We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 2012) and studies were approved by Institutional Animal Care and Use for Research Committees at Stony Brook University (#1618 and #1792).
1. vHipp-nAcc Synaptic Co-cultures
<ol>
	<li>Sacrifice mice (postnatal day 0 - 3, from wild-type (WT) or &#945;7 nAChRs transgenic mouse line) with CO2. Decapitate each pup and save the tail in a 1.5 ml centrifuge tube for post facto genotyping. Perform the following steps in a sterilized hood.</li>
	<li>Remove the top of the skull with scissors and forceps to expose the whole brain. Starting at the juncture where the cerebral cortices part from each other caudally, obtain a coronal section that contains the posterior cortices including the vHipp5,21 under a dissecting microscope.</li>
	<li>Dissect vHipp tissue strips containing the CA1-subiculum region out according to a developing mouse brain atlas22 (Figure 1A). Transfer to a 35 mm culture dish containing cold culture media (4 &#176;C), cut into small thin slices (around 100 &#181;m &#215; 100 &#181;m microslices).</li>
	<li>Pre-incubate 12 mm poly-D-lysine/laminin-coated glass coverslips inside 24 well tissue culture plates with culture media (~ 500 &#956;l) for at least 30 min in the incubator at 37 &#176;C. Remove media before plating microslices.</li>
	<li>Plate with a fire-polished Pasteur pipette at the center of the coverslip with minimal amount of media (~ 50 &#956;l, containing ~ 20 pieces of microslices) to facilitate attachment of the explants. Plate vHipp microslices originating from a single animal (either the +/+ or the -/- genotype of the transgenic &#945;7 line) on each coverslip.</li>
	<li>After the explants settle onto the coverslip, gently add additional media (~ 100 &#956;l) by the wall so that the level of the media is high enough to completely cover the explants on the periphery, but not those at the center of the coverslip.</li>
	<li>After an O/N incubation in 37 &#176;C, 5% CO2 incubator, disperse nAcc neurons from embryonic days 18 to postnatal day 1 WT mice and add to the explants.
	<ol>
		<li>Dissect out nAcc tissues according to a developing mouse brain atlas20 (Figure 1B).</li>
		<li>Cut into small pieces (around 500 &#181;m &#215; 500 &#181;m microslices) and transfer to a 15 ml tube.</li>
		<li>Treat with 0.25% trypsin (2 ml) for 15 min at 37 &#176;C. Wash tissue chunks three times with cold washing media (5 ml, 4 &#176;C) followed by one wash in cold culture media (5 ml, 4 &#176;C). Allow suspension to rest for 5 min in each wash step, and then pour off supernatant carefully.</li>
		<li>Dissociate cells by gentle trituration in 2 ml of culture media with a lightly fire-polished Pasteur pipette.</li>
		<li>Transfer cell suspension to another 15 ml tube and centrifuge at 2,000 x rpm for 5 min. Remove the supernatant and resuspend the cells in culture media at 1 ml per 6 pups. Disperse cells by gently pipetting in media several times with a fire-polished Pasteur pipette.</li>
		<li>Add 0.25 ml of dispersed nAcc cells to each coverslip with vHipp explants.</li>
		<li>Maintain the cultures in a humidified 37 &#176;C, 5% CO2 incubator. Place culture plates on dampened sterile gauze pads to mechanically stabilize and maintain humidity, facilitating synapse formation.</li>
	</ol>
	</li>
</ol>
2. Immunocytochemistry
<ol>
	<li>Fix cultures (5 - 7 days in vitro) in 4% paraformaldehyde/4% sucrose/PBS (~500 &#956;l per coverslip, 20 min, RT).</li>
	<li>Permeabilize cultures with 0.25% Triton X-100/ PBS (~500 &#956;l per coverslip, 5 min, RT).</li>
	<li>Block cultures with 10% normal donkey serum in PBS (~500 &#956;l per coverslip, 30 min, RT) prior to antibody staining. Incubate with primary antibodies O/N at 4 &#176;C. Use the following primary antibodies: anti- nAChRs (&#945;4-ECD 1:500; anti-&#945;5 subunits, 1:500), anti-vesicular glutamate transporter 1 (1:250), anti-GAD65 (1:100).</li>
	<li>Wash primary antibodies out with PBS (3 times/~500 &#956;l per coverslip, 5 min) and then incubate cultures in secondary antibodies conjugated to Alexa 488 (~500 &#956;l per coverslip, 1:500) or Alexa 594 (~500 &#956;l per coverslip, 1:500) for 1 hr at 37 &#176;C.</li>
	<li>Incubate cultures in &#945;BgTx conjugated to Alexa 594 (~500 &#956;l per coverslip, 1:1,000 in culture media) for 45 min at 37 &#176;C prior to fixation for labeling surface &#945;7*nAChRs.</li>
	<li>Mount and seal coverslips.</li>
	<li>Capture images with a fluorescence microscope equipped with Plan-Apochromat objectives (20X with 0.8 NA or 63X oil with 1.4 NA) and a CCD camera.</li>
</ol>
3. FM1-43 Based Vesicular Fusion
<ol>
	<li>Maintain cultures (5 - 7 days in vitro) in an imaging chamber, mount the chamber on spinning disk confocal microscope. Continuously perfuse (1 ml/min) with HBS cocktail at RT.</li>
	<li>Stop perfusion and load cultures with 10 &#181;M FM1-43 in 56 mM K+ ACSF (~500 &#956;l) for 90 sec (staining), wash external dye away for 15 min with Ca2+ free HBS containing ADVASEP-7 (0.1 mM) to scavenge membrane-bound FM1-43.</li>
	<li>Stimulate the cultures with 56 mM K+ ACSF (~500 &#956;l) without FM1-43 for 120 sec (destaining), reload cultures with FM1-43 using the same conditions, and wash again with Ca2+ free HBS containing ADVASEP-7 for 15 min.</li>
	<li>Acquire FM1-43 fluorescence images with spinning disk confocal microscope equipped with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and capture images with a CCD camera.</li>
	<li>Collect baseline FM1-43 fluorescence images (every 2 s for 1 min) as pre-nicotine control; apply nicotine (1 &#181;M) by rapid perfusion (2 ml/min) for 1 min, and then wash nicotine out with HBS cocktail. Keep capture time-lapse images before, during, and after nicotine application every 2 sec for 5 min.</li>
	<li>Quantify FM1-43 fluorescence intensity before (Fstaining) and after (Fdestaining) nicotine application at each synaptic bouton.</li>
	<li>Calculate total amount of releasable FM1-43 fluorescence along vHipp axons by quantifying the difference of FM1-43 fluorescence intensity before and after nicotine application (&#916;F = Fstaining-Fdestaining). Analyze fraction of fluorescence intensity decrease after nicotine with the following equation: Fdecrease% = &#916;F/Fstaining.</li>
</ol>
4. Calcium Imaging
<ol>
	<li>Rinse cultures (5 - 7 days in vitro) quickly with normal HBS, then load cultures with 5 &#181;M Fluo-4 Ca2+ indicator (AM ester) and 0.02% Pluronic F-127 in HBS (2 ml) for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Wash out Fluo-4 solution with HBS (3 times/5 min). Return cultures to incubator (37 &#176;C / 5% CO2) for at least 30 min.</li>
	<li>Maintain cultures in the same imaging chamber and same conditions as described in FM1-43 based vesicular fusion section (see step 3.1).</li>
	<li>Collect Fluo-4 fluorescence images of axonal projections from the vHipp micro-slices with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and a CCD camera.</li>
	<li>Collect baseline Fluo-4 fluorescence images (every 10 sec for 2 min) as pre-nicotine control, apply nicotine (1 &#181;M) by rapid perfusion (2 ml/min) for 1 min, and then wash nicotine out with HBS cocktail. Keep capture time-lapse images before, during, and after nicotine application every 10 sec for 30 min.</li>
	<li>Save all frames of the raw fluo-4 fluorescence images, export as a series of TIFF format images for further analyzing.</li>
	<li>Collect the integrated intensity of fluo-4 fluorescence along vHipp axons before and after nicotine application.</li>
	<li>Normalize integrated fluorescence intensity with the following equation: &#916;F/F0 =&#160; (F-F0)/F0, where F0 is the background-corrected pre-nicotine integrated fluorescence intensity and F is the integrated fluorescence intensity along vHipp axons at each time point after nicotine application. Analyze and plot normalized integrated fluorescence intensity.</li>
</ol>

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The authors have nothing to disclose.&#160;

The co-culture preparation described re-capitulates ventral hippocampal&#8211;accumbens circuits in vitro. This preparation permits relatively straightforward and reliable examination of the spatial and temporal profiles by which activation of pre-synaptic nAChRs elicit enhanced glutamatergic transmission5, 21.
Co-cultures are defined as the growth of different specific cell types in one dish which can provide physiological conditions in vitro to demonstrate in...

Cholinergic modulation of circuit excitability contributes to fundamental aspects of cognition, and altered cholinergic modulation is a feature of neurodegenerative and neuropsychiatric disorders including Alzheimer&#8217;s disease, Parkinson&#8217;s disease, schizophrenia and addiction1-4. An established mechanism of cholinergic facilitation of synaptic transmission in the CNS is via direct activation of nAChRs localized at pre-synaptic sites. Activation of these pre-synaptic receptors leads to increased intracellular Ca2+ ([Ca2+]i) in pre-synaptic terminals &#8211; both directly, due to the relatively high calcium conductance of certain nAChR subtypes, and indirectly, via intracellular signaling cascades5, thereby enhancing neurotransmitter release. In fact, the activation of pre-synaptic nAChRs has been linked with changes in release of a wide variety of neurotransmitters including glutamate, GABA, ACh, and dopamine6-10. Although this process has been studied indirectly using electrophysiological methods at various synapses, optical reporters of [Ca2+]i and synaptic vesicle recycling allow more direct and temporally precise measurement of pre-synaptic phenomena.
Pre-synaptic localization of nAChRs has been demonstrated convincingly with direct immuno-gold labeling of nAChRs at the electron microscopic (EM) level11,12. Several other techniques have also been used to address the nAChR localization indirectly, including detecting locations of nAChRs subunit- fluorescent protein chimeras in cultured neurons13,14, electrophysiological recording of nAChR currents in synaptic terminals15,16, monitoring nicotine induced changes in [Ca2+]i in synaptic nerve terminals by live cell imaging17, and indirect monitoring of neurotransmitter release at the synaptic terminal by live cell imaging techniques with fluorescent indicators, including exocytosis of synaptic vesicles viewed by styryl amphipathic FM dyes (FM1-43 and FM4-64) and/or synapto-pHluorin and by specific fluorescent neurotransmitter reporters, such as CNiFERs for ACh and iGluSnFr for glutamate18-20. Overall, these current approaches for identifying pre-synaptic localization of nAChRs are complicated, and require special systems and techniques to allow reliable identification and physiological monitoring of pre-synaptic activity.
Here we describe protocols and equipment for an in vitro co-culture system of a ventral hippocampal (vHipp) &#8211; nucleus accumbens (nAcc) circuit that provides direct access to identify and analyze both pre- and post-synaptic components of synaptic transmission. We show examples of pre-synaptic localization of nAChRs and the live cell imaging of nAChR mediated Ca2+ signaling and neurotransmitter release along vHipp axons. A natural (and straightforward) extension of the protocol presented here is the preparation of pre- and post-synaptic contacts comprised of neurons from different genotypes. In this manner the contribution of a particular gene product to the pre- and/or post-synaptic mechanisms of modulation can be assessed directly.

<table><tbody><tr><td colspan="4">1. Culture Media (50 ml)</td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>GIBCO</td><td>10888022</td><td>48 ml</td></tr><tr><td>B-27 Supplements</td><td>GIBCO</td><td>0080085-SA</td><td>1 ml</td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>10908-010</td><td>0.5 ml</td></tr><tr><td>GlutaMAX Supplement</td><td>GIBCO</td><td>35050-061</td><td>0.5 ml</td></tr><tr><td>Brain-derived neurotrophic factor (BDNF)</td><td>GIBCO</td><td>15140-122</td><td>20 ng/ml</td></tr><tr><td colspan="4">2. washing media (HBSS, 100 ml)</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red&amp;nbsp;</td><td>GIBCO</td><td>14175-095</td><td>99 ml</td></tr><tr><td>HEPES ( 1M)</td><td>GIBCO</td><td>15630-130</td><td>1 ml</td></tr><tr><td colspan="4">3. HEPES buffered saline&amp;nbsp; (HBS)&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">4. HBS Cocktail for live imaging pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td>tetrodotoxin&amp;nbsp;</td><td>Tocris</td><td>1078</td><td>2 &amp;micro;M</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>10 &amp;micro;M</td></tr><tr><td>D-AP-5</td><td>Tocris</td><td>105</td><td>50 &amp;micro;M</td></tr><tr><td>CNQX</td><td>Tocris</td><td>1045</td><td>20 &amp;micro;M</td></tr><tr><td>LY341495</td><td>Tocris</td><td>1209</td><td>10 &amp;micro;M</td></tr><tr><td colspan="4">5. Calcium-free&amp;nbsp; HBS&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">6. 56 mM Potassium ACSF pH=7.4</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td>119 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td>56 mM</td></tr><tr><td>MgSO4.7H</td><td>Sigma</td><td>M1880</td><td>1.3 mM</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C1016</td><td>2.5 mM</td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S8282</td><td>1 mM</td></tr><tr><td>NaHCO3</td><td>Sigma</td><td>S5761</td><td>26.2 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr></tbody></table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

The preparation employed consists of gene chimeric co-cultures of vHipp&#8211;nAcc circuits in vitro. Projections emanating from vHipp microslices, as pre-synaptic axonal input, can make synaptic contacts with post-synaptic targets, the dispersed neurons from nAcc. Nicotine induced a sustained (&#8805; 30 min) facilitation of glutamatergic transmission from nAcc neurons innervated by vHipp axons21 and prolonged calcium signaling along vHipp axons5 via pre-synaptic &#945;7*nAChRs.
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Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

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9.0K Views.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Video: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Behavior

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/61481/quantifying-spontaneous-ca2-fluxes-and-their-downstream-effects-in-primary-mouse-midbrain-neurons'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Bancroft, E. A.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Quantifying Spontaneous Ca<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:0.6em;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>2+<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2020)</a> &#160;This video demonstrates the real-time visualization of calcium influx in ventral midbrain neurons derived from mouse embryos. Using a viral vector to express a calcium indicator, the neurons are imaged under a confocal microscope to track changes in fluorescence intensity during spontaneous and neurotransmitter-induced calcium activity.

Source:&#160;Bancroft, E. A.,&#160;et al. Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. ...

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Neuroimaging

Structural & Functional Brain Imaging

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Source: Zhong, C., et al. <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neuronal Culture Techniques

Co-culture and Interaction Studies

In Vitro Calcium Imaging in Short-Term and Long-Term Rat Hippocampal Cultures

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/53330/fluorescence-and-bioluminescence-imaging-of-subcellular-ca2-in-aged-hippocampal-neurons'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Calvo-Rodr&#237;guez,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Fluorescence and Bioluminescence Imaging of Subcellular Ca<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:0.6em;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>2+<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> in Aged Hippocampal Neurons.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2015).</a> &#160;This video demonstrates the use of fluorescence imaging to measure cytosolic calcium levels in short-term and long-term rat hippocampal cultures. By comparing young and aged neurons, it highlights how increased L-type voltage-gated calcium channel density and prolonged activation in aged neurons result in calcium overloading, a hallmark of neuronal aging. Fluorescent calcium indicators reveal these differences through changes in fluorescence intensity upon stimulation.

Source:&#160;Calvo-Rodr&#237;guez,&#160;et al. Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons.&#160;J. Vis. Exp. ...

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

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Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

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