sfn2012

Talman WT, Jones S, Nitschke Dragon D, Lin L-H (2012) Selective damage to glia in the nucleus tractus solitarii attenuates cardiovascular reflexes. Neuroscience 2012 Abstracts 524.05. Society for Neuroscience, New Orleans, LA.

Summary: Lesions of the nucleus tractus solitarii (NTS) are known to attenuate or abolish cardiovascular reflex responses. We have previously reported that lesions produced by saporin (SAP) conjugates and focused on neurons that express the neurokinin-1 (NK1) receptor or on other neurons that express both tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), also attenuate baroreflex function in rats. We found that lesions of both types of neurons also led to loss of glia that stained with glial fibrillary acidic protein (GFAP). Further, we found that injection of SAP alone into the NTS led to loss of GFAP staining while leaving neurons in the region unaffected. Because both of the lesions directed at neurons were made by a toxic conjugate containing SAP, we sought to determine if SAP alone produced changes in cardiovascular reflex function. We found that injection of SAP (3 ng in 100 nl) into the NTS led to loss of the glial marker GFAP as well as connexin 43 (Cx43) immunofluorescent labeling in the NTS but did not affect the neuronal markers NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), TH, DBH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), NK1, and protein gene product 9.5 (PGP 9.5). In animals treated with bilateral injections of SAP into the NTS, reflex responses were decreased during testing of the baroreflex, the chemoreflex, or the von Bezold Jarisch reflex. Comparable decreases in baroreflex responses were seen in animals treated with SAP alone when compared with other animals treated with SAP conjugates that targeted and concentrated damage to TH/DBH neurons or NK1 neurons in NTS. In contrast, when TH/DBH neurons were targeted by the toxin 6-hydroxydopamine (6-OHDA) lability of arterial pressure did not occur as it did in the other SAP and SAP conjugate studies and reflex responses to the activation of the baroreflex, the chemoreflex, and the von Bezold Jarisch reflex did not differ from control. Furthermore, injections containing SAP or a SAP conjugate, but not those containing 6-OHDA, led to lability of arterial pressure as well as cardiac arrhythmias and cardiac myocytolysis. Our studies cannot exclude a physiological effect of SAP on neurons nor can it exclude an indirect effect of glial damage on NTS neurons. However, the similarity of responses when glia seem to have been targeted alone in contrast to those responses when select neuronal types seem to have been targeted suggests that each of the cardiovascular reflexes relies on intact glia in the NTS for full reflex expression.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11)

Mallory CS, Paolone G, Koshy Cherian A, Blakely RD, Sarter M (2012) Paying attention with a compromised cholinergic system: Attenuated activation of cholinergic neurotransmission in attentional task-performing CHT+/- mice. Neuroscience 2012 Abstracts 536.08. Society for Neuroscience, New Orleans, LA.

Summary: Prefrontal cholinergic neurotransmission is necessary for sustained attentional performance. In rats, prefrontal acetylcholine (ACh) release reaches 140% over baseline during the performance of a sustained attention task (SAT; St. Peters et al., 2011a). SAT performance also increases the density of choline transporters (CHT) in synaptic plasma membranes (Apparsundaram et al., 2005), which we hypothesize is needed to sustain elevations of cholinergic activity and behavioral responses. Here we employed the SAT recently adapted for use in mice (St. Peters et al., 2011b) and developed new techniques that permit monitoring of ACh release via microdialysis of mice performing the SAT in order to determine the impact of genetically manipulated levels of choline transporter capacity. First, reverse dialysis of atropine (50 µM) increased ACh release levels in naive WT mice. In contrast, CHT+/- mice could not sustain these increases, consistent with changes observed in levels of muscarinic receptors in the CHT +/- mice (Bazalakova et al., 2007). However, SAT performance did not differ significantly between WT controls and CHT+/- mice. Furthermore, basal (absolute) levels of ACh release were comparable between strains. However, performance-associated increases in ACh release were strikingly attenuated in CHT+/- mice, reaching 40% over basal levels versus 130% in WT. Performance-associated increases in ACh release in CHT+/- mice were TTX-sensitive, similar to release monitored in WT mice (1 µM via reversed dialysis). To determine whether cholinergic activity was necessary for SAT performance in CHT+/- mice we then removed basal forebrain cholinergic neurons by infusing murine-p75NTR-saporin obtaining similar impairment on SAT performance in both strains. Finally, and because cholinergic activity modulates cortical circuitry primarily via nAChR, mecamylamine (MEC; 50 µM) was reverse dialyzed during SAT performance. WT mice were only moderately impaired in the SAT task, whereas the performance of CHT+/- mice rapidly declined and the performance-associated ACh levels rapidly returned to the pre-task levels. In summary CHT+/- mice are able to perform the basic SAT, despite attenuated levels of cholinergic neurotransmission, likely as a result of compensatory postsynaptic mechanisms. However, their attentional performance and underlying cholinergic signaling exhibit heightened sensitivity to behavioral and pharmacological challenges. Together, these findings suggest that CHT+/- mice are an important model for the impaired cognitive control of attentional performance that is a common symptom of ADHD, schizophrenia and other cognitive disorders.

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Krajewski-Hall SJ, Mittelman-Smith MA, Williams H, Lafrance KJ, Mcmullen NT, Rance NE (2012) A role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the regulation of estrous cycles and the estrogen modulation of body temperature. Neuroscience 2012 Abstracts 585.02. Society for Neuroscience, New Orleans, LA.

Summary: We have recently described a method to selectively ablate kisspeptin/neurokinin B/dynorphin (KNDy) neurons using stereotaxic injections of NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (Mittelman-Smith, Endocrinology, 2012). These studies revealed a critical role for arcuate KNDy neurons in tonic gonadotropin secretion, the rise in serum LH after ovariectomy and estrogen modulation of body weight. Here we determine the effects of KNDy neuron ablation on estrous cycles and the estradiol modulation of body temperature. In the first study, stereotaxic injections of NK3-SAP or Blank-SAP were made in the arcuate nucleus of ovary-intact, adult female rats. Rats with nearly complete KNDy-neuron ablation (verified by NKB immunohistochemistry) exhibited constant diestrus and ovarian atrophy, confirming the importance of these neurons in reproductive regulation. In a second experiment, we evaluated the effects of KNDy neuron ablation on the thermoregulatory axis in rats that were ovariectomized (OVX) and then treated with 17β-estradiol (E2). Tail skin temperatures (TSKIN) and core temperatures (TCORE) were recorded in rats throughout the light/dark cycle and during exposure to different ambient temperatures (TAMBIENT) in an environmental chamber. Notably, the average TSKIN of KNDy-ablated rats was consistently lower than control rats, indicative of lower levels of cutaneous vasodilatation. Moreover, KNDy neuron ablation blocked the reduction of TSKIN by E2 that occurred during the light phase in the environmental chamber, but did not affect the E2 suppression of TSKIN during the dark phase. At a high TAMBIENT of 33 C, the mean TCORE of OVX control rats increased to 39.0 C, and was reduced by E2 replacement. In contrast, at this high TAMBIENT, the average TCORE of OVX, KNDy-ablated rats was lower than OVX control rats, and TCORE was not altered by E2 replacement. Because KNDy neurons exhibit dramatic changes in morphology and gene expression in postmenopausal women, we have hypothesized these neurons contribute to the generation of hot flushes. These studies support this hypothesis by providing the first evidence that KNDy neurons participate in the E2 modulation of body temperature and promote cutaneous vasodilatation, one of the cardinal signs of a hot flush.

Related Products: NKB-SAP (Cat. #IT-63), Blank-SAP (Cat. #IT-21)

Anderson ML, Govindaraju KP, Shors TJ (2012) Acetylcholine and Learning: Are they related and does it matter for associating events across time?. Neuroscience 2012 Abstracts 600.12. Society for Neuroscience, New Orleans, LA.

Summary: Decades ago, acetylcholine was considered intrinsic to processes related to attention and/or learning and memory. Much of this was based on its presumed role in dementia associated with Alzheimer’s disease. However, in the last decade or so, this relationship has been questioned and with good reason (Parent & Baxter, 2004). 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. This type of task is dependent on the hippocampus and is severely disrupted in both patients with Alzheimer’s disease and animal models of the disorder (Kishimoto, 2012; Waddell et al., 2008; Woodruff-Pak & Papka, 1996). In the present study, we hypothesized that animals with minimal Ach input to both hippocampi would not learn whereas those with input into one hippocampus could. The immunotoxin 192 IgG-Saporin was infused into the MSDB to selectively kill cholinergic neurons in Sprague-Dawley rats and then trained with either delay or trace eyeblink conditioning. Delay conditioning requires that the stimuli during training are contiguous in time and is not dependent on the hippocampus. Animals were given 200 trials for four days for 800 trials in total. A complete bilateral MSDB-cholinergic lesion was considered complete if the number of neurons that express choline acetyltransferase was reduced by 75 %. A bilateral lesion of this magnitude prevented early acquisition of the trace response (p<.05). Indeed, none of the animals so far trained reached a learning criterion of 60 % CRs during any session of training. In contrast, animals with a loss of ACh in just one hemisphere were able to learn the CR. Furthermore, preliminary data suggest delay conditioning was unaffected by the loss of ACh from the septum. Finally, animals with half the number of cholinergic neurons were still able to learn trace eyeblink conditioning regardless of whether the damage was bilateral or unilateral. Thus, it would appear that the progressive loss of ACh coincides with the loss of learning potential, especially when that learning requires associations across time. This approach and the experimental results may model the progressive nature of Alzheimer’s disease, in which the loss of neuronal function is slow but cumulative.

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

Grupe M, Paolone G, Jensen AA, Nielsen KS, Christensen JK, Grunnet M, Sarter M (2012) Positive allosteric modulation of 4 2* nicotinic acetylcholine receptors augments the amplitudes of prefrontal nicotine-evoked glutamatergic transients. Neuroscience 2012 Abstracts 696.15. Society for Neuroscience, New Orleans, LA.

Summary: α4β2* nicotinic acetylcholine receptors (nAChR) are a promising target for cognition enhancement. These receptors have been demonstrated to mediate the modulatory effects of the tonic component of cholinergic neurotransmission on fast prefrontal glutamatergic-cholinergic interactions. Specifically, α4β2* nAChR are expressed by thalamic glutamatergic afferents and amplify cue-evoked glutamatergic release events, thereby initiating a chain of neuronal events required for the detection of cues in attention tasks (Hasselmo & Sarter, 2011). In this study we investigated the effect of NS9283, a potent and selective positive allosteric modulator of low-sensitivity α4β2 nAChR (Timmermann et al., 2012), on nicotine-evoked glutamatergic release events in the mPFC of anaesthetized rats. Glutamatergic transients were recorded using amperometric measures of currents generated by the oxidation of glutamate and, subsequently, peroxide, on Platinum electrodes equipped with immobilized glutamate oxidase (see Parikh et al., 2010). Nicotine was pressure-ejected (0.040-2 nmol in 40-100 nL, respectively) into the vicinity of the recording electrode situated in the thalamic input layer of the prelimbic cortex. Systemic (i.p.) administration of NS9283 (3.0 mg/kg; administered 30 min prior to nicotine) enhanced the amplitude of glutamatergic transients evoked by the lowest dose of nicotine (40 pmol) by 72%. The modulator did not increase the efficacy of nicotine. Local pressure-ejections of NS9283 (400 pmol in 40 nL) per se were capable of evoking glutamatergic release events, presumably reflecting modulation of the effects of endogenous acetylcholine at these nAChRs. Accordingly, 192 IgG saporin-induced removal of cholinergic projections to the recording region abolished NS9283-evoked glutamatergic transients. Collectively, this evidence substantiates the identification of NS9283 as a positive modulator of nAChRs and its potency in vivo to modulate evoked glutamatergic release events. These results are consistent with the hypothesis that such compounds facilitate cue detection processes and thereby enhance attentional performance. Supported by NIH grant MH080332 and The Ministry of Science, Innovation and Higher Education, Denmark, PhD grant 10-084289.

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

Kalinchuk AV, Kim S, Mccarley RW, Basheer R (2012) Cholinergic basal forebrain neurons contribute to the biochemical and electrophysiological changes in the cortex during sleep deprivation. Neuroscience 2012 Abstracts 486.24. Society for Neuroscience, New Orleans, LA.

Summary: Short term sleep deprivation (SD) (2-3h) increases the levels of inducible nitric oxide (NO) synthase (iNOS)-mediated NO and adenosine (AD) in the basal forebrain (BF) (Basheer et al., 1999; Kalinchuk et al., 2006). We showed recently that the prolongation of SD for 5h triggers similar biochemical changes in the prefrontal/frontal cortex (PFC/FC), the area which receives projections from the BF (Kalinchuk et al., 2010). Lesion of the BF cholinergic cells using immunotoxin 192-IgG saporin attenuates SD-induced AD increase in the BF and recovery non-rapid eye movement (NREM) sleep response (Kalinchuk et al., 2008). However, it is not known whether the cholinergic cells play a role in SD-induced biochemical changes in the cortex. In the current study we lesioned BF cholinergic cells, and compared SD-induced biochemical changes simultaneously in the PFC and BF in the same animals before and after the lesion. We correlated the changes in the biochemical markers, NO and adenosine, with the changes in electrophysiological markers of homeostatic sleep pressure, encephalogram (EEG) theta power during SD and delta power during recovery NREM sleep after SD. Male rats were implanted with electrodes for EEG/electromyogram (EMG) recording and 2 guide cannulae for microdialysis probes targeting BF and PFC. Microdialysis samples were collected simultaneously from both areas every 30 min during 8h SD. Dialysates were analyzed for AD using high performance liquid chromatography (HPLC)/fluorescent detection and for NO metabolites nitrate and nitrite (NOx) using Fluorimetric Assay Kit (Cayman). The lesion of the BF cholinergic cells was performed using the local injections of 192-IgG saporin into the BF, and similar experiment was repeated 2 weeks after the injection. Histochemical analysis confirmed the localization of the probes in the BF and PFC and the quality of the lesion procedure. Before saporin injection, SD induced increases in the levels of NOx and AD, which became significant after 1h (NOx) and 2h (AD) of SD in the BF and after 4h (NOx) and 5h (AD) of SD in the FC. EEG recording detected increases in the intensity of theta power during SD and delta power during following recovery NREM sleep. 2 weeks after saporin injection, SD-induced changes in NOx and AD were significantly attenuated both in the BF and the PFC. Also the increases in theta and delta power were significantly attenuated. We conclude that cholinergic neurons of the BF, which provide strong activating input to the PFC, contribute to the generation of homeostatic sleep pressure during SD, including its biochemical and electrophysiological correlates.

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

Zushida K, Light K, Uchida S, Hevi C, Shumyatsky GP (2012) The GRP peptide and the GRPR-positive interneurons control fear acquisition and extinction. Neuroscience 2012 Abstracts 496.03. Society for Neuroscience, New Orleans, LA.

Summary: The gastrin releasing peptide (GRP) is the marker of the neural circuits relaying fear-related conditioned stimulus (CS) information to the amygdala. The GRP is expressed by principal cells and the GRP-receptor (GRPR) is expressed by interneurons. The GRPR is expressed in the amygdala and hippocampus. To examine the role of the GRPR-positive interneurons in these two brain areas, we performed local injections of the bombesin-saporin (SAP)-toxin, which selectively eliminates the GRPR-expressing cells. The intra-BLA [lateral (LA) and basal nuclei (BA) of amygdala] injection of bombesin-SAP before fear conditioning significantly enhanced cued, but not contextual fear memory. We did not observe any significant effect of post-training intra-BLA injections of bombesin-SAP on fear memory recall. Also, there were no significant effects of bombesin-SAP on acquisition of contextual and cued fear memory in mice injected bombesin-SAP into LA, BA and central amygdala (CeA), respectively. Also, we examined cued fear memory in the GRP knockout mice and found significant enhancement in their cued fear memory. These results support the idea that GRPR-expressing interneurons play an inhibitory role in acquisition of fear memory and suggested inhibitory effect by the GRPR-expressing GABA interneurons on fear memory requires both LA and BA but not CeA.

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ATS Poster of the Year Winner

Lee J, Jeong D, Chang J (2012) The effects of basal forebrain cholinergic neuron of recognition tests. Neuroscience 2012 Abstracts 345.10. Society for Neuroscience, New Orleans, LA.

Summary: The cholinergic neurons of the Medial septum and the basal nucleus areas of the basal forebrain project to the frontal cortex and the Hippocampus, and degeneration of the cholinergic basal forebrain neuron is a common feature of Alzheimer’s disease(AD) and vascular dementia and it has been correlated with cognitive decline. This research studied to verify the effects of cholinergic neuron in basal forebrain and the role of hippocampus and frontal cortex on recognition through recognition test and immunohistochemistry after damaging cholinergic neuron of the basal forebrain by intraventricular injection of 192 IgG-saporin. 192 IgG-saporin of 8ul (0.63ug/ul) was injected to the bilateral lateral ventricle of rats. After 2 weeks, Novel object recognition (NOR) test and Object in place (OIP) test was conducted to elucidate damage of cholinergic neuron. After completing the behavioral test, the ChAT cholinergic neuron in the brain was ascertained to confirm with immunohistochemistry if cholinergic neuron was damaged. In NOR test, the lesion group with 192 IgG-saporin showed 10% lower novel object preference than normal group. In OIP test, the normal group showed 50% novel object preference and the lesion group with 192 IgG-saporin showed 30% novel object preference in an hour delay test. On the other hand, the normal group and the lesion group with 192 IgG-saporin shoed 33% and 35% novel object preference respectively in a day delay test. However, this rate is not that significant value enough to elucidate behavioral difference between normal group and lesion group. In immunohistochemistry, the number of cholinergic neuron was remarkably decreased in basal forebrain. According to both of the behavioral tests, lesion group seem to less remember novel object than normal group. Also, they searched less the novel object that changed its location than normal group in the short term condition. However, there was no significant difference in the long term condition. These results suggest that the lesion with 192 IgG-saporin can damage spatial working memory.In the Immunohistochemistry result of the lesion condition, cholinergic input to hippocampus in basal forebrain affects recognition. However, the effect is not so essential.

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

Lee S-Y, Ma J, Chung C, Han J-S (2012) Effects of chronic stress on alterations of GR-PKA-NF-kappa B signaling and spatial learning in rats with cholinergic deafferentation. Neuroscience 2012 Abstracts 345.20. Society for Neuroscience, New Orleans, LA.

Summary: Aging and Alzheimer’s disease (AD) is associated with diminished integrity of the cholinergic innervations of the hippocampus and cortex. Previously, we demonstrated that removal of the cholinergic innervations impaired regulation of the HPA axis with response to acute stress and induced changes in the interaction among glucocorticoid receptor (GR), nuclear factor-κB (NF- κB) p65, and the cytoplasmic catalytic subunit of protein kinase A (PKAc) in the hippocampus. The current research examined effects of chronic stress on the altered signaling induced by cholinergic deafferentation. 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. Rats with only cholinergic deafferentation or sham-operated rats with chronic stress showed intact spatial learning. Rats with cholinergic deafferentation that received chronic stress showed impairments of spatial learning. And we examined that cholinergic deafferentation induced alterations in GR and NF- κB p65 expression in hippocampus and prefrontal cortex. Thus the loss of cholinergic integrity during aging and in AD may increase proneness to chronic stress.

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Fricks-Gleason AN, Keefe KA (2012) Poster: Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity. Neuroscience 2012 Abstracts 360.06. Society for Neuroscience, New Orleans, LA.

Summary: It is well established that exposure to multiple high doses of methamphetamine (METH) produces damage to central monoamine systems. A number of factors, including the production of nitric oxide (NO), have been implicated in this neurotoxicity. While it is relatively clear that NO contributes to METH-induced neurotoxicity to the dopamine (DA) nerve terminal, the source of this NO has not been clearly delineated. There is considerable evidence suggesting that the generation of NO arises a consequence of the activation of neuronal nitric oxide synthase (nNOS). In striatum, nNOS is located post-synaptic to the DA nerve terminal in a subpopulation of striatal interneurons. Thus, we have hypothesized that DA-mediated activation of the nNOS-containing striatal interneurons is necessary for METH-induced neurotoxicity. These interneurons, along with the cholinergic neurons of striatum, selectively express the neurokinin-1 (NK-1) receptor, which is activated by the neuropeptide Substance P. Consequently, toxins targeted to NK-1 receptor-containing neurons can be used to lesion this population of striatal interneurons. One such toxin, a conjugate of Substance P to the ribosome inactivating protein saporin (SSP-SAP), has been shown to be effective in selectively destroying neurons expressing the NK-1 receptor in striatum. Therefore, using targeted deletion of the nNOS-containing interneurons via SSP-SAP, we examined the extent to which impairing post-synaptic production of NO attenuates METH-induced neurotoxicity. The SSP-SAP lesions resulted in a significant and selective loss of nNOS-containing interneurons throughout the striatum, although it was not possible to completely eliminate all of the neurons. Surprisingly, however, this marked deletion of nNOS-containing interneurons did not confer resistance to METH-induced DA neurotoxicity, even in areas completely devoid of nNOS-positive cell bodies and histochemical detection of NOS activity with NADPH diaphorase histochemical staining. Furthermore, these lesions did not attenuate NO production, as assessed via nitrotyrosine immunohistochemistry, even in areas devoid of nNOS. Taken together, these data suggest that nNOS-containing interneurons either are not necessary for METH-induced DA neurotoxicity, leaving open the potential contribution of other sources of NO, such as endothelial NOS (eNOS), or produce NO/RNS that can diffuse extensively through striatal tissue and thereby still mediate the neurotoxicity.

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