AB143 Sigma-AldrichAnti-Choline Acetyltransferase (ChAT) Antibody

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

The striatum serves as the main input to the basal ganglia, and is key for the regulation of motor behaviors, compulsion, addiction, and various cognitive and emotional states. Its deterioration is associated with degenerative disorders such as Huntington's disease. Despite its apparent anatomical uniformity, it consists of intermingled cell populations, which have precluded straightforward anatomical sub-classifications adhering to functional dissections. Approximately 95% of the striatal neurons are inhibitory projection neurons termed medium spiny neurons (MSNs). They are commonly classified according to their expression of either dopamine receptor D1 or D2, which also determines their axonal projection patterns constituting the direct and indirect pathway in the basal ganglia. Immunohistochemical patterns have further indicated compartmentalization of the striatum to the striosomes and the surrounding matrix, which integrate MSNs of both the D1 and D2 type. Here, we present a transgenic mouse line, Gpr101-Cre, with Cre recombinase activity localized to matrix D1 and D2 MSNs. Using two Gpr101-Cre founder lines with different degrees of expression in the striatum, we conditionally deleted the vesicular inhibitory amino acid transporter (VIAAT), responsible for storage of GABA and glycine in synaptic vesicles. Partial ablation of VIAAT (in ~36% of MSNs) resulted in elevated locomotor activity compared to control mice, when provoked with the monoamine reuptake inhibitor cocaine. Near complete targeting of matrix MSNs led to profoundly changed motor behaviors, which increased in severity as the mice aged. Moreover, these mice had exaggerated muscle rigidity, retarded growth, increased rate of spontaneous deaths, and defective memory. Therefore, our data provide a link between dysfunctional GABA signaling of matrix MSNs to specific behavioral alterations, which are similar to the symptoms of Huntington's disease.

The basal forebrain (BF) plays a crucial role in cortical activation. Our previous study showed that activation of cholinergic BF neurons alone is sufficient to suppress slow-wave sleep (SWS) and promote wakefulness and rapid-eye-movement (REM) sleep. However, the exact role of silencing cholinergic BF neurons in the sleep-wake cycle remains unclear. We inhibitied the cholinergic BF neurons genetically targeted with archaerhodopsin (Arch) with yellow light to clarify the role of cholinergic BF neurons in the sleep-wake cycle. Bilateral inactivation of cholinergic BF neurons genetically targeted with archaerhodopsin prolonged SWS and decreased the probability of awakening from SWS in mice. However, silencing these neurons changed neither the duration of wakefulness or REM sleep, nor the probability of transitions to other sleep-wake episodes from wakefulness or REM sleep. Furthermore, silencing these neurons for 6 h within the inactive or active period increased the duration of SWS at the expense of the duration of wakefulness, as well as increasing the number of prolonged SWS episodes (120-240 s). The lost wakefulness was compensated by a delayed increase of wakefulness, so the total duration of SWS and wakefulness during 24 h was kept stable. Our results indicate that the main effect of these neurons is to terminate SWS, whereas wakefulness or REM sleep may be determined by co-operation of the cholinergic BF neurons with other arousal-sleep control systems.

Severe neuronal loss in the hippocampus, that is, hippocampal sclerosis (HS), can be seen in 3 main clinical contexts: dementia (particularly frontotemporal lobar degeneration [FTLD]), temporal lobe epilepsy (TLE), and hippocampal ischemic injury (H-I). It has been suggested that shared pathogenetic mechanisms may underlie selective vulnerability of the hippocampal subfields such as the CA1 in these conditions. We determined the extent of neuronal loss in cases of HS-FTLD (n=14), HS-TLE (n=35), and H-I (n=20). Immunohistochemistry for zinc transporter 3 was used to help define the CA3/CA2 border in the routinely processed human autopsy tissue samples. The subiculum was involved in 57% of HS-FTLD, 10% of H-I, and 0% of HS-TLE cases (pless than 0.0001). The CA regions other than CA1 were involved in 57% of HS-TLE, 30% of H-I, and 0% of HS-FTLD cases (p=0.0003). The distal third of CA1 was involved in 79% of HS-FTLD, 35% of H-I, and 37% of HS-TLE cases (p=0.02). The distal third of CA1 was the only area involved in 29% of HS-FTLD, 3% of HS-TLE, and 0% of H-I cases (p=0.019). The proximal-middle CA1 was the only area affected in 50% of H-I, 29% of HS-TLE, and 0% of HS-FTLD cases (p=0.004). These findings support heterogeneity in the pathogenesis of HS.

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (greater than 20 μm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

The basal forebrain cholinergic system modulates neuronal excitability and vascular tone throughout the cerebral cortex and hippocampus. This system is severely affected in Alzheimer's disease (AD), and drug treatment to enhance cholinergic signaling is widely used as symptomatic therapy in AD. Defining the full morphologies of individual basal forebrain cholinergic neurons has, until now, been technically beyond reach due to their large axon arbor sizes. Using genetically-directed sparse labeling, we have characterized the complete morphologies of basal forebrain cholinergic neurons in the mouse. Individual arbors were observed to span multiple cortical columns, and to have greater than 1000 branch points and total axon lengths up to 50 cm. In an AD model, cholinergic axons were slowly lost and there was an accumulation of axon-derived material in discrete puncta. Calculations based on published morphometric data indicate that basal forebrain cholinergic neurons in humans have a mean axon length of ∼100 meters.DOI: http://dx.doi.org/10.7554/eLife.02444.001.

Cholinergic inputs to the auditory cortex can modulate sensory processing and regulate stimulus-specific plasticity according to the behavioural state of the subject. In order to understand how acetylcholine achieves this, it is essential to elucidate the circuitry by which cholinergic inputs influence the cortex. In this study, we described the distribution of cholinergic neurons in the basal forebrain and their inputs to the auditory cortex of the ferret, a species used increasingly in studies of auditory learning and plasticity. Cholinergic neurons in the basal forebrain, visualized by choline acetyltransferase and p75 neurotrophin receptor immunocytochemistry, were distributed through the medial septum, diagonal band of Broca, and nucleus basalis magnocellularis. Epipial tracer deposits and injections of the immunotoxin ME20.4-SAP (monoclonal antibody specific for the p75 neurotrophin receptor conjugated to saporin) in the auditory cortex showed that cholinergic inputs originate almost exclusively in the ipsilateral nucleus basalis. Moreover, tracer injections in the nucleus basalis revealed a pattern of labelled fibres and terminal fields that resembled acetylcholinesterase fibre staining in the auditory cortex, with the heaviest labelling in layers II/III and in the infragranular layers. Labelled fibres with small en-passant varicosities and simple terminal swellings were observed throughout all auditory cortical regions. The widespread distribution of cholinergic inputs from the nucleus basalis to both primary and higher level areas of the auditory cortex suggests that acetylcholine is likely to be involved in modulating many aspects of auditory processing.

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.

Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic γ-aminobutyric acid (GABA)ergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the postcommissural putamen of rhesus monkeys. Postembedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, whereas 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of "direct" and "indirect" striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons.

In the outer retina, G protein-coupled receptor (GPCR) signaling mediates phototransduction and synaptic transmission between photoreceptors and ON bipolar cells. In contrast, the functions of modulatory GPCR signaling networks in the inner retina are less well understood. We addressed this question by determining the consequences of augmenting modulatory Gi/o signaling driven by endogenous transmitters. This was done by analyzing the effects of genetically ablating the R7 RGS-binding protein (R7BP), a membrane-targeting protein and positive allosteric modulator of R7-RGS (regulator of the G protein signaling 7) family that deactivates Gi/oα subunits. We found that R7BP is expressed highly in starburst amacrine cells and retinal ganglion cells (RGCs). As indicated by electroretinography and multielectrode array recordings of adult retina, ablation of R7BP preserved outer retina function, but altered the firing rate and latency of ON RGCs driven by rods and cones but not rods alone. In developing retina, R7BP ablation increased the burst duration of glutamatergic waves whereas cholinergic waves were unaffected. This effect on glutamatergic waves did not result in impaired segregation of RGC projections to eye-specific domains of the dorsal lateral geniculate nucleus. R7BP knockout mice exhibited normal spatial contrast sensitivity and visual acuity as assessed by optomotor reflexes. Taken together these findings indicate that R7BP-dependent regulation of R7-RGS proteins shapes specific aspects of light-evoked and spontaneous activity of RGCs in mature and developing retina.