneurons and serotonergic neurons within the ventrolateral medulla. Hypoxia (8% O2 – 3 hours) induced a robust increase in Fos expression within the catecholaminergic C1 region of the ventrolateral medulla. On the lesioned side, Fos expression was significantly reduced (53.4 ± 17.6 vs. control: 129.8 ± 22.3 neurons) within the C1 region after hypoxia challenge. Residual Fos expression seen in lesioned side in response to hypoxia provides a basis for probing additional circuits that may be recruited in hierarchical manner in response to hypoxia. In conclusion, the C1 neurons activate the ventrolateral pontine noradrenergic neurons (A5 region) possibly via the release of glutamate from monosynaptic C1 inputs.

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

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)

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.

Zhao Z, Wan L, Liu X, Huo F, Li H, Barry D, Krieger S, Kim S, Liu Z, Xu J, Rogers B, Li Y, Chen Z (2014) Cross-inhibition of NMBR and GRPR signaling maintains normal histaminergic itch transmission. J Neurosci 34:12402-12414. doi: 10.1523/JNEUROSCI.1709-14.2014

Summary: After itch detection, the itch pathway moves through an array of G-protein coupled receptors and transient receptor potential channels in dorsal root ganglion neurons into dorsal horn neurons which integrate and transduce these signals, sending them to the somatosensory cortex. The purpose of this work is to clarify whether gastrin-releasing peptide (GRP) or B-type natriuretic peptide regulates histaminergic itch. Several strains of knockout mice received 200, 300, or 400 ng intrathecal injections of bombesin-SAP (Cat. #IT-40). Blank-SAP (Cat. #IT-21) was used as a control. The data further define the respective functions of the neuromedin B receptor and GRP receptor in itch, and reveals a working relationship between the different interneuron populations.

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