Anti-choline Acetyltransferase Research Articles

The role of the Ventral Nucleus of the Trapezoid Body in the auditory prepulse inhibition of the acoustic startle reflex

Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways.

A Semi-Three-Dimensional Bioprinted Neurocardiac System for Tissue Engineering of a Cardiac Autonomic Nervous System Model.

In this study, we designed a tissue-engineered neurocardiac model to help us examine the role of neuronal regulation and confirm the importance of neural innervation techniques for the regeneration of cardiac tissue. A three-dimensional (3D) bioprinted neurocardiac scaffold composed of a mixture of gelatin-alginate and alginate-genipin-fibrin hydrogels was developed with a 2:1 ratio of AC16 cardiomyocytes (CMs) and retinoic acid-differentiated SH-SY5Y neuronal cells (NCs) respectively. A unique semi-3D bioprinting approach was adopted, where the CMs were mixed in the cardiac bioink and printed using an anisotropic accordion design to mimic the physiological tissue architecture in vivo. The voids in this 3D structure were methodically filled in using a NC-gel mixture and crosslinked. Confocal fluorescent imaging using microtubule-associated protein 2 (MAP-2) and anticholine acetyltransferase (CHAT) antibodies for labeling the NCs and the MyoD1 antibody for the CMs revealed functional coupling between the two cell types in the final crosslinked structure. These data confirmed the development of a relevant neurocardiac model that could be used to study neurocardiac modulation under physiological and pathological conditions.

Neuroprotective potential of sevoflurane against isoflurane induced cognitive dysfunction in rats via anti-inflammatory and antioxidant effect.

Intravenous anesthetics have excellent analgesic activity without inducing the side effect in the respiratory system. The aim and objective of the current experimental study was to access the neuroprotective effect of sevoflurane against isoflurane induced cognitive dysfunction in rats. Isoflurane was used for induction the neurodysfunction in the rats, and rats received the oral administration of sevoflurane (2.5, 5 and 10 mg/kg). Morris water test was carried out for the estimation of cognitive function. Neurochemical parameters, antioxidant parameters and pro-inflammatory cytokines were also estimated. Sevoflurane significantly (P < 0.001) altered the neurochemical parameters such as anti-choline acetyltransferase, acetylcholine esterase, acetylcholine, protein carbonyl, choline brain-derived neurotrophic factor, and amyloid β; antioxidant parameters such as glutathione, superoxide dismutase, and malondialdehyde; pro-inflammatory cytokines include interleukin (IL-2, IL-10, IL-4, IL-6, IL-10, IL-1β), and tumor necrosis factor-α. Sevoflurane significantly reduced the activity of caspase-3. Sevoflurane exhibited the neuroprotection against the cognitive dysfunction in rats via anti-inflammatory and antioxidant mechanism.

The Impact of Omega‐3 Fatty Acids on Cardiac Autonomic Nerve Fiber Innervation in High Fat‐Fed Mice

Peripheral neuropathy and cardiac autonomic neuropathy (CAN) are common and serious complications of diabetes, characterized by loss of peripheral nerves that innervate the skin and loss of parasympathetic and sympathetic nerves that innervate the heart, respectively. CAN is associated with increased risk of myocardial infarction and mortality, yet often goes undetected in patients. Both prediabetic and diabetic patients develop peripheral neuropathy and CAN, suggesting that the level of hyperglycemia at which peripheral neuropathy and CAN develop precedes the onset of diabetes. Although evidence from human and rodent studies indicate that omega-3-fatty acids prevent and/or reverse the development of peripheral neuropathy, the effects of omega-3-fatty acids on CAN has not been investigated. PURPOSE Assess the effects of omega-3-fatty acids on cardiac autonomic nerve fiber innervation in high fat diet-induced prediabetic mice. We hypothesize that omega-3 fatty acids will attenuate a reduction in cardiac autonomic nerve fiber innervation in high fat-fed mice. METHODS Male C57BL/6 mice were randomized into four diet groups (n = 12-14/group) for 14 weeks: 10% low fat fish oil (LFFO), 54% high fat-fish oil (HFFO), 10% low fat-lard (LFL), or 54% high fat-lard (HFL). The von Frey behavioral test for hindpaw mechanical sensitivity was used to characterize peripheral neuropathy at baseline then biweekly for 14 weeks. At study completion, fasting glucose and insulin values were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Mice were anesthetized, transcardially perfused with 1% PBS, and heart tissue was harvested and frozen in O.C.T. Heart was sectioned in three planes (anterior coronal, posterior coronal, and sagittal) and mounted on slides. Fluorescence immunohistochemistry was performed on heart cryosections using anti-choline acetyl-transferase and anti-tyrosine hydroxylase followed by Alexa Fluor secondary antibodies to visualize parasympathetic and sympathetic nerve fibers. For each mouse, nerve fibers were quantified on three slides (1 from each plane) and at least 3 areas with the highest nerve fiber density for each slide were examined. Nerve fiber density was determined using Nikon Elements and was calculated as the total length of all counted nerve fibers divided by the total area examined (µm/mm2). RESULTS Body weight, fasting insulin and glucose, and HOMA-IR were higher in HFL compared to all other groups. Fasting glucose was also higher in LFL compared to LFFO. Hindpaw withdrawal threshold was lower in HFL compared to HFFO, indicative of hindpaw hypersensitivity in HFL. CONCLUSIONS Omega-3 fatty acids prevented excess body weight, insulin resistance, elevated glucose, and hindpaw hypersensitivity in mice fed a high fat diet. Based on this data, we expect a diet rich in omega-3 fatty acids will attenuate cardiac autonomic nerve fiber denervation. Taken together, these findings would suggest that omega-3 fatty acids might hold therapeutic potential in the prevention of metabolic derangements as well as peripheral and CAN in individuals who consume a high fat, high calorie diet.

Anatomical study of the masseteric and obturator nerves: Application to face transplant and reanimation procedures.

The masseteric nerve (MN) and the anterior branch of the obturator nerve (ON) that innervate the transferred gracilis muscle have proved highly efficient for reanimating paralyzed facial muscles when muscle transfer is required. Previous researchers have published the total axonal load for myelinated fibers in both nerves. However, the real motor axonal load has not been established. We performed the study on 20 MN and 13 ON. The segments of the MN and the ON were embedded in paraffin, sectioned at 10 μm, and stained following a standard immunohistochemical procedure using anti-choline acetyltransferase to visualize the motor fibers. The MN has a higher axonal load than the ON. There were statistically significant differences between the axonal load of the proximal segment of the MN and the ON. These findings confirm that end-to-end anastomoses between the MN and the ON should preferably use the proximal segment. However, MN neurotomy should ideally be performed between the proximal and distal segments, preserving innervation to the deep fascicles. Our results show that the MN is ideal as a donor motor nerve for reinnervating transplanted muscle for dynamic reanimation of the paralyzed face. The neurotomy should ideally be performed between the first and second collateral branches of the MN. Clin. Anat. 32:612-617, 2019. © 2019 Wiley Periodicals, Inc.

Peri-arterial Autonomic Innervation of the Human Ear

Auricular vasomotor responses are considered to be signs of clinical conditions including migraine. The mechanisms of auricular vasomotor control are still debatable. This study aimed at investigating perivascular co-transmitters of vasomotor control in the auricle. Another aim was to provide three-dimensional arterial maps of the auricle, as a proxy of periarterial autonomic innervation. Twelve paired human auricles were used to visualize the arteries following Spalteholz clearing and μ-CT-based reconstruction. Perivascular innervation staining was conducted using anti-tyrosine hydroxylase (TH), anti-neuropeptide Y (NPY), anti-vasoactive intestinal peptide (VIP) and anti-choline acetyl transferase (ChAT). The combined Spalteholz technique and μ-CT revealed a highly consistent arrangement of the auricular vasculature. The superficial temporal (STA) and posterior auricular artery (PAA) supply the helical rim arcade and arcade, with the STA mainly forming the superior and the PAA forming the middle and inferior auricular artery. Co-existence of sympathetic NPY+ and TH+ terminals mediating vasoconstriction, and VIP+ and ACh+ indicating cholinergic vasodilatation, was found in the perivascular zone. The presence of both sympathetic vasoconstriction and cholinergic co-innervation for active vasodilatation was shown in the perivascular auricular zone. Assuming that the highly-consistent vasculature gives way to these terminals, this periarterial innervation may be found spread out across the helix.

Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington's Disease.

Poly (ADP-ribose) polymerases (PARPs) are enzymes that catalyze ADP-ribose units transfer from NAD to their substrate proteins. It has been observed that PARP-1 is able to increase both post-ischemic and excitotoxic neuronal death. In fact, we have previously shown that, INO-1001, a PARP-1 inhibitor, displays a neuroprotective effect in the R6/2 model of Huntington’s disease (HD). In this study, we investigated the effects of PARP-1-inhibition on modulation of phosphorylated c-AMP response element binding protein (pCREB) and CREB-binding protein (CBP) localization in the different striatal neuronal subsets. Moreover, we studied the neurodegeneration of those interneurons that are particularly vulnerable to HD such as parvalbuminergic and calretininergic, and of other subclasses of interneurons that are known to be resistant, such as cholinergic and somatostatinergic interneurons. Transgenic mice were treated with INO-1001 (10 mg/Kg daily) starting from 4 weeks of age. Double-label immunofluorescence was performed to value the distribution of CBP in ubiquitinated Neuronal intranuclear inclusions (NIIs) in the striatum. INO-1001-treated and saline-treated brain sections were incubated with: goat anti-choline acetyl transferase; goat anti-nitric oxide synthase; mouse anti-parvalbumin and mouse anti-calretinin. Morphometric evaluation and cell counts were performed. Our study showed that the PARP inhibitor has a positive effect in sparing parvalbumin and calretinin-containing interneurons of the striatum, where CREB was upregulated. Moreover, INO-1001 promoted CBP localization into the nuclei of the R6/2 mouse. The sum of our data corroborates the previous observations indicating PARP inhibition as a possible therapeutic tool to fight HD.

Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BackgroundRetinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.MethodsHigh intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.ResultsThe ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).ConclusionTreatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

Valsartan Reduced Atrial Fibrillation Susceptibility by Inhibiting Atrial Parasympathetic Remodeling through MAPKs/Neurturin Pathway

Background/Aims: Angiotensin II receptor blockers (ARBs) have been proved to be effective in preventing atrial structural and electrical remodelinq in atrial fibrillation (AF). Previous studies have shown that parasympathetic remodeling plays an important role in AF. However, the effects of ARBs on atrial parasympathetic remodeling in AF and the underlying mechanisms are still unknown. Methods: Canines were divided into sham-operated, pacing and valsartan + pacing groups. Rats and HL-1 cardiomyocytes were divided into control, angiotensin II (Ang II) and Ang II + valsartan groups, respectively. Atrial parasympathetic remodeling was quantified by immunocytochemical staining with anti-choline acetyltransferase (ChAT) antibody. Western blot was used to analysis the protein expression of neurturin. Results: Both inducibility and duration were increased in chronic atrial rapid-pacing canine model, which was significantly inhibited by the treatment with valsartan. The density of ChAT-positive nerves and the protein level of neurturin in the atria of pacing canines were both increased than those in sham-operated canines. Ang II treatment not only induced atrial parasympathetic remodeling in rats, but also up-regulated the protein expression of neurturin. Valsartan significantly prevented atrial parasympathetic remodeling, and suppressed the protein expression of neurturin. Meanwhile, valsartan inhibited Ang II -induced up-regulation of neurturin and MAPKs in cultured cardiac myocytes. Inhibition of MAPKs dramatically attenuated neurturin up-regulation induced by Ang II. Conclusion: Parasympathetic remodeling was present in animals subjected to rapid pacing or Ang II infusion, which was mediated by MAPKs/neurturin pathway. Valsartan is able to prevent atrial parasympathetic remodeling and the occurrence of AF via inhibiting MAPKs/neurturin pathway.

P.1.b.006 Role of striatal cholinergic interneurons in attention during learning in the rat

Aim of this study: Attentional set-shifting is a kind of flexible behaviour in which attention shifts from one stimulus dimension to another stimulus dimension. Many pieces of evidence suggest that the striatum plays a role in the set-shifting [1,2]. We investigated the hypothesis that cholinergic interneurons (CINs) play a role in such flexible behavior, using a set-shifting paradigm and immunotoxin-induced lesions of the CINs. Materials and Methods: We used Long-Evans rats (n = 42) weighing 250–300 g on arrival. All procedures were approved by OIST Animal Care and Use Committee. Control and lesion groups were prepared for each of three behavioural conditions (n = 7 in each group). Immunotoxin (anti-cholineacetyl transferase saporin) was bilaterally injected into the dorsomedial striatum (DMS), causing a specific lesion of the CINs while leaving other types of cells intact [3]. After recovery, instrumental conditioning was commenced in operant chambers. Throughout the experiments, each daily session consisted of 80 trials. In the initial 4 sessions, both left and right levers were inserted and animals learnt to press either the left or right lever to obtain a sucrose pellet, during which the light stimulus was differently presented as follows: condition 1, no light given; condition 2, a light presented always above the correct lever; condition 3, a light presented randomly on either side. In the subsequent 10 sessions, the light stimulus was randomly presented on either the left or right lever and animals learnt to choose a correct lever cued by the light (visual-cue learning). These procedures enabled us to examine different types of attentional shift to the light during visual-cue learning based on attention to: a novel stimulus; a previously relevant stimulus; or a previously irrelevant stimulus. Percentages of correct responses in each session were analyzed. After completion of the behavioural experiments, we analyzed the lesion histologically. Results: Comparing the percentages of correct responses in visual-cue learning between control groups showed that learning was faster in the condition when the light cue was previously relevant than when there was no light cue or the light was previously irrelevant. Lesions of the CINs impaired switching during visual-cue learning in the condition when the light cue was previously irrelevant, which required the animals to attend to a previously ignored stimulus. On the other hand, the lesion did not have a prominent effect on visual-cue learning under previously no-cue or relevant-cue conditions. Lesioned rats finally acquired visual-cue learning at 10th session in all conditions. Conclusions: The present results suggest that the CINs in DMS play an important role in attending to a previously irrelevant stimulus during set-shifting. However, these neurons do not strongly contribute to attentional processes when attending to a previously relevant or a novel stimulus for shifting attentional set during learning.

THE TIME COURSE OF ELECTRICAL REMODELLING, STRUCTURAL REMODELLING, NEURAL REMODELLING AND ENDOTHELIN-1 LEVEL IN ATRIAL FIBRILLATION

ObjectivesThe consistency between electrical remodelling, structural remodelling, neural remodelling and endothelin-1 level in atrial fibrillation (AF) has not been reported. The present study aimed to investigate the time course of...

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.

Hypocretin1/OrexinA Axon Targeting of Laterodorsal Tegmental Nucleus Neurons Projecting to the Rat Medial Prefrontal Cortex

Cortical activation and goal-directed behaviors characterize wakefulness. One cortical region especially involved in these phenomena is the medial prefrontal cortex (mPFC), which receives many inputs from cholinergic-containing neurons in brain stem structures implicated in arousal and wakefulness, such as the laterodorsal tegmental nucleus (LDT). Hypocretins/orexins (Hcrt/Ox), whose dysfunction is linked to narcolepsy, maintains arousal and stabilizes sleep-wakefulness states. We aim to determine if Hcrt1/OxA axons (1) innervate LDT neurons projecting to the mPFC, a target that would allow them to sustain arousal and wakefulness, and (2) target preferentially cholinergic versus noncholinergic LDT neurons. The retrograde tracer Fluorogold (FG) was injected in the rat mPFC, and dual immunolabeling of anti-FG and either anti-choline acetyltransferase (ChAT) or anti-Hcrt1/OxA antisera was determined in LDT. Also, actual Hcrt1/OxA targeting of cholinergic LDT neurons was ascertained by dual anti-Hcrt1/OxA and anti-ChAT detection in additional noninjected animals. Many LDT FG-labeled neurons were cholinergic (52.05 ± 3.72%). Hcrt1/OxA immunoprecipitate was observed in cytoplasm and granular vesicles within axons. Some Hcrt1/OxA-containing axons established asymmetric excitatory-type synapses with either unlabeled (46/438) or FG-labeled (7/438) dendrites. One-third of the target neurons were ChAT labeled. Hcrt1/OxA excitatory input to LDT neurons projecting to mPFC probably contributes to the wakefulness-enhancing actions of Hcrt/Ox impaired in narcoleptics.

SK-PC-B70M alleviates neurologic symptoms in G93A-SOD1 amyotrophic lateral sclerosis mice

SK-PC-B70M, an oleanolic-glycoside saponins fraction extracted from the root of Pulsatilla koreana, carries active ingredient(s) that protects the cytotoxicity induced by Aβ(1–42) in SK-N-SH cells. It was recently demonstrated that SK-PC-B70M improved scopolamine-induced deficits of memory consolidation and spatial working memory in rats, and reduced Aβ levels and plaque deposition in the brains of the Tg2576 mouse model of Alzheimer disease. In the present study, we investigated whether SK-PC-B70M produces helpful effects on the pathology of the G93A-SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS). Administration of SK-PC-B70M (100 or 400 mg/kg/day) from 8 weeks to 16 weeks of age attenuated neurological deficits of G93A-SOD1 mice in several motor-function-related behavioral tests. SK-PC-B70M treatment significantly suppressed the accumulation of the by-products of lipid peroxidation, malonedialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), in the spinal cord of G93A-SOD1 mice. Moreover, histologic analysis stained with cresyl violet or anti-choline acetyltransferase (ChAT) revealed that SK-PC-B70M suppressed neuronal loss in the ventral horn of the spinal cords of G93A-SOD1 mice. These results suggest that SK-PC-B70M affords a beneficial effect on neurologic deficits of G93A-SOD1 ALS mice.

Physiological properties of cholinergic and non-cholinergic magnocellular neurons in acute slices from adult mouse nucleus basalis.

BackgroundThe basal forebrain is a series of nuclei that provides cholinergic input to much of the forebrain. The most posterior of these nuclei, nucleus basalis, provides cholinergic drive to neocortex and is involved in arousal and attention. The physiological properties of neurons in anterior basal forebrain nuclei, including medial septum, the diagonal band of Broca and substantia innominata, have been described previously. In contrast the physiological properties of neurons in nucleus basalis, the most posterior nucleus of the basal forebrain, are unknown.Methodology/Principal FindingsHere we investigate the physiological properties of neurons in adult mouse nucleus basalis. We obtained cell-attached and whole-cell recordings from magnocellular neurons in slices from P42-54 mice and compared cholinergic and non-cholinergic neurons, distinguished retrospectively by anti-choline acetyltransferase immunocytochemistry. The majority (70–80%) of cholinergic and non-cholinergic neurons were silent at rest. Spontaneously active cholinergic and non-cholinergic neurons exhibited irregular spiking at 3 Hz and at 0.3 to 13.4 Hz, respectively. Cholinergic neurons had smaller, broader action potentials than non-cholinergic neurons (amplitudes 64±3.4 and 75±2 mV; half widths 0.52±0.04 and 0.33±0.02 ms). Cholinergic neurons displayed a more pronounced slow after-hyperpolarization than non-cholinergic neurons (13.3±2.2 and 3.6±0.5 mV) and were unable to spike at high frequencies during tonic current injection (maximum frequencies of ∼20 Hz and >120 Hz).Conclusions/SignificanceOur results indicate that neurons in nucleus basalis share similar physiological properties with neurons in anterior regions of the basal forebrain. Furthermore, cholinergic and non-cholinergic neurons in nucleus basalis can be distinguished by their responses to injected current. To our knowledge, this is the first description of the physiological properties of cholinergic and non-cholinergic neurons in the posterior aspects of the basal forebrain complex and the first study of basal forebrain neurons from the mouse.

Projections from auditory cortex to cholinergic cells in the midbrain tegmentum of guinea pigs

Anterograde and retrograde tracing techniques were used to characterize projections from the auditory cortex to the pedunculopontine and laterodorsal tegmental nuclei (PPT and LDT, respectively) in the midbrain tegmentum in guinea pigs. For anterograde tracing, tetramethylrhodamine dextran (FluoroRuby) was injected at several sites within auditory cortex. After sufficient time for transport, the brain was processed for immunohistochemistry with anti-choline acetyltransferase to reveal presumptive cholinergic cells. Anterogradely labeled axons were observed ipsilaterally and, in smaller numbers, contralaterally, in both the pedunculopontine and laterodorsal tegmental nuclei. In all four nuclei, tracer-labeled boutons appeared to contact immunolabeled (i.e., cholinergic) cells. The contacts occurred on cell bodies and dendrites. The results were similar following injections that spread across multiple auditory cortical areas or injections that were within primary auditory cortex. In order to confirm the anterograde results, in a second series of experiments, retrograde tracers were deposited in the pedunculopontine tegmental nucleus. These injections labeled layer V pyramidal cells in the auditory cortex. The results suggest an excitatory projection from primary auditory cortex bilaterally to cholinergic cells in the midbrain tegmentum. Such a pathway could allow auditory cortex to activate brainstem cholinergic circuits, possibly including the cholinergic pathways associated with arousal and gating of acoustic stimuli.

Efferent innervation to the auditory basilar papilla of scincid lizards

Hair cells of the inner ear of vertebrates are innervated by afferent neurons that transmit sensory information to the brain as well as efferent neurons that receive feedback from the brainstem. The function of the efferent feedback system is poorly understood and may have changed during evolution when different tetrapod groups acquired sensitivity to airborne sound and extended their hearing ranges to higher frequencies. Lizards show a unique subdivision of their basilar papilla (homologous to the mammalian organ of Corti) into a low-frequency (<1 kHz) and a high-frequency (approximately 1-5 kHz) region. The high-frequency region was reported to have lost its efferent innervation, suggesting it was insignificant or even functionally detrimental at higher frequencies. We re-examined the innervation to the basilar papilla of five species of Australian scincid lizards, by using immunohistochemistry. Anti-choline acetyltransferase (ChAT) was used as an efferent marker. Co-localization with anti-synaptic vesicle protein 2 confirmed the synaptic identity of label. Cholinergic terminals were observed along the whole length of the basilar papilla, including the regions that had previously been described as devoid of efferent innervation. However, there was a clear decrease in terminal density from apical, low-frequency to basal, high-frequency locations. Our findings suggest that efferent innervation is a general feature of the hair cells in the basilar papilla of lizards, irrespective of tonotopic location. This re-enforces the notion that efferent feedback control of hair cells is a fundamental and important property of all vertebrate hearing organs.

Chronic systemic injection of D-galactose impairs the septohippocampal cholinergic system in rats

Accumulated evidence indicates chronic systemic injection of D-galactose mimics aging progress induced by oxidative stress. We addressed whether memory impairment in this model was associated with the cholinergic septohippocampal degeneration. Rats injected with D-galactose for 6 weeks showed impairment of spatial learning and memory as measured by the water maze test. Correspondingly, anti-choline acetyltransferase immunohistochemistry demonstrated a severe loss of cholinergic terminals in the hippocampus accompanied by a mild cholinergic neuronal atrophy and loss in the medial septum and the nucleus of the vertical limb of the diagonal band of Broca. A major synaptic degeneration in the hippocampus was confirmed by ultrastructural analysis. These findings provide neuropathological evidence for rodents with chronic injection of D-galactose as a promising model for brain aging and age-related neurodegeneration.

Spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) Treatment Rescues Olfactory Bulbectomy-Induced Memory Impairment by Activating Ca2+/Calmodulin Kinase II and Protein Kinase C in Mouse Hippocampus

Olfactory bulbectomy (OBX) in mice elicits impaired memory and cognitive functions. Here, we found that chronic oral administration of spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) (0.1-1 mg/kg/day), a novel cognitive enhancer, significantly improved memory deficits as assessed by Y-maze and novel object recognition tasks in OBX mice. Immunostaining of cholinergic neurons in the medial septum by using an anti-choline acetyltransferase antibody indicated that chronic ZSET1446 treatment did not rescue cholinergic neurons. However, chronic treatment significantly restored OBX-induced decreases both in calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) phosphorylation without improving decreased extracellular signal-regulated kinase phosphorylation in the hippocampal CA1 region. Consistent with enhanced CaMKII and PKC phosphorylation, ZSET1446 treatment improved glutamate receptor 1 (Ser-831) phosphorylation in the hippocampal CA1 region. ZSET1446 treatment also significantly rescued impaired long-term potentiation (LTP) in the hippocampal CA1 region of OBX mice. Taken together, the cognition-enhancing effect of ZSET1446 is probably mediated in part by stimulation of CaMKII and PKC activities, which in turn rescue impaired hippocampal LTP in OBX mice.

Up-Regulation of Cation-Independent Mannose 6-Phosphate Receptor and Endosomal-Lysosomal Markers in Surviving Neurons after 192-IgG-Saporin Administrations into the Adult Rat Brain

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons.