targeted-toxins

Köppen JR, Stuebing SS, Sieg M, Blackwell AA, Blankenship P, Grisley ED, Cheatwood JL, Wallace DG (2014) Is selective hippocampal cholinergic deafferentation sufficient to produce temporally graded retrograde amnesia?. Neuroscience 2014 Abstracts 749.20. Society for Neuroscience, Washington, DC.

Summary: Dementia of the Alzheimer’s type (DAT) is a neurodegenerative disorder marked by degeneration of basal forebrain structures and is associated with significant mnemonic deficits. The current study used a rat string-pulling task to evaluate whether selective cholinergic deafferentation of the hippocampus is sufficient to produce temporally graded retrograde amnesia. Female rats were pre-trained to pull strings to obtain reinforcement (cashew). Subsequently, rats were trained to discriminate between two scented strings. One scented string was consistently reinforced (+A), while the other scented string was never reinforced (B). After rats met criterion, they either waited two weeks (recent) or six weeks (remote) prior to receiving a sham surgery or infusion of 192-IgG-Saporin into the medial septum. Two weeks later rats were given four days of reversal training during which they experienced the same scented strings; however, the cashew was at the end of the string that was not previously reinforced. Following reversal training, rats were trained on a novel discrimination (+C/D). The results of the current study are consistent with selective cholinergic deafferentation of the hippocampus being sufficient to produce retrograde amnesia that was not temporally graded. First, all rats met criterion in a similar number of days. Rats receiving infusion of 192-IgG-Saporin into the medial septum had a higher number of correct responses during reversal training, relative to sham rats; however, no group differences were observed between recent and remote groups. Next, there were no group differences in the ability to learn a new discrimination. Finally, no group differences we observed in the latency to approach and pull up the string. The results were not caused by deficits in motivation or motor function, but they do reflect impairments in mnemonic function. The current study provides a novel behavioral assessment technique that models the retrograde amnesia characteristics observed in DAT.

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

Van Kampen JM, Kay DG, Maelicke A (2014) The galantamine prodrug, Memogain®, reverses deficits in hippocampal neurogenesis associated with the loss of basal forebrain cholinergic neurons. Neuroscience 2014 Abstracts 789.21. Society for Neuroscience, Washington, DC.

Summary: Loss of basal forebrain cholinergic innervation of the hippocampus and severe neuronal loss within the hippocampal CA1 region are early hallmarks of Alzheimer’s disease (AD), and are strongly correlated with cognitive status. This loss of cholinergic innervation is a key factor underlying alterations in hippocampal neurogenesis, which are also characteristic of AD. We have previously reported the effects of various cholinergic compounds on hippocampal neurogenesis indicating that acetylcholine serves as a potent neurogenic regulator. Memogain® (GLN 1062) is an inactive galantamine pro-drug with 15 fold higher brain availability than galantamine. It is designed to provide improved blood brain barrier penetration, greater potency, and fewer side effects than the cholinesterase inhibitors currently used for the treatment of Alzheimer’s dementia. This would serve both to promote patient adherence and permit the use of higher doses. Galantamine is unique among the cholinesterase inhibitors in that it also has allosteric actions at α-7 nicotinic receptors, activation of which has been linked to both disease-modifying and cognitive enhancing effects, as well as effects on hippocampal cell proliferation. Here, we describe the neurogenic actions of Memogain® in a rodent model of cholinergic depletion. Infusion of the immunotoxin, 192IgG saporin (SAP), used to induce selective basal forebrain cholinergic cell loss reminiscent of that found in AD, resulted in a pronounced loss of basal forebrain cholinergic neurons and hippocampal ChAT fiber density. Consistent with earlier reports, SAP-lesioned animals had significantly fewer BrdU+ and PCNA+ cells in both the dentate gyrus and CA1 region of the hippocampus, when compared to sham-operated control animals. These animals also displayed significant impairments in spatial working memory, as assessed by a T-maze and the radial arm maze. By contrast, animals treated with Memogain® displayed a restoration of hippocampal cell proliferation, increased neuronal cell counts, normalized neuronal migration, and improvements in cognitive function. Thus, the beneficial effects of Memogain® may extend beyond acute cognitive enhancement, to include disease modification through support of hippocampal neurogenesis.

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

Becker A, Goldberg M (2014) The effects of targeted intracerebral saporin injection on recovery from stroke. Neuroscience 2014 Abstracts 800.09. Society for Neuroscience, Washington, DC.

Summary: It is well known that the diffuse neuromodulatory systems of the brain play a role in cortical plasticity that may extend to cortical reorganization after brain injury. The basal forebrain cholinergic system in particular is necessary for both cortical plasticity and behavioral recovery from cortical electrolytic lesions. The role of the cholinergic system in recovery from stroke has never been directly investigated. In this experiment, we asked the question: is the basal forebrain cholinergic system in the mouse necessary for behavioral recovery from stroke? To answer this question, we administered intracerebral injections of the selective immunotoxin mu p75-saporin bilaterally to the cholinergic nucleus basalis in young adult mice. Using choline acetyltransferase immunohisochemistry, cresyl violet staining, and fluoro-jade B staining we discovered a dose at which these injections eliminate local cholinergic neurons while leaving other cell types and cholinergic cells outside the nucleus basalis unharmed. We report the effects of these injections on behavioral recovery from a subsequently induced photothrombotic cortical ischemic stroke.

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

Hulsey D, Hays S, Khodaparast, N, Casavant R, Ruiz A, Das P, Nutting E, Carrier X, Iyengar M, Quareshi I, Sultana S, Rennaker R, Kilgard M (2014) Vagus nerve stimulation dependent enhancement of cortical plasticity requires cholinergic innervation of the cortex. Neuroscience 2014 Abstracts 542.20. Society for Neuroscience, Washington, DC.

Summary: Primary motor cortex (M1) transiently reorganizes in response to motor skill learning. Pairing forelimb movements with Vagus Nerve Stimulation (VNS) drives enhanced and robust analogous plasticity within M1. These changes occur outside of the typical period for motor plasticity and are independent of new skill learning. The mechanism by which VNS enhances M1 plasticity is not well understood. Skill learning and the associated cortical plasticity is dependent on cholinergic innervation of the cortex. VNS may enhance plasticity by engaging neuromodulatory systems necessary for plasticity. We hypothesize that cholinergic innervation of M1 is necessary for motor plasticity associated with VNS pairing. To test this hypothesis, we trained female Sprague Dawley rats on a skilled lever pressing task emphasizing use of the proximal forelimb. After task acquisition, one group of rats received a lesion to the cholinergic neurons of the basal forebrain using 192-IgG-Saporin, while another group received a control injection. All subjects also received a VNS cuff implant during the surgery. After one week of recovery, all subjects receive VNS paired to successful task performances for five days. Intracortical microstimulation was performed to derive M1 maps of each group 24 hours after their final VNS paired session. Subjects with an intact cholinergic system show significant expansion of proximal forelimb representation over naïve animals within the cortex. Subjects without cholinergic innervation of the cortex show no difference in M1 organization when compared to naïve animals. We conclude that cholinergic innervation is necessary for the effects of VNS on motor plasticity.

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

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)