Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Nat Neurosci
2010 Jan 01;131:127-132. doi: 10.1038/nn.2469.
Show Gene links
Show Anatomy links
An in vivo biosensor for neurotransmitter release and in situ receptor activity.
Nguyen QT
,
Schroeder LF
,
Mank M
,
Muller A
,
Taylor P
,
Griesbeck O
,
Kleinfeld D
.
???displayArticle.abstract???
Tools from molecular biology, combined with in vivo optical imaging techniques, provide new mechanisms for noninvasively observing brain processes. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. We devised cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, use the G(q) protein-coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca(2+)] and report [Ca(2+)] with a genetically encoded fluorescent Ca(2+) sensor. The initial realization of CNiFERs detected acetylcholine release via activation of M1 muscarinic receptors. We used chronic implantation of M1-CNiFERs in frontal cortex of the adult rat to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We found that these drugs potently inhibited in situ muscarinic receptor activity.
Figure 2. In vivo characterization of acutely implanted M1-CNiFERs(a) Stimulating electrodes are implanted in NBM to recruit the cortical afferent cholinergic system, and electrocorticogram wires are placed to detect NBM-evoked cortical activation (Methods in Supplemental Material). M1-CNiFERs and control-CNiFERs are implanted in separate columns in neocortex, where cholinergic terminals are widely distributed, and imaged acutely or chronically using two-photon laser scanning microscopy. To the right, a two-photon microscopy image of M1-CNiFERs (cyan) and control-CNiFERs (red) implanted in rat motor cortex in 25 – 50 μm diameter columns. Data represent a Z-projection from 40 – 60 μm below the cortical surface. There are ~ 10 – 20 CNiFER cells per site in this field of view. (b) M1-CNiFER FRET responses (lower) and ECoG activity (upper) evoked by increasing levels of NBM electrical stimulation. Cortical activation appears as a shift from large to small amplitude waves. Control-CNiFERs are non-responsive. (c) M1-CNiFER response to NBM stimulation is strongly correlated to loss of power in the electrocorticogram δ-band, quantified as the Z-score normalized logarithm of the reciprocal of the power, -log[power in ECoG δ-band], for each animal (Methods; Supplemental Material). CNiFER responses are defined as the area under the curve of ΔR/R for 10 s after the stimulus normalized to that of 10 s before the stimulus (n = 55 trials with 4 animals). (d) Subcutaneous physostigmine salicylate at 200 μg/kg enhances the amplitude and duration of M1-CNiFER response to NBM stimulation. The response disappears by ~ 8000 s. Data in bottom trace represent the fractional change of 1/3 the area under the curve of ΔR/R for 30 seconds after the stimulus as compared to that of 10 s before the stimulus. Top traces are examples of raw data used to calculate bottom trace; NBM stimulation occurs every 300 s and vehicle in PBS (n = 3). (e) Subcutaneous physostigmine salicylate at 300 μg/kg causes an increase in baseline M1-CNiFER FRET fluorescence over ~ 8000 s. This appears to result from modulation of background levels of acetylcholine in cortex. M1-CNiFERs (cyan) and control-CNiFERs (red) measured as an average over 10 s every 300 s (top). All measurements are normalized to first 3 measurements before vehicle injection, and not to internal baselines, thus preserving the tonic response. Data from 4 consecutive 300 s epochs for each animal (n = 4), and plotted in black at each time point (bottom). All bars are standard errors.
Figure 3. Chronic implantation of CNiFERs(a) Chronically implanted M1- and control-CNiFER sites are shown on the left, a X-Z time series from the M1-CNiFERs in response to a single-train NBM stimulation is shown in the center and the average intensity (mean ± standard error; n = 4 animals) of the M1-CNiFER response as a function of depth is shown on the right. (b) Electrocorticogram and FRET responses in M1- and control-CNiFERs in response to NBM stimulation; 300 ms train of 300 μA pulses (Methods; Supplemental Material). (c) Atropine antagonism. Left traces: M1-CNiFER responses to single-train NBM stimulation are inhibited by reverse dialysis of intracortical atropine sulfate. Bar graph: average peak inhibition of CNiFER response due to 1 – 5 μM atropine (23 ± 4 %) (n = 4 rats) or 100 μM atropine (87 ± 16 %) (n = 3 rats). (d) Temporal resolution of acutely implanted M1-CNiFERs. Top pane: stimulation protocol. Traces: Each trace represents the mean response of M1-CNiFERs to two consecutive stimulations of NBM (n = 5 for each condition, repeated over 3 animals). (e) Response of M1-CNiFERs versus stimulation current. Top pane: stimulation protocol. Graph: average M1-CNiFER response normalized to that at 500 μA (n = 6 rats). (f) Response versus duration of the stimulation train. Top pane: stimulation protocol. Graph: average M1-CNiFER response normalized to that at 500 ms (n = 6 rats). The black curves in (e) and (f) are visual aids.
Figure 4. In vivo pharmacology of chronically implanted M1-CNiFERs(a) Olanzapine intraperitoneal injection at 3 mg/kg suppresses the M1-CNiFER response elicited by repetitive NBM stimulation (500 μA; black vertical dashed lines). The M1-CNiFER response is partially recovered by increasing the amplitude of NBM stimulation (1 mA; red vertical dashed lines). (b-e) Atypical, but not conventional, antipsychotics suppress the M1-CNiFER response elicited by NBM stimulation. Graphs: M1-CNiFER peak response normalized to those averaged during vehicle injection; olanzapine at 3-5 mg/kg (n = 4); clozapine at 5 mg/kg (n = 4); chlorpromazine at 5 mg/kg (n = 4); and haloperidol at 1 mg/kg (n = 3). Grey: vehicle. Black: antipsychotic. (f) Olanzapine, injected i.p. at a dose of 10 mg/ml, does not elicit a response in M1-CNiFERs while nicotine ditartrate (green), injected i.p. at a dose of 1 mg/ml elicits a response. (g) Composite results (n = 4 rats) of the maximum response, measured between 120 and 720 s after injection. The response of M1-CNiFERs to nicotine is significantly greater than that for controls, while the response of M1-CNiFERS to olanzapine is at chance. **Significantly different values by t-test (p < 0.05).
Agnati,
Volume transmission and wiring transmission from cellular to molecular networks: history and perspectives.
2006, Pubmed
Agnati,
Volume transmission and wiring transmission from cellular to molecular networks: history and perspectives.
2006,
Pubmed
Beaudet,
The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals.
1978,
Pubmed
Berg,
Activation of nucleus basalis facilitates cortical control of a brain stem motor program.
2005,
Pubmed
Bolden,
Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells.
1992,
Pubmed
Bymaster,
Muscarinic mechanisms of antipsychotic atypicality.
2003,
Pubmed
Chew,
A model of anticholinergic activity of atypical antipsychotic medications.
2006,
Pubmed
Coward,
Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors.
1999,
Pubmed
Davies,
The highly efficacious actions of N-desmethylclozapine at muscarinic receptors are unique and not a common property of either typical or atypical antipsychotic drugs: is M1 agonism a pre-requisite for mimicking clozapine's actions?
2005,
Pubmed
Day,
Application of in vivo microdialysis to the study of cholinergic systems.
2001,
Pubmed
Denk,
Two-photon laser scanning fluorescence microscopy.
1990,
Pubmed
Drobizhev,
Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins.
2009,
Pubmed
Duff Davis,
In vivo spectrometric calcium flux recordings of intrinsic Caudate-Putamen cells and transplanted IMR-32 neuroblastoma cells using miniature fiber optrodes in anesthetized and awake rats and monkeys.
2000,
Pubmed
Everitt,
Central cholinergic systems and cognition.
1997,
Pubmed
Frostig,
In Vivo Two-Photon Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation Principles and Hardware Realization
2009,
Pubmed
Frostig,
MPScope 2.0: A Computer System for Two-Photon Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation and Electrophysiology
2009,
Pubmed
Gray,
Molecular targets for treating cognitive dysfunction in schizophrenia.
2007,
Pubmed
Haas,
Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.
2006,
Pubmed
Hires,
Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.
2008,
Pubmed
Horowitz,
Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current.
2005,
Pubmed
Ichikawa,
Atypical, but not typical, antipsychotic drugs increase cortical acetylcholine release without an effect in the nucleus accumbens or striatum.
2002,
Pubmed
Johnson,
The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release.
2005,
Pubmed
Kawaja,
Morphological and neurochemical features of cultured primary skin fibroblasts of Fischer 344 rats following striatal implantation.
1992,
Pubmed
Kostenis,
Techniques: promiscuous Galpha proteins in basic research and drug discovery.
2005,
Pubmed
Krasel,
Kinetics of G-protein-coupled receptor signalling and desensitization.
2004,
Pubmed
Levey,
Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies.
1991,
Pubmed
Mank,
A genetically encoded calcium indicator for chronic in vivo two-photon imaging.
2008,
Pubmed
Meltzer,
What's atypical about atypical antipsychotic drugs?
2004,
Pubmed
Metherate,
Ionic flux contributions to neocortical slow waves and nucleus basalis-mediated activation: whole-cell recordings in vivo.
1993,
Pubmed
Nguyen,
Effects of atypical antipsychotics on vertebrate neuromuscular transmission.
2002,
Pubmed
Nishimura,
Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke.
2006,
Pubmed
Okumoto,
Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.
2005,
Pubmed
Parada,
Selective action of acute systemic clozapine on acetylcholine release in the rat prefrontal cortex by reference to the nucleus accumbens and striatum.
1997,
Pubmed
Raedler,
Towards a muscarinic hypothesis of schizophrenia.
2007,
Pubmed
Rasmusson,
Frequency-dependent increase in cortical acetylcholine release evoked by stimulation of the nucleus basalis magnocellularis in the rat.
1992,
Pubmed
Rasmusson,
Modification of neocortical acetylcholine release and electroencephalogram desynchronization due to brainstem stimulation by drugs applied to the basal forebrain.
1994,
Pubmed
Schaffer,
Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion.
2006,
Pubmed
Shaner,
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
2004,
Pubmed
Shirazi-Southall,
Effects of typical and atypical antipsychotics and receptor selective compounds on acetylcholine efflux in the hippocampus of the rat.
2002,
Pubmed
Snyder,
Schizophrenia therapy: beyond atypical antipsychotics.
2008,
Pubmed
Snyder,
Antischizophrenic drugs and brain cholinergic receptors. Affinity for muscarinic sites predicts extrapyramidal effects.
1974,
Pubmed
Tani,
Pharmacological characterization of nicotinic receptor-mediated acetylcholine release in rat brain--an in vivo microdialysis study.
1998,
Pubmed
Tsien,
Building and breeding molecules to spy on cells and tumors.
2005,
Pubmed
Umbriaco,
Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections.
1994,
Pubmed
Vilardaga,
Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
2003,
Pubmed
Vögler,
Receptor subtype-specific regulation of muscarinic acetylcholine receptor sequestration by dynamin. Distinct sequestration of m2 receptors.
1998,
Pubmed
Willars,
Quantitative comparisons of muscarinic and bradykinin receptor-mediated Ins (1,4,5)P3 accumulation and Ca2+ signalling in human neuroblastoma cells.
1995,
Pubmed
Young,
Spontaneous release of transmitter from growth cones of embryonic neurones.
,
Pubmed
,
Xenbase
Zhang,
Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes.
2005,
Pubmed
