Molecular cloning of a putative vesicular transporter for acetylcholine.
ABSTRACT: Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.
Project description:Basal forebrain cholinergic neurons, which innervate the hippocampus and cortex, have been implicated in many forms of cognitive function. Immunolesion-based methods in animal models have been widely used to study the role of acetylcholine (ACh) neurotransmission in these processes, with variable results. Cholinergic neurons have been shown to release both glutamate and ACh, making it difficult to deduce the specific contribution of each neurotransmitter on cognition when neurons are eliminated. Understanding the precise roles of ACh in learning and memory is critical because drugs that preserve ACh are used as treatment for cognitive deficits. It is therefore important to define which cholinergic-dependent behaviors could be improved pharmacologically. Here we investigate the contributions of forebrain ACh on hippocampal synaptic plasticity and cognitive behavior by selective elimination of the vesicular ACh transporter, which interferes with synaptic storage and release of ACh. We show that elimination of vesicular ACh transporter in the hippocampus results in deficits in long-term potentiation and causes selective deficits in spatial memory. Moreover, decreased cholinergic tone in the forebrain is linked to hyperactivity, without changes in anxiety or depression-related behavior. These data uncover the specific contribution of forebrain cholinergic tone for synaptic plasticity and behavior. Moreover, these experiments define specific cognitive functions that could be targeted by cholinergic replacement therapy.
Project description:BACKGROUND:In cholinergic neurons, the hydrolysis of phosphatidylcholine (PC) by a phospholipase D (PLD)-type enzyme generates some of the precursor choline used for the synthesis of the neurotransmitter acetylcholine (ACh). We sought to determine the molecular identity of the relevant PLD using murine basal forebrain cholinergic SN56 cells in which the expression and activity of the two PLD isoforms, PLD1 and PLD2, were experimentally modified. ACh levels were examined in cells incubated in a choline-free medium, to ensure that their ACh was synthesized entirely from intracellular choline. RESULTS:PLD2, but not PLD1, mRNA and protein were detected in these cells and endogenous PLD activity and ACh synthesis were stimulated by phorbol 12-myristate 13-acetate (PMA). Introduction of a PLD2 antisense oligonucleotide into the cells reduced PLD2 mRNA and protein expression by approximately 30%. The PLD2 antisense oligomer similarly reduced basal- and PMA-stimulated PLD activity and ACh levels. Overexpression of mouse PLD2 by transient transfection increased basal- (by 74%) and PMA-stimulated (by 3.2-fold) PLD activity. Moreover, PLD2 transfection increased ACh levels by 26% in the absence of PMA and by 2.1-fold in the presence of PMA. Overexpression of human PLD1 by transient transfection increased PLD activity by 4.6-fold and ACh synthesis by 2.3-fold in the presence of PMA as compared to controls. CONCLUSIONS:These data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in cholinergic cells, and indicate that in a model system choline generated by PLD1 may also be used for this purpose.
Project description:Nerve growth factor (NGF) stimulates the expression of the cholinergic gene locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), the proteins necessary for the synthesis and storage of the neurotransmitter acetylcholine (ACh). To determine whether this action of NGF is mediated by the p140TrkA NGF receptor (a member of the Trk tyrosine kinase family) we used a murine basal forebrain cholinergic cell line, SN56, stably transfected with rat trkA cDNA. Treatment of these transfectants with NGF activated mitogen-activated protein kinase and increased cytosolic free calcium concentrations, confirming the reconstitution of TrkA-mediated signalling pathways. The expression of ChAT and VAChT mRNA, as well as ACh content, were coordinately up-regulated by NGF in SN56-trkA transfectants. None of these responses occurred in the parental SN56 cells, which do not express endogenous TrkA, indicating that these actions of NGF required TrkA. We previously reported that ciliary neurotrophic factor (CNTF) upregulates the expression of ChAT and VAChT, as well as ACh production, in SN56 cells. The combined treatment of SN56-trkA cells with CNTF and NGF revealed a complex interaction of these factors in the regulation of cholinergic gene locus expression. At low concentrations of CNTF (<1 ng/ml), the upregulation of ACh synthesis evoked by these factors was additive. However, at higher concentrations of CNTF (>1 ng/ml), NGF attenuated the stimulatory effect of CNTF on ChAT and VAChT mRNA and ACh content. This attenuation was not due to interference with early steps of CNTF receptor signalling, as pre-treatment of SN56-trkA cells with NGF did not affect the nuclear translocation of the transcription factor, Stat3, evoked by CNTF.
Project description:The distribution and (patho-)physiological role of neuropeptides in the adult and aging brain have been extensively studied. Galanin is an inhibitory neuropeptide that can coexist with ?-aminobutyric acid (GABA) in the adult forebrain. However, galanin's expression sites, mode of signaling, impact on neuronal morphology, and colocalization with amino acid neurotransmitters during brain development are less well understood. Here, we show that galaninergic innervation of cholinergic projection neurons, which preferentially express galanin receptor 2 (GalR2) in the neonatal mouse basal forebrain, develops by birth. Nerve growth factor (NGF), known to modulate cholinergic morphogenesis, increases GalR2 expression. GalR2 antagonism (M871) in neonates reduces the in vivo expression and axonal targeting of the vesicular acetylcholine transporter (VAChT), indispensable for cholinergic neurotransmission. During cholinergic neuritogenesis in vitro, GalR2 can recruit Rho-family GTPases to induce the extension of a VAChT-containing primary neurite, the prospective axon. In doing so, GalR2 signaling dose-dependently modulates directional filopodial growth and antagonizes NGF-induced growth cone differentiation. Galanin accumulates in GABA-containing nerve terminals in the neonatal basal forebrain, suggesting its contribution to activity-driven cholinergic development during the perinatal period. Overall, our data define the cellular specificity and molecular complexity of galanin action in the developing basal forebrain.
Project description:The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.
Project description:The basal forebrain cholinergic projection system to the cortex mediates essential aspects of visual attention performance, including the detection of cues and the response to performance challenges (top-down control of attention). Higher levels of top-down control are mediated via elevated levels of cholinergic neuromodulation. The neuronal choline transporter (CHT) strongly influences the synthesis and release of acetylcholine (ACh). As the capacity of the CHT to import choline into the neuron is a major, presynaptic determinant of cholinergic neuromodulation, we hypothesize that genetically-imposed CHT capacity variation impacts the balance of bottom-up versus top-down control of visual attention. Following a brief review of the cognitive concepts relevant for this hypothesis, we describe the key results from our research in mice and humans that possess genetically-imposed changes in choline uptake capacity. CHT subcapacity is associated with poor top-down attentional control and attenuated (cholinergic) activation of right frontal regions. Conversely, mice overexpressing the CHT, and humans expressing a CHT variant hypothesized to enhance choline transporter function, are relatively resistant to challenges of visual attention performance. Genetic or environmental modulation of CHT expression and function may be associated with vulnerabilities for cognitive disorders.
Project description:Neocortical choline acetyltransferase (ChAT)-expressing interneurons are a subclass of vasoactive intestinal peptide (ChAT-VIP) neurons of which circuit and behavioural function are unknown. Here, we show that ChAT-VIP neurons directly excite neighbouring neurons in several layers through fast synaptic transmission of acetylcholine (ACh) in rodent medial prefrontal cortex (mPFC). Both interneurons in layers (L)1-3 as well as pyramidal neurons in L2/3 and L6 receive direct inputs from ChAT-VIP neurons mediated by fast cholinergic transmission. A fraction (10-20%) of postsynaptic neurons that received cholinergic input from ChAT-VIP interneurons also received GABAergic input from these neurons. In contrast to regular VIP interneurons, ChAT-VIP neurons did not disinhibit pyramidal neurons. Finally, we show that activity of these neurons is relevant for behaviour and they control attention behaviour distinctly from basal forebrain ACh inputs. Thus, ChAT-VIP neurons are a local source of cortical ACh that directly excite neurons throughout cortical layers and contribute to attention.
Project description:Cholinergic neurons originating from the basal forebrain innervate the entire cortical mantle. Choline-sensitive microelectrodes were used to measure the synaptic release of cortical acetylcholine (ACh) at a subsecond resolution in rats performing a task involving the detection of cues. Cues that were detected, defined behaviorally, evoked transient increases in cholinergic activity (at the scale of seconds) in the medial prefrontal cortex (mPFC), but not in a nonassociational control region (motor cortex). In trials involving missed cues, cholinergic transients were not observed. Cholinergic deafferentation of the mPFC, but not motor cortex, impaired cue detection. Furthermore, decreases and increases in precue cholinergic activity predicted subsequent cue detection or misses, respectively. Finally, cue-evoked cholinergic transients were superimposed over slower (at the timescale of minutes) changes in cholinergic activity. Cortical cholinergic neurotransmission is regulated on multiple timescales to mediate the detection of behaviorally significant cues and to support cognitive performance.
Project description:The simian immunodeficiency virus (SIV) macaque model resembles human immunodeficiency virus-acquired immunodeficiency syndrome (AIDS) and associated brain dysfunction. Altered expression of synaptic markers and transmitters in neuro-AIDS has been reported, but limited data exist for the cholinergic system and lipid mediators such as prostaglandins. Here, we analyzed cholinergic basal forebrain neurons with their telencephalic projections and the rate-limiting enzymes for prostaglandin synthesis, cyclooxygenase isotypes 1 and 2 (COX1 and COX2) in the brains of SIV-infected macaques with or without encephalitis and antiretroviral therapy and uninfected controls.Cyclooxygenase isotype 1, but not COX2, was coexpressed with markers of cholinergic phenotype, that is, choline acetyltransferase and vesicular acetylcholine transporter (VAChT), in basal forebrain neurons of monkey, as well as human, brain. Cyclooxygenase isotype 1 was decreased in basal forebrain neurons in macaques with AIDS versus uninfected and asymptomatic SIV-infected macaques. The VAChT-positive fiber density was reduced in frontal, parietal, and hippocampal-entorhinal cortex. Although brain SIV burden and associated COX1- and COX2-positive mononuclear and endothelial inflammatory reactions were mostly reversed in AIDS-diseased macaques that received 6-chloro-2',3'-dideoxyguanosine treatment, decreased VAChT-positive terminal density and reduced cholinergic COX1 expression were not. Thus, COX1 expression is a feature of primate cholinergic basal forebrain neurons; it may be functionally important and a critical biomarker of cholinergic dysregulation accompanying lentiviral encephalopathy. These results further imply that insufficiently prompt initiation of antiretroviral therapy in lentiviral infection may lead to neurostructurally unremarkable but neurochemically prominent irreversible brain damage.
Project description:Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.