targeted-toxins

Fraley GS (2014) Immunolesions of melanopsin receptive neurons in the adult Pekin drake attenuates the hormonal reproductive axis. Neuroscience 2014 Abstracts 543.01. Society for Neuroscience, Washington, DC.

Summary: Several light sensitive receptors have been described in the avian brain that are thought to regulate the reproductive axis independently from the eyes and pineal gland. Recently, my lab has described the presence of three of these photoneuroendocrine systems in the Pekin duck: opsin, opsin 5, and melanopsin. I set out to test the hypothesis that melanopsin receptive neurons are necessary to maintain seasonal reproductive status in the Pekin drake. To accomplish this, 50-week-old Pekin drakes were housed in the aviary at Hope College under long day length (18 hrs lights on) conditions in floor pens (5 drakes per pen). To specifically lesion melanopsin-receptive neurons, drakes were anethestized (8 mg/kg Propofol, IV), given analgesics (2 mg/kg ketfen, SC) skin incised and a trephine hole drilled 10 mm caudal to bony orbits and 1 mm to the left of midline. A 33 gauge stainless steel needle attached to a Hamilton syringe was lowered stereotactically 3.5 mm ventral to dura into the lateral ventricle. Three microliters of an anti-melanopsin-saporin conjugate (MSAP, 100 ng/ul) was injected into the lateral ventricle (n = 10). Control drakes were injected with 3 ul of equimolar unconjugated anti-melanopsin and saporin (SAP, n = 10). The incision was closed with VetBond, and the drakes returned to the aviary after complete recovery from anesthesia. After 4 weeks, birds were euthanized (400 mg/kg FatalPlus, IP) and body weight measured, and brains, pituitaries, and testes collected and stored for analyses. MSAP-treated drakes had significantly (p < 0.001) reduced relative teste weights compared to SAP controls. qRT-PCR analyses (n = 5 per treatment) of anterior pituitary showed a significant reduction (p < 0.001) in both LH-beta and FSH mRNA’s. Immunoctyochemical analyses (n = 5 per treatment) showed a significant reduction in melanopsin and GnRH-immunoreactivities. These data underscore the importance of the photoneuroendocrine system in maintaining the reproductive axis in seasonally breeding birds.

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

Helena CV, Toporikova N, Kalil B, Stathopoulos AM, Anselmo-Franci JA, Bertram R (2014) Involvement of kndy neurons in luteinizing hormone surges induced by steroids. Neuroscience 2014 Abstracts 543.11. Society for Neuroscience, Washington, DC.

Summary: A subset of hypothalamic arcuate neurons that coexpress kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been postulated to be critical for puberty onset and regulation of luteinizing hormone (LH) secretion. A method for targeted ablation of KNDy neurons was recently developed using the molecular neurotoxin saporin conjugated to the selective NK3R agonist [MePhe7]Neurokinin B (Nk3-SAP). Ovariectomized rats were microinjected bilaterally into the arcuate nucleus with Blank-SAP or Nk3-SAP. One set of rats was transcardiacally perfused 1, 2 or 3 weeks after the injections and immunocytochemistry for kisspeptin was performed in the arcuate nucleus region. The number of KNDy neurons was significantly decreased after 1 week of the toxin injection, however maximal fiber ablation was only achieved 3 weeks after the microinjections. Another group of rats was treated with oil (OVO), estradiol (OVE) or estradiol plus progesterone (OVEP). One week later, rats had their jugular vein cannulated and blood samples were taken at 10am and hourly from 3 until 6pm. Selective ablation of KNDy neurons of OVO rats significantly reduced basal LH levels at all time points studied. Basal LH levels in OVE and OVEP animals did not differ between groups, yet KNDy ablation increased peak LH levels in the afternoon of OVE and OVEP rats. A third group of OVE animals was microinjected with norbinaltorphimine (nor-BNI), a kappa opioid receptor antagonist, directly into the anteroventral periventricular nucleus (AVPV) one hour before the expected LH surge. The blockage of dynorphin receptors intra-AVPV significantly increased the LH surge, similar to the effect of KNDy ablation in OVE rats. Our results suggest that KNDy neurons provide inhibition to AVPV kisspeptin neurons through dynorphin and thus regulate the size of the LH surge induced by estradiol or estradiol plus progesterone.

Related Products: NKB-SAP (Cat. #IT-63)

Kohls MD, Lappi DA, Ancheta LR (2014) Properties of recombinant isolectin B4 (IB4): Binding and immunostaining. Neuroscience 2014 Abstracts 627.07. Society for Neuroscience, Washington, DC.

Summary: Isolectin B4 (IB4) is a protein found in the seeds of Griffonia simplicifolia, a woody climbing shrub native to western and central Africa. Although initially used as an identifier and agglutination agent for B-type red blood cells, it has since become widely used in the neurosciences as a neuronal tracer, for labeling specific populations in the spinal cord, and as a targeting moiety for delivering toxins to specific cells. Recent developments in response to competition from the nutritional supplement industry have reduced the available supply of seeds from which the native protein is purified. In order to create a consistent supply of pure and active IB4 we have determined the full nucleotide sequence of the IB4 gene, cloned it from Griffonia genomic DNA, and expressed recombinant IB4 in E. coli. The recombinant IB4 (rIB4) was purified and tested in several activity assays against the native protein. A fusion protein of rIB4 and GFP was created to demonstrate the use of this protein in immunostaining. Griffonia also contains isolectin A that agglutinates A-type red blood cells – the A and B lectins form tetramers with varying subunit combinations. These tetramers are potential sources of contamination in preparations of the native protein. rIB4 is completely free of any A lectin contamination. The rIB4 is highly pure, and has identical activity to the native protein.

Related Products: IB4-SAP (Cat. #IT-10)

Li J, Nelson D, Gibbs R (2014) Cholinergic regulation of aromatase in brain. Neuroscience 2014 Abstracts 640.10. Society for Neuroscience, Washington, DC.

Summary: Our goal is to understand mechanisms by which estrogens can influence brain function and cognition. Estrogens have been shown to influence neuronal plasticity and cognitive performance. Recent studies suggest that, in some cases, local estrogen synthesis can have a greater impact on neuronal survival and plasticity than systemic estrogen administration. Cholinergic projections also have a significant impact on neuronal plasticity in the brain, and recent studies demonstrate critical links between effects of estrogens and effects mediated by cholinergic inputs. In this project we are investigating whether aromatase expression and activity in specific regions of the adult brain are regulated by cholinergic activity. In one experiment, ovariectomized (OVX) rats were treated with the cholinesterase inhibitors donepezil (3 mg/Kg) or galantamine (5 mg/Kg) daily for one week prior to tissue collection. In a second experiment, OVX rats received intraseptal infusions of 192IgG-saporin (SAP) to selectively destroy cholinergic inputs to the hippocampus. Tissues were collected two weeks following the infusions. Different groups of rats were used to evaluate effects on aromatase mRNA and aromatase activity. Effects on aromatase mRNA were evaluated using qRT-PCR. Effects on aromatase activity were evaluated using a novel microsomal assay in which brain tissue microsomes were extracted and activity was measured in vitro by measuring conversion of testosterone to estradiol. Results show an increase in aromatase mRNA in the preoptic area following treatment with galantamine, but no effect in the hippocampus, frontal cortex, or amygdala. Galantamine also produced an increase in aromatase activity in the amygdala, but no significant effect in other brain regions. Donepezil had no significant effects on either aromatase mRNA or activity. Effects of the cholinergic lesions are still being evaluated; however, preliminary results suggest no significant effect on relative levels of aromatase mRNA in the hippocampus. These results indicate that cholinergic manipulations can affect aromatase expression and activity in specific regions of the brain such as the preoptic area and amygdala, with little or no effect in the hippocampus and frontal cortex. This could have important implications for the effects of cholinergic and anticholinergic medications on local estrogen production in the brain.

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

Patrone LA, Biancardi V, Bicego KC, Gargaglioni LH (2014) Medullary catecholaminergic (CA) neurons modulate hypoxic ventilatory response in neonatal rats (P7-8). Neuroscience 2014 Abstracts 643.10. Society for Neuroscience, Washington, DC.

Summary: It is known that catecholaminergic (CA) neurons are involved in autonomic and respiratory regulation during low O2 conditions in adult mammals. We evaluated the participation of medullary CA neurons of male and female neonatal rats (P7-8) in mediating the hypoxic ventilatory response (HVR) by specifically lesioning them with antidopamine beta-hydroxylase-saporin (DBH-SAP, 42ng / 100nL) injected into the 4th ventricle. We also quantified rates of O2 consumption (VO2) of control and lesioned neonates (P7-8) exposed to hypoxia. Minute ventilation (VE) of neonates was recorded by pressure-plethysmography from the body chamber during normoxia and hypoxia (10% O2), and the VO2 measurement by open flow respirometry. The mammalian HVR typically results in increased VE upon exposure to acute hypoxia. HVR was significantly reduced in male and female lesioned neonatal rats by about 23 and 15%, respectively, (male- control group: 137.3±7.9 (% of baseline) vs. lesioned group: 105.3±2.4 (% of baseline), p<0.01; female- control group: 127.0±3.0 (% of baseline) vs. lesioned group: 108.6±1.7 (% of baseline) p<0.02). The VO2 was decreased in the lesioned newborns, but only the lesioned male group was significantly lower (control group: 76.8±12.14 (% of baseline) vs. lesioned group: 45.3±13.3 (% of baseline) p<0.03). These results suggest that catecholaminergic neurons, specifically from medullary nuclei, exert an excitatory modulation of O2 chemosensitivity in neonatal rats.

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

Shim H, Park H-J, Lee H, Shim I (2014) The role of the supramammillary area in spatial learning and memory. Neuroscience 2014 Abstracts 652.05. Society for Neuroscience, Washington, DC.

Summary: The supramammillary area (SuM) of the hypothalamus, although small in size, has wide spread connection with numerous brain structures. It is known that the SuM can control the frequency of the hippocampal theta rhythm, which plays a role in the cognitive functions of the hippocampal formation. In order to examine the role of the specific cells of the SuM in learning and memory, selective cholinergic neurotoxic or excitotoxic lesioned rats of the SuM were tested for spatial memory on the Morris water maze (MWM) test. After the behavior tests, the expression of acetylcholine esterase (AChE) in the hippocampus was studied using the immunohistochemistry. In the MWM test, both lesion of the SuM with 192 IgG-saporin and ibotenic acid produced the impairment of spatial learning and memory. In the immunohistochemistry, the SuM-lesioned rat model by selective cholinergic neurotoxin showed decrease in the AChE expression in the hippocampal CA3. These findings suggest that cholinergic cells of the SuM area play a critical role in the process of consolidation of memory.

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

Tiwari D, Haynes J, Short J, Pouton C (2014) Investigating the potential of stem cell based therapy in an immunotoxin mouse model of Alzheimer’s disease. Neuroscience 2014 Abstracts 295.14. Society for Neuroscience, Washington, DC.

Summary: Purpose: To characterize a dual reporter embryonic stem (ES) cell line and validate an immunotoxin mouse model of Alzheimer’s disease for future transplantation experiments. Methods: A dual (mcherry and Lhx8+) reporter ES cell line was derived from E14Tg2a mouse ES cells assessed for differentiation capability and characterized using immunocytochemistry. For the immunotoxin model, 6-8 weeks C57BL/6 male mice (n = 12) were treated with bilateral intracerebroventricular injections of saline or mu-p75-saporin toxin (0.4µg/µl/mouse) to cause cholinergic neuronal lesions. Mice were cognitively assessed using a novel water maze (WM) protocol and novel object recognition (NOR) paradigm. Immunohistochemistry was performed to detect toxin dependent neuronal loss. Results: A significant difference in learning the WM task was observed during cued and spatial trials, with toxin-treated mice showing longer latency to platform than controls (two way ANOVA; p<0.01). Also performance during probe trial was significantly reduced in treated mice (t-test; p<0.05), indicating memory loss by toxin. No memory impairment was detected using the NOR test. Immunohistochemistry for choline acetyltransferase (ChAT) confirmed a significant loss (p<0.001; t test) of cholinergic neurons in the medial septum. These data indicate that the model is appropriate for future transplantation studies. FACS analysis of reporter cell line showed a small population of Lhx8+ cells at day 6 and 10 of differentiation. Immunocytochemistry for ChAT on day 18 cells revealed few cholinergic positives neurons as compared to wild type controls. Conclusion: Literature suggests a possible role of Lhx8 in cholinergic development and these cells are being further investigated by transplantation.

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

Llorente A, Gonzalez De San Roman E, Moreno M, Manuel I, Giralt M, Rodriguez R (2014) Activity mediated by neurolipid (CB1 and LPA1) and neuropeptide (GAL1) receptors in a rat model with cholinergic basal forebrain lesion. Neuroscience 2014 Abstracts 307.25. 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. An intraparenquimal injection of the 192IgG-saporin (SAP) immunotoxin, specifically eliminates CBFN. The present study examined the activity of endocannabinoid (CB1), lysophosphatidic acid (LPA1), galanin (GAL1) and muscarinic (MR) receptors, measuring Gi/o protein activation by [35S]GTPγS autoradiography in rats after the selective cholinergic basal forebrain lesion. CB1 immunoreactivity (CB1-ir) was also analyzed in the SAP administration area. We observed a high CB1-ir in the basal forebrain of the lesioned rats. The autoradiographic assays revealed that WIN55,212-2 (10 μM) evoked stimulation (CB1 activity) was increased in lateral olfactory tract (data expressed in % stimulation over basal; CSF vs SAP; 55 ± 11% vs 128 ± 13%, p<0.05) and in entorhinal cortex (156 ± 17% vs 277 ± 30%, p<0.01), but decreased in hippocampal dentate gyrus (299 ± 37% vs 166 ± 21%, p<0.05) and in medial amygdala (116 ± 20% vs 50 ± 7%, p<0.05). LPA (10 μM) induced stimulation (LPA1 activity) showed an increase in the internal capsule (60 ± 10% vs 137 ± 19%, p<0.01). The MR activity that was measured using carbachol (100 μM) was increased in hippocampal dentate gyrus (27 ± 6% vs 62 ± 7%, p<0.05) and in entorhinal cortex (55 ± 10% vs 94 ± 11%, p<0.05) but decreased in the nucleus basalis of Meynert (nbM) (46 ± 10% vs 15 ± 6%, p<0.05). Finally, there was an increased stimulation (GAL1 activity) of galanin (1 μM) in the nbM (45 ± 13% vs 80 ± 12%, p<0.05). The CB1-ir and GAL1 activity was increased in the lesioned area where the cholinergic neurotransmission was impaired, indicating a possible neuroprotective action on the surviving CBFN.

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

Leduc-Pessah HL, Weilinger N, Fan C, Thompson R, Trang T (2014) Development of morphine analgesic tolerance is modulated by spinal P2X7 receptors. Neuroscience 2014 Abstracts 327.08. Society for Neuroscience, Washington, DC.

Summary: Growing evidence suggests that microglia, which are immune cells in the central nervous system, are causally involved in the development of opioid analgesic tolerance. Here, we investigated the importance of spinal microglial ATP-gated P2X7 receptors (P2X7Rs) in morphine tolerance. In adult male Sprague Dawley rats, we found that seven days of systemic morphine treatment resulted in a progressive decline in morphine anti-nociception and a loss in analgesic potency, the two key features of analgesic tolerance. The development of morphine tolerance correlated with an increase in spinal Iba-1 expression, a marker indicative of microglial activation. To assess whether spinal microglia are required for morphine tolerance, we depleted microglia in the spinal cord of morphine treated rats using intrathecal injections of a saporin-conjugated antibody to Mac1 (Mac1-saporin). We found that Mac1-saporin attenuated the decline in morphine anti-nociception in rats that received chronic morphine treatment. In contrast, intervention with Mac1-saporin failed to restore morphine analgesia in rats with established tolerance. Thus, spinal microglia are causally involved in the development, but they are not required for the ongoing expression, of morphine tolerance. In addition, we found that P2X7R protein expression was markedly increased in the spinal cord of morphine tolerant animals. Pharmacological blockade of P2X7Rs with the selective antagonist A740003 attenuated the development of tolerance but did not reverse established tolerance. In BV2 microglial cells, repeated morphine treatment increased total P2X7R protein expression, an effect recapitulated by the mu-opioid receptor agonist DAMGO, and suppressed by the mu-receptor antagonist, CTAP. The morphine-induced increase in P2X7R protein expression was concomitant with a potentiation of BzATP evoked P2X7R calcium responses and inward current. Collectively, our findings suggest that spinal microglia are causally involved in the development, but not expression, of morphine analgesic tolerance. We also determined that the expression and function of P2X7R in microglia are critically modulated by mu-opioid receptors.

Related Products: Mac-1-SAP rat (Cat. #IT-33)

Lecrux C, Sandoe C, Neupane S, Kropf P, Toussay X, Tong X-K, Shmuel A, Hamel E (2014) Correlation between hemodynamics and neuronal activity during altered brain states. Neuroscience 2014 Abstracts 352.03. Society for Neuroscience, Washington, DC.

Summary: Introduction: Changes in neuronal activity are spatially and temporally replicated by concurrent alterations in cerebral blood flow (CBF) under physiological conditions, a phenomenon known as neurovascular coupling (NVC) which forms the basis of several brain imaging techniques. However, virtually nothing is known about NVC under conditions of altered brain states. Using the whisker-to-barrel pathway, we tested whether the coupling between neuronal activity and cerebral perfusion would be affected by changes in cortical states triggered by varying acetylcholine (ACh) tone. Methods: Sensory stimulation was induced in rats by mechanical whisker stimulation. In the barrel cortex, CBF was measured by laser-Doppler flowmetry and somatosensory evoked potentials (SEPs) were recorded with a multichannel electrode. Activated neurons were identified by double-immunohistochemistry for c-Fos and markers of pyramidal cells or GABA interneurons. ACh tone was increased pharmacologically (physostigmine 0.1mg/kg, subcutaneous; linopirdine, 10mg/kg, intraperitoneal) or through basal forebrain (BF) electrical stimulation. ACh tone was decreased using a selective cholinotoxin (saporin, 4mg/2mL, icv). Under enhanced ACh tone, muscarinic (scopolamine, 0.1mg/kg, intravenous) or central nicotinic (chlorisondamine dichloride, 12mg/5mL, icv) receptors were selectively blocked. Results: Whisker-evoked CBF responses were altered by changes in ACh tone induced by linopirdine (+31±4%, p<0.001), physostigmine (+40±8%, p<0.01), BF stimulation (+52±18%, p<0.05) or saporin (-41%, p<0.001) compared to their respective controls. These changes reflected alterations in the activity or extent, but not in the identity, of the neuronal network of cortical pyramidal cells and specific GABA interneurons selectively recruited by whisker stimulation. Under enhanced or decreased ACh tone, whisker-evoked CBF responses accurately mirrored changes in neuronal activity, and correlated with corresponding changes in the amplitude of the SEPs in cortical layers II/III and IV. Moreover, a positive correlation was observed between hemodynamic changes and band-limited power in the high gamma band in cortical layers II/III. The enhanced CBF response under high ACh tone required muscarinic ACh receptor activation. Conclusions: Changes in neuronal and vascular signals upon sensory stimulation remain tightly correlated under enhanced or reduced ACh tone, and reflect altered activity of the neuronal network selectively recruited by sensory stimulation. These subtle changes are reliably captured by superficial hemodynamic signals.

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