sfn2016

Friedman CA, Russell BJ, Kohls MD, Ancheta LR, Shramm PA, Lappi DA (2016) Targeting vesicular gaba transporter (vGAT)-expressing cells with a polyclonal antibody to the lumenal domain of vGAT: results with a saporin conjugate. Neuroscience 2016 Abstracts 124.06 / E30. Society for Neuroscience, San Diego, CA. PMID: 0

Summary: The vesicular GABA transporter (vGAT) mediates the accumulation of GABA into synaptic vesicles and the release from these vesicles. vGAT is expressed in nerve endings of GABAergic neurons throughout the CNS. The GABAergic system is crucial for the development and functional maturation of the nervous system, as well as the maintenance of balance between excitation and inhibition required for normal neural circuit function. A panel of research tools has been created that target the lumenal domain of vGAT. Antiserum was raised against a peptide from the C-terminus of rat vGAT and resulted in an affinity-purified antibody and an immunotoxin specific for vGAT-expressing cells. The antigen sequence is identical among human, rat, mouse, pig and guinea pig. A stably-transfected clone of HEK293 cells (2E11HEK) that expresses vGAT on the cell surface shows excellent results for western blot, ICC and flow cytometry using both the antiserum and affinity-purified antibody. The affinity-purified antibody was used to create an immunotoxin by conjugating it to the ribosome-inactivating protein, saporin. Saporin irreversibly inactivates ribosomes, blocking protein synthesis, when it is escorted into a cell. Saporin cannot enter a cell on its own, but when escorted by something that binds to a cell surface marker it is internalized along with the binding moiety and causes cell death. The immunotoxin (Anti-vGAT-SAP) is 1000-fold more cytotoxic to 2E11HEK cells than non-conjugated saporin, based on the EC50 in a cytotoxicity assay. The affinity-purified vGAT antibody binds specifically to cells that express vGAT, and delivers a payload to the interior of these cells. Anti-vGAT-SAP could be an important tool in studying diseases involving dysfunction of GABAergic neurons. GABAergic neuron dysfunction is thought to be an underlying factor in Epilepsy, Down Syndrome, Fragile X Syndrome, Schizophrenia and Autism. In vivo, elimination of vGAT-expressing cells in a particular area (rather than knocking out vGAT systemically) makes it possible to study the functions of those regional cells. Animals can then be tested behaviorally before and after injections of Anti-vGAT-SAP to demonstrate the effects of loss of cells in a particular region of interest.

Related Products: vGAT Rabbit Polyclonal (Cat. #AB-N44), Anti-vGAT-SAP (Cat. #IT-71)

Bernabe CS, Caliman IF, Abreu ARR, Shekhar A, Johnson PL (2016) A unique subdivision of serotonergic neurons in the dorsal raphe nucleus projects to the basolateral amygdala complex to enhance fear-conditioned behaviors. Neuroscience 2016 Abstracts 74.23 / GGG14. Society for Neuroscience, San Diego, CA.

Summary: The basolateral and lateral amygdala nuclei complex (BLC) is implicated in a number of emotional responses including fear and anxiety. Previous studies have shown that increased serotonin release in the BLC enhances fear conditioned behaviors, and we recently demonstrated that pharmacologically depleting serotonin in the BLC using 5,7-dihydroxytryptamine (5,7,DHT) injections disrupted fear conditioned behaviors. In 2005 Abrams and colleagues determined that there were robust BLC projections that originate from the midline dorsal (DRD) and ventral (DRV) subdivisions of the dorsal raphe nucleus (DRN), but it was not determined that they were serotonergic. Here we injected a saporin (SAP) toxin coupled to a serotonin transporter (SERT) into the BLC to selectively lesion local serotonergic fibers which replicated disrupted fear conditioning behaviors that was observed in the BLC 5,7DHT study. Since the SERT-SAP can retrogradely lesion the associated cell bodies (Shen et al., 2007) via fast retrograde microtubule associated transport, we also injected the retrograde tracer cholera toxin B (CtB) into the BLC via same the cannula that SERT-SAP was injected. This was done to not only verify loss of serotonergic neurons in DRN subdivisions, but also to specifically verify BLC projecting serotonergic neurons. We later used immunohistochemistry (IHC) to detect SERT in the BLC and observed a 90% decrease in local SERT-immunoreactive fibers. We also verified that almost all CtB-immunoreactive BLC projecting neurons in DRN were also positive for tryptophan hydroxylase (TPH: a serotonergic specific enzyme). We further determined that BLC projecting neurons immunoreactive for both CtB and TPH were primarily located within the midline DRD and DRV divisions of the DRN, and not in the lateral wing (DRVL) divisions of DRN. Regardless of location, the SERT-SAP group had 72% to 74% less CtB/TPH-double immunoreactive neurons than control-SAP group. These data elucidate the roles of serotonergic networks in the pathophysiology of fear, and especially focus on the origins of these pathways as a way to identify potential novel therapeutic targets.

Related Products: Anti-SERT-SAP (Cat. #IT-23)

Abudukeyoumu N, Garcia-Munoz M, Jaidar OP, Arbuthnott G (2016) Striatal cholinergic interneurons: their depletion and its progression. Neuroscience 2016 Abstracts 245.09 / RR4. Society for Neuroscience, San Diego, CA.

Summary: Even before the discovery that Parkinson’s was produced by the loss of dopaminergic neurons, this neurological disease was treated with anticholinergic drugs. A balance between cholinergic and dopaminergic activity in striatum is not only important in PD but for the normal function of the nucleus (i.e., behavior, reward, memory and cognitive functions). An important source of striatal acetylcholine (Ach) comes from giant and sparsely distributed cholinergic interneurons (ChI). However, their study has been hampered by a concentration of only 1-3 % of the whole striatal cell population. We performed a stereological systematic random sampling of striatal tissue from 21 days old C57BL/6J male mice. To selectively deplete ChI we performed a stereotaxic injection of saporin ribosome inactivating immunotoxin that targets choline acetyltransferase (0.3µl). Following survival periods of 2, 4 or 6 weeks, animals were sacrificed and brain sections immunostained against ChAT to identify ChI, or against vesicular acetylcholine transporter (vAChT) to identify synaptic boutons. For each of the three survival periods, we counted and compared the number of ChIs between the intact and the lesioned hemispheres and the change in the number of vesicular acetylcholine transporters (vAChT). Compared to striatal sections from naïve controls and sham injections, we observed a decrease in ChIs according to each survival period of 24.4% (week 2, n=9), 33.74% (week 4, n= 11) and 19.89% (week 6, n=10). In contrast, we observed a percent increase in vAChT positive boutons of 42.3, 21.6 and 28.3% for each of the respective survival periods (n=9, n=11 and n=10). We are investigating whether the increase in vAChT positive terminals is due to an indirect upregulation produced by compensatory axonal sprouting from surviving ChI, or from afferent axonal terminal fields of cholinergic mesopontine neurons.

Related Products: Anti-ChAT-SAP (Cat. #IT-42)

Sharma A, Sharma R, Sahota P, Thakkar M (2016) Basal forebrain cholinergic neurons are vital for sleepiness observed after alcohol consumption. Neuroscience 2016 Abstracts 254.12 / AAA18. Society for Neuroscience, San Diego, CA.

Summary: Purpose: Corticopetal wake-promoting basal forebrain region (BF) is implicated to mediate sleepiness following alcohol intake. However, the specific phenotype of the neuronal population mediating this effects is unknown. Since the BF cholinergic neurons are sole supplier of cholinergic inputs to several forebrain regions, including the cortex, we hypothesized that the cholinergic neurons of the BF may have a critical role in alcohol induced sleepiness. Methods: To test our hypothesis, adult male Sprague-Dawley rats were instrumented with sleep recording electrodes and bilateral guide cannulas targeted toward the cholinergic zone of the BF. To verify alcohol induced sleep promotion, rats were administered alcohol [35% (v/v); 3 g/Kg; intragastric] at dark onset (pre-lesion). Subsequently, the animals were divided into two groups. Lesions: Selective lesion of the BF cholinergic neurons was performed by bilateral administration of immunotoxin, 192-IgG-Saporin (0.28 µg/0.5µL/side) in the BF. Shams: Bilaterally infusion with artificial cerebrospinal fluid (0.5µL/side). Rats were left undisturbed for 3 weeks. Subsequently, alcohol induced sleepiness was re-examined (post-lesion) as described above. On completion, rats were euthanized, brains removed and processed for ChAT immunohistochemistry in the BF to verify selective lesion of the cholinergic neurons. Results: Pre-Lesion: Robust sleep promotion was observed following alcohol administration. Post-Lesion: As compared to controls, rats in the lesion group took significantly (p<0.05) longer time to fall asleep and spent significantly less time asleep following alcohol administration. Conclusions: Our results suggest that the cholinergic neurons are the mediators of sleepiness following alcohol intake.

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

Jokiaho A, Watts AG (2016) Catecholaminergic innervation and the neuronal activation of hypothalamic glucose sensitive regions during rapid- and slow-onset hypoglycemia in adult male rats.. Neuroscience 2016 Abstracts 256.21 / CCC20. Society for Neuroscience, San Diego, CA.

Summary: Hypoglycemic counterregulation is mediated by glucosensors located in the hypothalamus, hindbrain, and portal-mesenteric veins. But which are engaged is rate-dependent, with portal vein sensors being obligatory for slow- but not rapid-onset hypoglycemia. Slow-onset hypoglycemia is particularly prevalent with insulin therapy in type 1 diabetes. We have previously shown that hindbrain-to-hypothalamus catecholaminergic (CA) projections are required for sympathoadrenal responses to slow- but not rapid-onset hypoglycemia, and that rapid- but not slow-onset hypoglycemia significantly increases CA/Fos colocalization in the ventrolateral medulla. These results show that the organization of a hypoglycemia-responsive brain networks is rather complex, and involves a set of what are likely parallel but interactive networks, each of which is responsible for controlling epinephrine, glucagon, and glucocorticoid responses. We now examine how various forebrain cell groups known to be important for glycemic regulation respond to , and how these responses are impacted by removing hindbrain-to-hypothalamus CA projections using injections of the immunotoxin, saporin conjugated to anti-DBH (DSAP) into the hypothalamic paraventricular nucleus (PVH). These injections remove CA inputs to the PVH and other regions within the medial hypothalamus. We then examined whether DSAP lesions affected Fos responses to slow- and rapid-onset insulin-induced hypoglycemia in key forebrain regions. We found that removing CA innervation differentially influences regional hypothalamic Fos responses to slow- and rapid-onset insulin-induced hypoglycemia. Rapid-onset hypoglycemia produced significantly greater Fos activations in the medial and lateral parvocellular and lateral parts of the PVH, parts of the lateral hypothalamus (LHA), the bed nucleus of the stria terminalis that was significantly reduced in all these regions with DSAP lesions. Of particular interest was the altered Fos in LHA regions that contain orexin neurons. We found that 27% of Fos activated neurons colocalized with orexin neurons in rapid-onset hypoglycemia, but this colocalization was significantly reduced by DSAP lesions. Furthermore we used a retrogradely transported polysynaptic neurotropic virus (PRV-152) injected into adrenal gland to show that 25% of PRV-labeled neurons in the LHA colocalized with orexin neurons. These results show that hindbrain-to-hypothalamus CA projections provide hypoglycemia-related information to regions of the forebrain in a rate-dependent way, with orexin neurons playing a particularly prominent role for sympathoadrenal responses.

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

Prater C, Harris B, Merrill A, Aliyas A, Anderson K, Carr J (2016) Tectal CRF R1 receptors modulate food intake. Neuroscience 2016 Abstracts 257.08 / DDD9. Society for Neuroscience, San Diego, CA.

Summary: The optic tectum (OT) and superior colliculus (SC) rapidly inhibit food intake when a visual threat is present. Previous work from our laboratory indicates that CRF, acting on CRF R1 receptors, may play a role in tectal inhibition of prey capture. Here we test the hypothesis that tectal CRF neurons modulate food intake in juvenile Xenopus laevis. We tested five predictions: 1) Does tectal CRF injection decrease food intake? 2) Does a selective CRF R1 antagonist block CRF effects on feeding? 3) Does a selective CRF R1 antagonist block stressor-induced inhibition of feeding? 4) Does eliminating tectal cells expressing CRF R1 increase feeding? 5) Does food deprivation increase food intake and, if so, can this be reversed with CRF? X. laevis were administered oCRF alone or in combination with the selective CRF R1 antagonist NBI27914 or antagonist vehicle. Test agents were bilaterally injected into the tecta of juvenile frogs. CRF conjugated to the ribosomal toxin saporin (CRF-SAP) was administered 2 wk prior to testing to eliminate tectal cells expressing CRF R1. oCRF administered bilaterally into the tecta significantly reduced food intake compared to sham and vehicle injected juveniles. When frogs were injected with oCRF and antagonist vehicle, food intake was significantly reduced. When injected with both NBI27914 and oCRF, food intake was maintained at baseline levels. Frogs ate significantly less when exposed to a reactive stressor (ether vapors) and when pre-treated with antagonist vehicle prior to exposure. NBI27914 reversed stressor-induced inhibition of food intake. Neither CRF-SAP injection nor food deprivation (2 wk) significantly changed food intake. No significant differences in food intake were noted between males and females across all studies. Overall, we found support for questions 1-3 and conclude that activation of the tectal CRF R1 inhibits food intake in frogs. Furthermore, tectal CRF R1 receptors appear to be involved in the reduction of food intake that occurs in response to a reactive stressor. However, elimination of tectal CRF R1 neurons did not increase feeding suggesting that this system may be more important for stress-related vs. baseline feeding. This work was done in partial completion of requirements for the doctoral degree at Texas Tech University (C.P.)

Related Products: CRF-SAP (Cat. #IT-13)

Staib JM, Knox D (2016) The role of cholinergic input from the medial septum in cued and contextual fear extinction memory. Neuroscience 2016 Abstracts 262.11 / III7. Society for Neuroscience, San Diego, CA.

Summary: In classical fear conditioning, a neutral stimulus (CS) is paired with an aversive stimulus (US), causing the animal to associate the US with CS, and display a fear response to the CS. Fear extinction occurs when the CS is presented without the US and the animals learn that the CS no longer predicts the US, thus learning to no longer show fear with CS presentation. Ventral medial prefrontal cortex inhibition of neural activity in basolateral and central amygdala nuclei is critical for extinction memory formation. Recently, we observed that cholinergic lesions in the Medial Septum and Diagonal Bands of Broca (MS/DBB), induced with 192-IgG saporin results in fear extinction memory deficits and contextual fear memory generalization between the conditioning and extinction contexts. While this suggests that MS/DBB cholinergic neurons may be a component of the fear extinction circuit, these neurons project to many brain regions. As a result, the MS/DBB cholinergic efferents that are critical for mediating extinction memory and contextual fear memory discrimination are unknown. The goal of the present study is to isolate the exact MS/DBB efferents that mediate extinction memory and contextual fear memory discrimination. While the study is in progress, some results have been collected. Cholinergic lesions in the dorsal hippocampus, ventral hippocampus, and medial prefrontal cortex have no effects on fear extinction memory or contextual fear memory discrimination. This is surprising because all of these regions are components of the fear extinction circuit and the dorsal hippocampus is critical for contextual learning during acquisition of fear and extinction memory. The MS/DBB also projects to habenula nuclei, and there are cholinergic interneurons in the MS/DBB as well. For the remainder of the study, we explore the potential role of MS/DBB cholinergic input to the habenula and MS/DBB cholinergic interneurons in mediating extinction memory and contextual fear memory discrimination. Isolating a region that has a direct role in mediating extinction memory could help focus future research in fear memory disorders like post traumatic stress disorder.

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

Cho J, Lee J, Jeong D, Kim H, Chang W, Moon J, Chang J (2016) Placenta-derived mesenchymal stem cells facilitate neural and cognitive recovery in dementia rat model. Neuroscience 2016 Abstracts 38.10 / G29. Society for Neuroscience, San Diego, CA.

Summary: Introduction: Dementia is a term that encompasses various types of neurodegenerative diseases of the brain that cause a gradual decline in mental abilities. Loss of cholinergic neurons in the brain cholinergic system including the hippocampus is a hallmark of many dementia cases. In this study, we report the therapeutic effects of administration of human placenta-derived mesenchymal stem cells (pMSCs) in dementia model Sprague-Dawley (SD) rats using two different cell injection methods: intracerebroventricular (ICV) and intravenous (IV) injections. Methods: Dementia modeling was carried out by intraventricular injection of 192 IgG saporin, which causes lesion of cholinergic neurons. Fifty male SD rats were divided into four groups: normal (n=9), lesion (n=9), ICV (n=12) and IV (n=12). All rats were then subject to Morris water maze test and subsequent immunostaining analyses using markers for human cytoplasm, acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and microglial cells at the hippocampus. Results: Lesioned rats showed poor performance in the Morris water maze test compared to the normal rats. Both ICV and IV pMSC administration allowed significant cognitive recovery compared to the lesioned rats. AChE was also significantly recovered back to normal levels at the hippocampus in rats injected with pMSCs post-lesion. ChAT did not co-localize with pMSCs, showing that pMSCs did not directly differentiate into cholinergic cells. Stem cell count showed a significantly greater number of pMSCs at the hippocampal dentate gyrus in IV group rats compared to ICV group rats. Number of microglial cells increased in lesioned rats, and was significantly reduced back to normal levels after pMSC injection. Discussion: Our results demonstrate that injection of pMSCs facilitates recovery of cholinergic neuronal population and function, as well as cognitive behavior. The mechanism through which such recovery happens does not seem to be direct differentiation of injected pMSCs into cholinergic neurons, but rather seems to be through paracrine effects that resemble microglial function. Further research will be necessary for elucidation of the exact mechanisms involved and establishment of optimal parameters for successful cell homing. Acknowledgements: This study was supported by the grant from the Yonsei University Future-leading Research Initiative (Yonsei Challenge) of 2015 (2015-22-0137) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1C1A1A02036851).

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

Urquhart M, Reyes BAS, Thomas SA, Mackie K, Van Bockstaele EJ (2016) Diacylglycerol lipase-α expression increases in the coeruleo-cortical pathway in dopamine-β-hydroxylase knockout mice as well as rats treated with DSP-4. Neuroscience 2016 Abstracts 77.09 / AAA24. Society for Neuroscience, San Diego, CA.

Summary: Endocannabinoids are involved in the regulation of many physiological processes including behavioral responses to stress. Endocannabinoids modulate norepinephrine (NE) signaling primarily via involvement of CB1 cannabinoid receptors (CB1r). Our previous studies have shown that acute and repeated administration of a CB1r agonist increases multiple indices of noradrenergic activity involving the locus coeruleus (LC)-frontal cortex (FC) pathway. Diacylglycerol lipase-α (DGL-α), a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) is localized to both the FC and the LC. Using electron microscopy, we have recently shown that in the rat FC DGL-α is localized in postsynaptic profiles that are targeted by dopamine-β-hydroxylase (DβH), the enzyme that converts dopamine to norepinephrine and represents a marker of noradrenergic neurons (Hartman et al., 1972). In this study, we also described interactions between DGL-α, CB1r and DβH in the FC using confocal microscopy. In the present study, we investigated expression levels of DGL-α under two conditions of NE deletion: in a rat model using a systemic injection of saporin conjugated with antibody against DβH (DSP-4) and in a genetically engineered mouse that lacked the enzyme DβH (DβH-knockout, KO). We compared expression levels of DGL-α to either control rats or wild type (WT) mice using Western blot analysis. Protein extracts from micropunches of FC and LC were obtained and probed for DGL-α. Results showed that DGL-α expression was significantly increased in FC (P < 0.05) of both DSP-4 treated rats and DβHKO mice when compared to WT mice. DGL-α expression was also significantly increased in the LC (P < 0.05) of DβHKO when compared to WT mice. These data add to the accumulating evidence that dysregulation of NE transmission results in significant adaptations in the brain endocannabinoid system.

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