targeted-toxins

Takakura AC, Barna BF, Cruz JC, Colombari E, Moreira TS (2014) Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats. Exp Physiol 99(3):571-585. doi: 10.1113/expphysiol.2013.076752

Summary: Previous work has shown that lesions to the retrotrapezoid nucleus (RTN) have at least a modest effect on breathing, but it is unclear whether those lesions affected the entire nucleus or were incomplete. The authors used bilateral lesions of the RTN with 0.3 to 1.2 ng total of SSP-SAP (Cat. #IT-11) to eliminate neurokinin-1 receptor-expressing neurons; these are also Phox2b+TH- neurons. The results indicate that loss of Phox2b(+)TH(-) neurons may cause deficits seen after RTN lesion, and help define the ways in which these cells are involved in controlling central and peripheral chemoreflexes.

Related Products: SSP-SAP (Cat. #IT-11)

Freiria-Oliveira AH, Blanch GT, De Paula PM, Menani JV, Colombari DS (2013) Lesion of the commissural nucleus of the solitary tract/A2 noradrenergic neurons facilitates the activation of angiotensinergic mechanisms in response to hemorrhage. Neuroscience 254:196-204. doi: 10.1016/j.neuroscience.2013.09.017

Summary: Previous work has generated conflicting data on the role of catecholaminergic A2 neurons in the nucleus of the solitary tract (NTS) in control of arterial pressure lability. The authors used Anti-DBH-SAP (Cat. #IT-03) to lesion these neurons in a hypotensive hemorrhage model. Rats received two injections of 12.6 ng into the commissural NTS. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The lesioned animals quickly recovered from hypotension, but were impaired by the icv administration of losartan.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Mouse IgG-SAP (Cat. #IT-18)

Guyenet PG, Stornetta RL, Bochorishvili G, Depuy SD, Burke PG, Abbott SB (2013) C1 neurons: the body’s EMTs. Am J Physiol Regul Integr Comp Physiol 305(3):R187-204 . doi: 10.1152/ajpregu.00054.2013

Summary: Although mainly known for their involvement in the control of arterial pressure, C1 neurons are also suspected to participate in numerous other physiological processes such as neuroendocrine response, glucose homeostasis, food consumption, and others. This review discusses the role of these neurons as ’emergency medical technicians’ – cells that produce and modulate physiological survival responses to acute physical stress. The use of Anti-DBH-SAP (Cat. #IT-03) to delineate C1 neurons in the rostral ventrolateral aspect of the medulla oblongata is discussed.

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

Grupe M, Paolone G, Jensen AA, Sandager-Nielsen K, Sarter M, Grunnet M (2013) Selective potentiation of (alpha4)3(beta2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats. Biochem Pharmacol 86(10):1487-1496. doi: 10.1016/j.bcp.2013.09.005

Summary: Nicotinic acetylcholine receptors (nAChR) are involved in a wide range of processes in the central nervous system, many having to do with higher cognitive functions. In order to better understand how these receptors mediate attentional performance, the authors investigated glutamate release under varying conditions. In one series of experiments rats received a 160-ng injection of 192-IgG-SAP (Cat. #IT-01) into the right medial prefrontal cortex. The resulting decrease in glutamate release after the cholinergic lesion adds to the data indicating that positive modulation of nAChR may help alleviate attentional impairments caused by some brain disorders.

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

Romano A, Potes CS, Tempesta B, Cassano T, Cuomo V, Lutz T, Gaetani S (2013) Hindbrain noradrenergic input to the hypothalamic PVN mediates the activation of oxytocinergic neurons induced by the satiety factor oleoylethanolamide. Am J Physiol Endocrinol Metab 305(10):E1266-73. doi: 10.1152/ajpendo.00411.2013

Summary: Feeding behavior and energy balance are in part controlled by signals from the gut. Oleoylethanolamide (OEA) is an acylethanolamide that is thought to play a role in this network. Since peripheral administration of OEA has effects on the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN) the authors investigated the role of noradrenergic afferent input to these areas. Rats received bilateral 84-ng injections of Anti-DBH-SAP (Cat. #IT-03) into the PVN. Mouse IgG-SAP (Cat. #IT-18) was used as a control.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Mouse IgG-SAP (Cat. #IT-18)

Khan D, Owens E, Zaben M, Dunnett SB, Gray WP (2013) CD4+ T lymphocytes interact with microglia to modulate hippocampal neurogenesis. Neuroscience 2013 Abstracts 699.04. Society for Neuroscience, San Diego, CA.

Summary: Hippocampal neurogenesis occurs within the subgranular zone of the dentate gyrus and is important for learning and memory. Neurogenesis is impaired in patients with chronic temporal lobe epilepsy, an observation that may account for the learning and memory deficits that these patients commonly have. Emerging literature demonstrates that CD4+ T lymphocytes increase neurogenesis and enhance cognition; however, the exact mechanisms remain undetermined. Vasoactive Intestinal Peptide (VIP) receptors are expressed on T lymphocytes, microglia and hippocampal progenitor cells, hence this study was designed to investigate VIP’s role in mediating neuro-immune modulation. Hippocampal cultures (P7-10 Sprague Dawley rats) were generated and maintained for 3 days in vitro (DIV) and treated with 5% supernatant generated from C57/Bl6 mouse spleen using a CD4+ T lymphocyte isolation kit. BrdU and experimental conditions were added for the terminal 6 hours before fixation and then processed for BrdU and nestin. For phenotype analysis, experimental conditions were added at 3DIV and fixed at 6DIV to be processed for nestin and TuJ1. To deplete microglia, Mac-1-SAP was added at 2DIV for 24 hours before experimental conditions were added. 5% T lymphocytes supernatant increased proliferation of hippocampal nestin-expressing cells; an effect that is further enhanced under VIP treatment via VPAC1 receptor subtype. Examining potential cytokine mediators of this effect, PCR analysis showed 6-fold increase in IL-4 mRNA expression, and IL-4 antagonist abolished VIP proliferative effects. Using Mac-1-SAP to account for microglial involvement by depleting microglia, VIP proliferative effects were abolished. Our phenotyping studies also demonstrated an additional neurogenic effect under VIP treated supernatant compared to standard control conditions. Taken together, these results show VPAC1 receptor subtype expressed by CD4+ T lymphocytes mediates VIP proliferative effects on hippocampal cells via IL-4 cytokine release. Microglia mediates VIP proliferative effects. While we demonstrated before that VPAC2 mediates hippocampal progenitor cell survival, the findings of this study strongly implicate VPAC1 receptor as a neuro-immune mediator of hippocampal neurogenesis, and from a therapeutic perspective, shows that the effect can be pharmacologically manipulated.

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

ATS Poster of the Year Winner

Tiwari D, Warden H, Haynes JM, Nicolazzo JA, Pouton CW, Short JL (2013) Investigating the potential of stem cell based therapy in an immunotoxin mouse model of Alzheimer’s disease. Neuroscience 2013 Abstracts 712.19. Society for Neuroscience, San Diego, CA.

Summary: Purpose: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by reduced cognitive function. Stem cell based therapeutic approaches are a potential therapeutic option. In order to investigate this possibility the study focuses on the characterization of a dual reporter embryonic stem (ES) cell line and validation of an immunotoxin mouse model of AD for future transplantation experiments. Methods: A dual (mcherry and Lhx8+) reporter ES cell line was derived from the E14Tg2a mouse ES cell line. The ES cells were assessed for their differentiation capability and characterized using mmunocytochemistry. For the immunotoxin model, 6-8 week old C57BL/6 male mice (n = 12) were treated with bilateral intracerebroventricular injections of saline (control) or mu-p75-saporin toxin (0.4µg/µl/mouse) to cause cholinergic neuronal lesions. Mice were cognitively assessed using a novel three day water maze (WM) protocol and the novel object recognition (NOR) paradigm. Immunohistochemistry was done to detect the toxin dependent loss of cholinergic neurons. Results and conclusions: A significant difference in learning the WM task was observed during cued and spatial trials, with toxin-treated mice taking longer to reach the platform than control mice (two way ANOVA; p<0.01). Performance in the WM during the probe trial was also significantly reduced in toxin-treated mice, compared to control mice (t-test; p<0.05), indicating memory loss in toxin-injected mice and better learning in the saline-treated controls. However, no memory impairment was detected using the NOR test. Immunohistochemistry for choline acetyltransferase (ChAT) confirmed a significant loss (p<0.0001; t test) of cholinergic neurons in the medial septum. These data indicate that the toxin model is appropriate for use in subsequent transplantation studies. FACS analysis of the reporter cell line showed the presence of a small population of Lhx8+ cells at day 6 and 10 of differentiation. Immunocytochemistry for ChAT on day 18 cells revealed the presence of a few cholinergic positives neurons as compared to wild type controls. Literature suggests a possible role of Lhx8 in cholinergic development and these cells will be investigated further in order to select cholinergic progenitors for transplantation.

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

Lee S-Y, Kim M-S, Han J-S (2013) Activation of NF-κB signaling in the hippocampus without cholinergic input was aggravated by chronic stress. Neuroscience 2013 Abstracts 717.18. Society for Neuroscience, San Diego, CA.

Summary: Previous studies have demonstrated that loss of cholinergic input to hippocampus contributes dysfunction of HPA axis and alters GR-PKA-NF-κB signaling in hippocampus. In the hippocampus without cholinergic input, interactions of GR and PKA are decreased, whereas interactions of PKA and NF-κB are increased and phosphorylations on Ser276 of NF-κB p65 are increased. On the other hand, activation of NF-κB p65 is associated with behavioral action of stress and depression. The present research was conducted to examine whether NF-κB activation induced by cholinergic lesions is aggravated in response to chronic stress. Young adult rats received immunotoxic lesions of basal forebrain cholinergic neurons by intracranial injections of 192 IgG-saporin into the medial septum/vertical limb of the diagonal band and substantia innominata/nucleus basalis. After 2 weeks recovery from surgery, rats with cholinergic lesions and vehicle-injected control rats were subjected to 1 hr restraint stress per day for 2 weeks. We examined that cholinergic deafferentation induced alterations in GR and NF-κB p65 expression in hippocampus and prefrontal cortex. Rats with cholinergic deafferentation and chronic stress showed more activation of NF-κB p65 signaling in the hippocampus compared with rats with cholinergic deafferentation only. Thus the loss of cholinergic integrity during aging and in AD might increase proneness to chronic stress.

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

Vargas SL, Watts AG (2013) Compensatory feeding after reversing dehydration-anorexia: Is it analogous to glucoprivic or food deprivation-induced feeding?. Neuroscience 2013 Abstracts 757.04. Society for Neuroscience, San Diego, CA.

Summary: We use dehydration (DE)-anorexia to identify the neural networks associated with feeding behavior. How these networks are organized and interact to control ingestive behavior in both the normal and anorexic states allows us to determine how they function in health and disease. DE-anorexia involves replacing drinking water with hypertonic saline (HS) for up to 5 days. This leads to cellular dehydration, and a reduction in food and body weight. Reversing DE-anorexia by removing HS and reinstating drinking water leads to a robust feeding episode. Here we used two experiments to determine whether this water-activated compensatory feeding is functionally related to 2-deoxyglucose (2DG)-activated (glucoprivic) or to food deprivation-induced feeding. First, we determined whether forebrain-projection catecholamine (CA) neurons in the hindbrain are required for water-activated compensatory feeding. These neurons project to the paraventricular nucleus of the hypothalamus (PVH) and are required for 2DG feeding. To do this we lesioned this pathway with the retrogradely-transported immunotoxin, anti-dopamine beta-hydroxylase (DBH) Saporin (DSAP). Anesthetized adult male Sprague-Dawley rats (300g) were injected into the PVH with either a control SAP (MIgSAP) or DSAP. Three weeks later animals were housed in BioDaq monitoring cages to record their feeding behavior. All animals were given HS for 5 days. They were then given drinking water back on the 5th day, and euthanized 75 minutes later. Lack of immunohistochemical staining (IHC) for DBH in the PVH confirmed complete lesions. DSAP lesions had no significant effect on the amount eaten or the latency to begin feeding. Forebrain-projecting CA neurons are therefore not required for water-activated compensatory feeding. Second, we compared the feeding behavior of DE-rats given back water to that of food-deprived rats given food. We also examined the neuronal activity in the hindbrain of these animals using Fos. Meal pattern analysis showed little difference between groups, once feeding was initiated. Dual IHC labeling for Fos and DBH showed no colocalization following drinking water and deficit induced feeding. This contrasts with 2DG stimulated feeding. Furthermore, we show that the Fos expression in particular parts of the nucleus of the solitary tract and the parabrachial nucleus is consistent their roles in projecting visceral and gustatory information to the hypothalamus to coordinate feeding. Thus water-activated compensatory feeding engages mechanisms similar to those used during food deprivation-induced feeding rather than glucoprivic feeding.

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

Zajo KN, Fadel JR, Burk JA (2013) Assessment of the contributions of baseline performance and prefrontal cortical cholinergic projections to orexin A-induced attentional enhancement. Neuroscience 2013 Abstracts 854.02. Society for Neuroscience, San Diego, CA.

Summary: Orexinergic neurons innervate several brain regions including the basal forebrain, a structure known to be crucial for normal attentional performance in rats. Our previous research demonstrated that orexin receptor blockade impairs attention and that infusions of orexin A into the lateral ventricle enhance attentional performance in animals that have just reached criteria for stable performance levels on a sustained attention task. Our current research investigated whether more highly trained animals show orexin A-induced enhancement of attentional performance and whether basal forebrain cholinergic inputs to the medial prefrontal cortex were necessary for orexin A-induced attentional enhancement. Male FBNF1 hybrid rats were trained in a sustained attention task that required discrimination of visual signals (500, 100 or 25-ms illumination of a central panel light) from trials when no signal was presented. After stable performance levels were established, rats received both intraventricular guide cannula implantation and infusions of either the immunotoxin 192IgG-saporin or vehicle into the medial prefrontal cortex. Postsurgically, rats were retrained to stable performance levels and then received infusions of 0 (vehicle), 10, 100 or 1000pM orexin A in a counterbalanced order prior to task performance. On infusion days, rats were exposed to a version of the task which increased attentional demands by presenting a visual distracter during the middle block of trials within a testing session. In rats trained to higher performance levels, intraventricular orexin A infusions did not significantly enhance attentional performance. Loss of cholinergic projections to the medial prefrontal cortex decreased attentional performance, particularly when a visual distracter was presented. Attentional performance was unaffected in lesioned rats when orexin A was infused into the lateral ventricle. Our findings suggest that orexin A-induced attentional enhancement may be dependent upon baseline performance levels and possibly the integrity of the basal forebrain cholinergic projections to the medial prefrontal cortex.

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