Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|R||IHC, WB||Gp||Affinity Purified||Polyclonal Antibody|
|Description||Anti-Vesicular Acetylcholine Transporter Antibody|
|Presentation||Affinity purified immunoglobulin. Lyophilized from 0.1 M PBS with 1.0% BSA and 0.1% sodium azide. Reconstitute with 100 μL of sterile distilled water.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain lyophilized material at 2-8°C for up to 12 months. Maintain reconstituted material at -20°C in undiluted aliquots for up to 6 months. Avoid repeated freeze/thaw cycles.|
|Material Size||20 µg|
|Reference overview||Application||Species||Pub Med ID|
|A major external source of cholinergic innervation of the striatum and nucleus accumbens originates in the brainstem.|
Dautan, D; Huerta-Ocampo, I; Witten, IB; Deisseroth, K; Bolam, JP; Gerdjikov, T; Mena-Segovia, J
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 4509-18 2014
Cholinergic transmission in the striatal complex is critical for the modulation of the activity of local microcircuits and dopamine release. Release of acetylcholine has been considered to originate exclusively from a subtype of striatal interneuron that provides widespread innervation of the striatum. Cholinergic neurons of the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei indirectly influence the activity of the dorsal striatum and nucleus accumbens through their innervation of dopamine and thalamic neurons, which in turn converge at the same striatal levels. Here we show that cholinergic neurons in the brainstem also provide a direct innervation of the striatal complex. By the expression of fluorescent proteins in choline acetyltransferase (ChAT)::Cre(+) transgenic rats, we selectively labeled cholinergic neurons in the rostral PPN, caudal PPN, and LDT. We show that cholinergic neurons topographically innervate wide areas of the striatal complex: rostral PPN preferentially innervates the dorsolateral striatum, and LDT preferentially innervates the medial striatum and nucleus accumbens core in which they principally form asymmetric synapses. Retrograde labeling combined with immunohistochemistry in wild-type rats confirmed the topography and cholinergic nature of the projection. Furthermore, transynaptic gene activation and conventional double retrograde labeling suggest that LDT neurons that innervate the nucleus accumbens also send collaterals to the thalamus and the dopaminergic midbrain, thus providing both direct and indirect projections, to the striatal complex. The differential activity of cholinergic interneurons and cholinergic neurons of the brainstem during reward-related paradigms suggest that the two systems play different but complementary roles in the processing of information in the striatum.
|Isl1 directly controls a cholinergic neuronal identity in the developing forebrain and spinal cord by forming cell type-specific complexes.|
Cho, HH; Cargnin, F; Kim, Y; Lee, B; Kwon, RJ; Nam, H; Shen, R; Barnes, AP; Lee, JW; Lee, S; Lee, SK
PLoS genetics 10 e1004280 2014
The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.
|Motor axon synapses on renshaw cells contain higher levels of aspartate than glutamate.|
Richards, DS; Griffith, RW; Romer, SH; Alvarez, FJ
PloS one 9 e97240 2014
Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.
|Severe drug-induced repetitive behaviors and striatal overexpression of VAChT in ChAT-ChR2-EYFP BAC transgenic mice.|
Crittenden, JR; Lacey, CJ; Lee, T; Bowden, HA; Graybiel, AM
Frontiers in neural circuits 8 57 2014
In drug users, drug-related cues alone can induce dopamine release in the dorsal striatum. Instructive cues activate inputs to the striatum from both dopaminergic and cholinergic neurons, which are thought to work together to support motor learning and motivated behaviors. Imbalances in these neuromodulatory influences can impair normal action selection and might thus contribute to pathologically repetitive and compulsive behaviors such as drug addiction. Dopamine and acetylcholine can have either antagonistic or synergistic effects on behavior, depending on the state of the animal and the receptor signaling systems at play. Semi-synchronized activation of cholinergic interneurons in the dorsal striatum drives dopamine release via presynaptic nicotinic acetylcholine receptors located on dopamine terminals. Nicotinic receptor blockade is known to diminish abnormal repetitive behaviors (stereotypies) induced by psychomotor stimulants. By contrast, blockade of postsynaptic acetylcholine muscarinic receptors in the dorsomedial striatum exacerbates drug-induced stereotypy, exemplifying how different acetylcholine receptors can also have opposing effects. Although acetylcholine release is known to be altered in animal models of drug addiction, predicting whether these changes will augment or diminish drug-induced behaviors thus remains a challenge. Here, we measured amphetamine-induced stereotypy in BAC transgenic mice that have been shown to overexpress the vesicular acetylcholine transporter (VAChT) with consequent increased acetylcholine release. We found that drug-induced stereotypies, consisting of confined sniffing and licking behaviors, were greatly increased in the transgenic mice relative to sibling controls, as was striatal VAChT protein. These findings suggest that VAChT-mediated increases in acetylcholine could be critical in exacerbating drug-induced stereotypic behaviors and promoting exaggerated behavioral fixity.
|Genetic dissection of retinal inputs to brainstem nuclei controlling image stabilization.|
Dhande, OS; Estevez, ME; Quattrochi, LE; El-Danaf, RN; Nguyen, PL; Berson, DM; Huberman, AD
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 17797-813 2013
When the head rotates, the image of the visual world slips across the retina. A dedicated set of retinal ganglion cells (RGCs) and brainstem visual nuclei termed the "accessory optic system" (AOS) generate slip-compensating eye movements that stabilize visual images on the retina and improve visual performance. Which types of RGCs project to each of the various AOS nuclei remain unresolved. Here we report a new transgenic mouse line, Hoxd10-GFP, in which the RGCs projecting to all the AOS nuclei are fluorescently labeled. Electrophysiological recordings of Hoxd10-GFP RGCs revealed that they include all three subtypes of On direction-selective RGCs (On-DSGCs), responding to upward, downward, or forward motion. Hoxd10-GFP RGCs also include one subtype of On-Off DSGCs tuned for forward motion. Retrograde circuit mapping with modified rabies viruses revealed that the On-DSGCs project to the brainstem centers involved in both horizontal and vertical retinal slip compensation. In contrast, the On-Off DSGCs labeled in Hoxd10-GFP mice projected to AOS nuclei controlling horizontal but not vertical image stabilization. Moreover, the forward tuned On-Off DSGCs appear physiologically and molecularly distinct from all previously genetically identified On-Off DSGCs. These data begin to clarify the cell types and circuits underlying image stabilization during self-motion, and they support an unexpected diversity of DSGC subtypes.
|B6eGFPChAT mice overexpressing the vesicular acetylcholine transporter exhibit spontaneous hypoactivity and enhanced exploration in novel environments.|
Nagy, PM; Aubert, I
Brain and behavior 3 367-83 2013
Cholinergic innervation is extensive throughout the central and peripheral nervous systems. Among its many roles, the neurotransmitter acetylcholine (ACh) contributes to the regulation of motor function, locomotion, and exploration. Cholinergic deficits and replacement strategies have been investigated in neurodegenerative disorders, particularly in cases of Alzheimer's disease (AD). Focus has been on blocking acetylcholinesterase (AChE) and enhancing ACh synthesis to improve cholinergic neurotransmission. As a first step in evaluating the physiological effects of enhanced cholinergic function through the upregulation of the vesicular acetylcholine transporter (VAChT), we used the hypercholinergic B6eGFPChAT congenic mouse model that has been shown to contain multiple VAChT gene copies. Analysis of biochemical and behavioral paradigms suggest that modest increases in VAChT expression can have a significant effect on spontaneous locomotion, reaction to novel stimuli, and the adaptation to novel environments. These observations support the potential of VAChT as a therapeutic target to enhance cholinergic tone, thereby decreasing spontaneous hyperactivity and increasing exploration in novel environments.
|Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry.|
Beier, KT; Borghuis, BG; El-Danaf, RN; Huberman, AD; Demb, JB; Cepko, CL
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 35-51 2013
The use of neurotropic viruses as transsynaptic tracers was first described in the 1960s, but only recently have such viruses gained popularity as a method for labeling neural circuits. The development of retrograde monosynaptic tracing vectors has enabled visualization of the presynaptic sources onto defined sets of postsynaptic neurons. Here, we describe the first application of a novel viral tracer, based on vesicular stomatitis virus (VSV), which directs retrograde transsynaptic viral spread between defined cell types. We use this virus in the mouse retina to show connectivity between starburst amacrine cells (SACs) and their known synaptic partners, direction-selective retinal ganglion cells, as well as to discover previously unknown connectivity between SACs and other retinal ganglion cell types. These novel connections were confirmed using physiological recordings. VSV transsynaptic tracing enables cell type-specific dissection of neural circuitry and can reveal synaptic relationships among neurons that are otherwise obscured due to the complexity and density of neuropil.
|Caudo-rostral brain spreading of α-synuclein through vagal connections.|
Ulusoy, A; Rusconi, R; Pérez-Revuelta, BI; Musgrove, RE; Helwig, M; Winzen-Reichert, B; Di Monte, DA
EMBO molecular medicine 5 1051-9 2013
α-Synuclein accumulation and pathology in Parkinson's disease typically display a caudo-rostral pattern of progression, involving neuronal nuclei in the medulla oblongata at the earliest stages. In this study, selective expression and accumulation of human α-synuclein within medullary neurons was achieved via retrograde transport of adeno-associated viral vectors unilaterally injected into the vagus nerve in the rat neck. The exogenous protein progressively spread toward more rostral brain regions where it could be detected within axonal projections. Propagation to the pons, midbrain and forebrain followed a stereotypical pattern of topographical distribution. It affected areas such as the coeruleus-subcoeruleus complex, dorsal raphae, hypothalamus and amygdala ipsilateral and, to a lesser extent, contralateral to the injection side. Spreading was accompanied by evidence of neuritic pathology in the form of axonal varicosities intensely immunoreactive for human α-synuclein and containing Thioflavin-S-positive fibrils. Thus, overexpression of human α-synuclein in the lower brainstem is sufficient to induce its long-distance caudo-rostral propagation, recapitulating features of Parkinson's disease and mechanisms of disease progression.
|Alterations in the motor neuron-renshaw cell circuit in the Sod1(G93A) mouse model.|
Wootz, H; Fitzsimons-Kantamneni, E; Larhammar, M; Rotterman, TM; Enjin, A; Patra, K; André, E; Van Zundert, B; Kullander, K; Alvarez, FJ
The Journal of comparative neurology 521 1449-69 2013
Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin-related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1(G93A) mouse model. Most calbindin-immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN-IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449-1469, 2013. © 2012 Wiley Periodicals, Inc.
|EGFR inhibitor erlotinib delays disease progression but does not extend survival in the SOD1 mouse model of ALS.|
Le Pichon, CE; Dominguez, SL; Solanoy, H; Ngu, H; Lewin-Koh, N; Chen, M; Eastham-Anderson, J; Watts, R; Scearce-Levie, K
PloS one 8 e62342 2013
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive paralysis due to motor neuron death. Several lines of published evidence suggested that inhibition of epidermal growth factor receptor (EGFR) signaling might protect neurons from degeneration. To test this hypothesis in vivo, we treated the SOD1 transgenic mouse model of ALS with erlotinib, an EGFR inhibitor clinically approved for oncology indications. Although erlotinib failed to extend ALS mouse survival it did provide a modest but significant delay in the onset of multiple behavioral measures of disease progression. However, given the lack of protection of motor neuron synapses and the lack of survival extension, the small benefits observed after erlotinib treatment appear purely symptomatic, with no modification of disease course.
|GUNIEA PIG ANTI-VESICULAR ACETYLCHOLINE TRANSPORTER|