Analysis of Neurite and Spine Formation in Neurons In Vitro

Abstract

Primary neuronal cultures are commonly used to study genetic and exogenous factors influencing neuronal development and maturation. During development neurons undergo robust morphological changes involving expansion of dendritic arbor, formation of dendritic spines, and expression of synaptic proteins. In this chapter, we will cover methodological approaches allowing quantitative assessment of in vitro cultured neurons. Various quantitative characteristics of dendritic arbor can be derived based immunostaining against anti-microtubule-associated protein 2 followed by dendrite tracing with the SNT plug-in of the FIJI software package. The number and subtypes of dendritic spines can be assessed by double labeling with DiI and Phalloidin iFluor448 followed by laser scanning confocal microscopy analysis. Finally, expression of presynaptic and postsynaptic proteins can be determined by immunohistochemistry and quantification using several available software packages including FIJI and Imaris, which also allows for 3D rendering and statistical displaying of the expression level of synaptic proteins.

1. Introduction

Primary neuronal cultures, and most often cultures of primary hippocampal neurons are an in vitro experimental model, which is commonly used to characterize wide range of factors modulating neuronal development, synaptic plasticity, neurodegeneration, and to test neuroprotective agents in rescue experiments where neuronal toxicity is introduced beforehand [1-3]. Quantitative readouts of dendritic arbor morphology, dendritic spine density, frequency analysis of dendritic spine subtypes, and expression of various synaptic proteins are typical outcome measures used in such experiments. This chapter aims to familiarize the reader with detailed methodological approaches allowing for quantitative assessment of in vitro cultured neurons.

Neuronal morphology plays an essential role in neuronal communication. Establishment of effective neuronal connectivity relies on a number of parameters including dendritic complexity, number and maturation of dendritic spines, and expression of pre-and post-synaptic proteins. Changes in any of these parameters are associated with different neurological diseases [4]. Dendrites receive and integrate signals from neighboring neurons and propagate them towards the perikaryon [5]. The number of all dendrites and complexity of their ramification is collectively named the dendritic arbor. The size of the dendritic arbor closely corelates with capacity to form neuronal networks [6]. Microtubule-associated protein 2 (MAP2) is involved in microtubule assembly and stabilization of dendrites [7]. Because MAP2 is localized only in dendrites, anti-MAP2 immunocytochemistry can be reliable used to visualize morphology of the dendritic arbor. Several quantitative characteristics can be collected to analyze and compare the complexity of the dendritic arbor. For a given neuron we routinely collect three different quantitative parameters: the combined length of all dendrites, the total arbor surface, and the number of intersections between dendrites and a series of concentric circles, which have a common center point in the center of the perikaryon [8,6,9]. The last analysis is also known as Sholl’s analysis from the name of its inventor. It is a commonly used measure of the dendritic arbor complexity, while the first two measures characterize the actual arbor size.

Dendritic spines are miniscule dendritic protrusions, which have highly plastic nature. They are formed by sideways elongations of the dendritic cytoskeleton and are known to assume a spectrum of different shapes, which differ by organization of the actin cytoskeleton within them and represent various stages of maturation [10]. Immature forms of dendritic spines take shapes of filopodia or thin spines, while mature forms have stubby and mushroom appearance. Mature forms of dendritic spines form functional synaptic connections between dendrites and axons of other neurons or are pruned when they fail to form such connections [5]. The Golgi silver stain was originally developed as a method to visualize dendritic spines. Though very elegant, this silver staining techniques is arduous to perform and yields inconsistent and irreproducible results, hence it has limitation when used as a basis for quantitative analysis [11]. Instead, we recommend using the combination of DiI ([1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) and phalloidin labeling, as a convenient and reproducible method allowing to visualize the entire spectrum of dendritic spine forms. DiI is a lipophilic fluorescent dye, which diffuses through the cell membrane, including that of dendritic spines rendering the outline of the spine discernible [12]. DiI labels particularly well matures spine forms (stubby and mushroom) and can be visualized using a standard rhodamine filter set. Phalloidin is a naturally occurring bicyclic peptide, with inherent affinity toward actin filaments in many different species of animals and plants [13]. In contrast to DiI, phalloidin primarily labels immature spine forms including filipodia and thin spines. Phalloidin can be labeled with numerous fluorescent dyes including iFluor 488, which allows for its application in tandem with DiI [10]. Quantification of dendritic spine density can be done along secondary and tertiary dendrites, within test areas of pre-defined length. Main readouts from dendritic spine quantitative analysis include the total number of spines per unit of length, and relative incidence of various spine types.

There is a large number of functional proteins expressed along the dendrites, which contribute to establishing functional synaptic connections. Perhaps the best known are subunits of glutamate receptors such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and N-methyl-d-aspartate (NMDAR), which are implicated in synaptic plasticity [14,15]. The postsynaptic density protein 95 (PSD-95) is a major component of postsynaptic densities and is structurally and functionally associated with AMPAR and NMDAR [16,17]. Another commonly used synaptic protein marker is synaptophysin (SYP), which is a vesicle protein involved in neurotransmitter release, synaptic connectivity, and synaptic plasticity [18]. Aforementioned proteins can be very selectively immunodetected and their expression along secondary and tertiary dendrites can be densitometrically quantified.

2. Materials

All solutions should be prepared using deionized water on the day of the experiments unless exemptions are otherwise stated. All tools and reagents used to culture neurons should be sterile.

2.1. Cell culture

1. Sulfuric acid 70%

2. Dulbecco’s phosphate-buffered saline (DPBS)

3. Poly-D lysine solution. Dilute poly-D-lysine (molecular weight 70,000-150,000) to the final concentration of 0.2 mg/mL using sterile, deionized water to obtain Poly-D lysine 2X stock. Sterilize the 2X stock by filtering it through a 0.2 μm syringe filter, makes aliquots, and store the aliquots at −20°C until use. Thaw an aliquot immediately prior to use and mix it 1:1 with DPBS).

4. Hank's Balanced Salt Solution (HBSS) Ca²⁺ and Mg²⁺ free

5. Papain stock solution. Reconstitute the entire content of a Papain vial (containing 100 units of Papain in 5 mL of Neurobasal medium. Incubate the vial in a 37°C water bath until the Papain is entirely dissolved and the solution becomes clear. This can take up to ten minutes. The resulting stock solution will contain 1 U of Papain in 50 μL volume (or 0.02 U/μL).

6. DNase I stock solution. Reconstitute the entire content of a DNAse I vial containing a 1,000 units of DNAse I in 500 μL of Neurobasal medium. The concentration of DNAse in the resulting stock solution will be 2U/μL.

7. Set of polished pipettes with gradually reducing aperture size. Reduce diameter tip of glass Pasteur pipettes by burning their tips in a burner flame while rapidly rotating the pipettes so the aperture will narrow evenly. For the first pipette, leave the pipette tip end in contact with the flame for around two to three seconds. For subsequent pipettes gradually increase the time of contact with the flame to achieve gradual reduction in the aperture size, obtaining pipettes 2-4 from bigger to lower aperture. Leave the polished pipettes under UV or autoclave them being careful not to break the tips.

8. Neurobasal medium for neuronal plating. Supplement Neurobasal medium with 2% B-27 supplement, 2 mmol/L glutamine, 50 mg/mL of streptomycin, and 100 U/mL penicillin.

9. Neurobasal medium for neuronal growth. Supplement Neurobasal medium with 2% B-27 supplement, 50 mg/mL of streptomycin, and 100 U/mL penicillin.

10. Cytosine-β-D-arabinoside (AraC) stock solution. Dilute AraC to 5 mmol/L using sterile, deionized water. Aliquot AraC solution and store the aliquots at −20°C protected from light.

11. Paraformaldehyde 2% and 4% solutions. Dilute paraformaldehyde in 0.1 M phosphate buffered pH 7.4. Aliquot and store the aliquots at −20°C until use.

2.2. Immunocytochemistry

1. 0.01 M phosphate buffered saline (PBS) pH 7.4. Dilute 10X commercially obtained PBS stock in sterile deionized water.

2. PBS-Triton. Dilute Triton in 0.01 M PBS pH 7.4 to the final concertation of 0.25%.

3. PBS-Tween. Dilute Tween-20 in 0.01 M PBS pH 7.4 to the final concertation of 0.05%.

4. Avidin block (kit). Add 100 μl of Avidin solution to 2.5 mL of PBS-Tween.

5. Biotin block (kit). Add 100 μl of Biotin solution to 2.5 mL of PBS-Tween.

6. Bovine serum albumin (BSA) blocking solution. Dilute lyophilize BSA in PBS-Tween to obtain 10% w/v solution.

2.2.1. Antibodies and dyes

1. Mouse monoclonal antibody against MAP2 diluted 4 μg/mL in PBS-Tween.

2. Mouse monoclonal antibody against (PSD-95) diluted 10 μg/mL in PBS-Tween.

3. Rabbit monoclonal antibody against NR1 subunit of the NMDAR diluted 1 μg/mL in PBS-Tween.

4. Rabbit monoclonal antibody against SYP diluted 0.8 μg/mL in PBS-Tween.

5. Biotinylated secondary antibody against mouse or rabbit IgG diluted 2 μg/mL in PBS-Tween.

6. Cy3 conjugated Streptavidin. Prepare 1 μg/mL dilution of Streptavidin/Cy3 in PBS-Tween. (see Note 1).

7. DAPI solution. Dilute DAPI in 80% methanol 1/1000. Store DAPI solution at 4°C in a light-tight vial.

8. DiI working solution. Dissolve DiI in DMSO to the final concentration of 0.2 μg/mL to prepare the stock solution. Once the solution is homogeneous dilute DiI stock in DPBS 1:500 to obtain working solution.

9. Phalloidin-iFluor 488 working solution. Dilute 1μL of Phalloidin-iFluor 488 in 1 mL of PBS with 1% BSA. Mix well by pipetting.

2.3. Equipment and software.

AmScope Stereoscopic Microscope

Fiji Software (NIH ImageJ2)

Fisherbrand^™ Multi-Platform Shaker

Imaris V 9.0 Software

Micro-Bead Sterilizer with Glass Beads

NU-4750 Water Jacketed CO₂ Incubator

NU-540 Series F Class II, Type A2 Biosafety Cabinet

Thermo IEC Centra CL2 Centrifuge

Zeiss 880 Laser Scanning Confocal Microscope with ZEN Black 2.3 SP1 Acquisition Software (v. 14.0.18.201)

80i Nikon Epi-fluorescent Microscope with DS-Qi1Mc High-Sensitivity, Cooled, Monochrome Camera

3. Methods

All procedures should be carried out in room temperature unless otherwise specified.

3.1. Cleaning of coverslips for neuronal plating.

1. Place 18 mm diameter glass coverslips in a closed glass container filled with 70% sulfuric acid. Incubate overnight while slowly shaking using orbital shaker.

2. Recover sulfuric acid the following day. The acid can be reused up to three times.

3. Wash the coverslips four times with distilled water, while gently shaking. Each wash should last approximately two 2 hrs.

4. Let the coverslips dry on the filter paper.

5. Place the coverslips in a Petri dish for sterilization. If there is more than one layer of coverslips, separate individual layers with the filter paper to avoid coverslips sticking to each other. Sterilize coverslips through autoclaving. Once sterile, the coverslips can be stored for prolonged time. Open the Petri dish container with sterilized coverslips only under the hood.

3.2. Culturing of primary hippocampal neurons

1. Coat the coverslips with poly-D-lysine by placing them in poly-D-lysine solution for 2 hr. at 37°C. We suggest using a 12-well plate and placing one coverslip per well. Wash the poly-D-lysine coated coverslips in sterile distilled water three times for 5 min. each. Let the coverslip dry completely under the hood before plating the cells.

2. Sterilize all instruments before use by submerging them in 70% ethanol for 15 min. followed by posterior sterilization at 100°C using a Micro-Bead Sterilizer for 30 sec. Let the instruments cool down before use.

3. Extract brains from the skulls of instantaneously decapitated P0 mouse pups. Place the brains in a Petri dish and cover them well with ice-cold Ca²⁺ and Mg²⁺ free HBSS. Maintain the Petri dish on ice.

4. Remove meninges under a stereoscope, dissect the hemispheres, and isolate the hippocampi. Place the hippocampi in a 35 mm Petri dish filled with 1 mL of Ca²⁺ and Mg²⁺ free HBSS. Transfer the Petri dish under a cell culture hood, and carry all subsequent steps there under aseptic conditions.

5. Transfer the hippocampi along with the entire volume of the HBSS solution into a 15 mL tube using a pipette.

6. Add 30 units of Papain (1,500 μL of the Papain stock solution) and 200 units of DNAse I (100 μL of the DNAse I stock solution) and incubate for 30 min. at 37°C. During the incubation time, manually dissociate the hippocampal tissue by pipetting up and down ten times with three fire-polished glass pipettes as follows: after 5 min. of incubation, pipette up and down with pipette 1. After 15 min. of incubation, pipette up and down with pipette 2. After 25 min of incubation, pipette up and down with pipette 3. (see Note 2).

7. Add 7 mL of plain Neurobasal medium to the triturated cell suspension. Transfer the entire volume to 15 mL tube and centrifuge for 6 min at 150 x g.

8. Gently remove the supernatant from above the pellet and resuspend the pellet in 5 mL of Neurobasal media with 100 μL DNase I by pipetting up and down with the fire-polished glass pipette 4 (see Note 3).

9. Let the 15 mL tube stand undisturbed for 2-5 min. to allow tissue debris to settle.

10. Transfer the supernatant to a 40-μm cell strainer presoaked with Neurobasal medium Place the strainer in a 50 mL tube and centrifuge for 6 min at 150 x g to obtain the neuronal pellet.

11. Resuspend the neurons in 1 mL of Neurobasal plating medium (see Note 4). Count the cells using hemocytometer and carefully plate 50,000 cells as a single drop on a coverslip placed in the bottom of a 12-well plate. Place the 12-well plate in an incubator at 37°C and 5% CO₂ for 1 hr. and let the neurons attach to the coverslips. Add 1.5 mL Neurobasal growth medium (see Note 5).

12. After 48 hr., remove 500 μl of medium, and replace it with the same amount a fresh Neurobasal growth medium containing 1.5 μL of AraC stock solution. AraC is added to inhibit growth of non-neural cells and its final concentration should be 5 μmol/L.

13. Change 1/3 of medium every three days with fresh Neurobasal growth medium (see Note 6).

3.3. Fixation of Neurons

1. At 17 day in vitro (DIV) carefully aspirate all medium and wash the neurons with DPBS one time for 5 min. (see Notes 7 and 8).

2. Add 2% or 4% paraformaldehyde depending on the staining that will be performed (see below) and incubate at room temperature for 15 min.

3. Wash the neurons with PBS twice by adding 2 mL of PBS to each well for 5 min. each time and then carefully aspirate PBS (see Note 9). Neurons, which are fixed and attached to the coverslips can be stored on the bottom of 12-well plate filled with PBS at 4°C until further use. The 12-well plate should be sealed with parafilm to prevent evaporation of the PBS, which should be replenish if needed.

3.4. Dendritic arbor analysis

3.4.1. Visualization of the dendritic arbor by MAP2 immunocytochemistry

For MAP2 immunocytochemistry we suggest using neurons fixed with 4% paraformaldehyde. At the beginning of the staining protocol, we place coverslips with affixed neuronal cultures cells up on the bottoms of the wells in a standard 12-well cell culture plate. All steps of the staining protocol are then carried out by exchanging reagents and washes in individual wells (see Note 7). We routinely use 500 μL of a reagent per well / step and 1,000 μL of PBS or PBS-Tween wash per well / step unless otherwise stated. All reagents are prepared immediately before use. All steps of the protocol should be performed on a shaker rotating at low speed and at room temperature, unless otherwise stated.

1. Wash three times with PBS for 15 min. each time.

2. Permeabilize neuronal membranes using PBS-Triton for 15 min.

3. Block with the BSA blocking solution for 1 hr.

4. Wash twice with PBS-Tween for 5 min. each time.

5. Block with the Avidin block for 15 min.

6. Wash three times with PBS-Tween for 15 min. each time.

7. Block with the Biotin block for 15 minutes.

8. Wash three times with PBS-Tween for 15 min. each time.

9. Incubate with anti-MAP2 primary antibody for 1 hr.

10. Wash three times with PBS-Tween for 15 min. each time.

11. Incubate with anti-mouse IgG biotinylated secondary antibody for 30 min.

12. Wash three times with PBS-Tween for 15 min. each time.

13. Incubate with Cy3/Streptavidin for 30 min.

14. Wash three times with PBS-Tween for 15 min. each time.

15. Stain neuronal nuclei with DAPI solution for 40 sec. (see Notes 10 and 11).

16. Wash twice with cold Methanol 80% for 2 min each time (see Note 12).

17. Place the coverslips neurons down on acid cleaned histological slides and affix them using a Fluoromount g mounting medium.

18. Store the slides in a light-tight box at 4°C.

3.4.2. Quantitative analysis of the dendritic arbor

1. Take photographs of MAP2 immunostained neurons under 40x objective using a fluorescent microscope.

2. Take photographs of neurons immunostained with the omission of the primary antibody (anti-MAP2) to adjust background fluorescence (see Note 13).

3. Open FIJI [19].

4. Open the NeuronJ plugin for neurite tracing and analysis. Open the image in an 8-bit format and trace each dendrite. Point the cursor to the start of the dendrite right next to the soma. Using the “+” command start tracing and follow the path of the dendrite with the mouse. Trace all the dendrites of the neuron and click save tracing (Fig 1B).

5. To measure the total arbor surface area, draw the outline of the dendritic arbor by joining tips of the most far outreaching dendritic branches using the freehand draw tool (Fig. 1C). Use the measure tool to quantify the area occupied by the outlined dendritic arbor.

6. For Sholl’s analysis open the SNT plugin (see Note 14). Create the Sholl’s grid of concentric circles spaced 3 μm (or as desired) over previously traced neuron. SNT enumerates the number of intersections between the dendrites and the Sholl grid.

Fig. 1. Analysis of dendritic arbor analysis.

A. Representative microphotograph of a hippocampal neuron at 17 day in vitro, which was immunostained against MAP2. B. Shown is tracing of the dendritic arbor using NeuronJ plugin. Combined dendrite length is calculated from the sum of the lengths of all traced dendritic branches for a given neuron. C. Shown is measurement of the total arbor surface. The measurable surface is created by manually joining the tips of the most far out reaching dendrites using the FIJI freehand tool. D. Sholl's analysis of dendrite branching. Shown is the schematic of the Sholl’s grid superimposed on an analyzed neuron. The Sholl’s grid represents a set of concentric circles, which are evenly spaced apart from the common center point and. In this case the distance between each pair of circles is set at 3 μm. Red dots indicate the cross points made between selected dendritic branches and the concentric circles. Results of the Sholl's analysis can be reported as a function between the distance from the center point (the soma) and the number intersection at a given distance, the total number of intersection per neuron, or both. Scale bar 40 μm in (A).

3.5. Dendritic spines analysis

3.5.1. Visualization of dendritic spines by double staining with DiI and Phalloidin-iFluor 488

For DiI/Phalloidin-iFluor 488 staining we suggest using neurons fixed with 2% paraformaldehyde. Using higher concentrations of paraformaldehyde or methanol as the fixative can impair DiI staining (see Note 15). At the beginning of the staining protocol, we place coverslips with affixed neuronal cultures cells up on the bottoms of the wells in a standard 12-well cell culture plate. All steps of the staining protocol are then carried out by exchanging reagents and washes in individual wells (see Note 7). We routinely use 500 μL of a reagent per well / step and 1,000 μL of PBS or PBS-Tween wash per well / step unless otherwise stated. All reagents are prepared immediately before use. All steps of the protocol should be performed on a shaker rotating at low speed and at room temperature, unless otherwise stated. When executing the staining protocol, the 12-well plate should be covered using a light-tight material e.g., aluminum foil.

1. Wash three times with DPBS for 15 min. each time.

2. Stain with DiI working solution for 15 min.

3. Wash twice with DPBS for 2 min. each time.

4. Leave the neurons in fresh DPBS overnight to allow for free-diffusion of DiI. This step is done without rotational shaking (see Note 16).

5. Wash three times with PBS for 5 min. each time.

6. Stain with Phalloidin-iFluor 488 working solution for 90 min.

7. Wash three times with PBS for 5 min. each.

8. Place the coverslips neurons down on acid cleaned histological slides and affix them using a Fluoromount g mounting medium.

9. Store the slides in a light-tight box at 4°C.

10. Visualize the cells after 72 hr. to allow dyes to equilibrate across neuronal membranes and diffuse well within the spine structures.

3.5.2. Image acquisition and analysis

1. Image DiI/Phalloidin-iFluor 488 stained neurons using a LSCM. Z-stack of images should be acquired through the entire thickness of the neuronal layer. Our verified imaging parameters include the 63 × 1.4 N.A. objective, a capture speed of 5 and a pinhole diameter of 1 Airy unit. In the z-axis, each two consecutively acquired slices have a 54% overlap between each other.

2. Process acquired Z-stacks using the Fiji software. Collapse Z-stacks into a two-dimensional image using the maximum intensity projection function. Subtract the background with a rolling ball radius of 35 pixels and adjust gamma to 1.1. Proportionally increase the picture resolution by a factor of 5 in the X- and Y-axis using the Transform J FIJI plugin in software.

3. Place four to five rectangular test area (4 μm × 20 μm) over secondary and tertiary dendrites aligning the long axis of the test area with the axis of the dendrite (Fig. 2A). Save the image of a neuron with superimposed test areas.

4. Identify dendritic spine subtype based on their distinct morphology: filopodium, thin, stubby, and mushroom-shaped and enumerate them [20] (Fig. 2B, C).

5. Average the total number of dendritic spines and their subtypes across all test areas taken from a single neuron.

Fig. 2. Analysis of dendritic spines.

A. Shown is a micrograph of a representative hippocampal neuron at 17 day in vitro, which was co-labeled with DiI (red) and phalloidin-iFluor 488 (green). Rectangular test areas (20 μm x 10 μm) are superimposed on primary and secondary dendrites with the long axis of the test area oriented parallel to the long axis of the dendrite. B. Shown are high magnification images of a representative test area captured under Fluorescein (top) and Rhodamine (center) filter sets and their merge (bottom). Particular types of dendritic spines are labeled with number, which correspond to the legend given in C. Filopodia and thin spines represent immature spine forms and stain stronger with Phalloidin, while stubby and mushroom spines denote more mature spine forms and more intensely labeled with DiI. C. Shown are morphological features used for classification of dendritic spine types. Scale bar 40 μm in (A) and 5 μm in (B).

3.6. Analysis of synaptic protein expression in primary and secondary dendrites

3.6.1. PSD-95, NR1 and SYP immunocytochemistry.

For synaptic protein immunocytochemistry we suggest using neurons fixed with 4% paraformaldehyde. At the beginning of the staining protocol, we place coverslips with affixed neuronal cultures cells up on the bottoms of the wells in a standard 12-well cell culture plate. All steps of the staining protocol are then carried out by exchanging reagents and washes in individual wells (see Note 7). We routinely use 500 μL of a reagent per well / step and 1,000 μL of PBS or PBS-Tween wash per well / step unless otherwise stated. All reagents are prepared immediately before use. All steps of the protocol should be performed on a shaker rotating at low speed and at room temperature, unless otherwise stated.

1. Wash three times with PBS for 15 min. each time.

2. Permeabilize neuronal membranes using PBS-Triton for 15 min.

3. Block with the BSA blocking solution for 1 hr.

4. Wash twice with PBS-Tween for 5 min. each time.

5. Block with the Avidin block for 15 min.

6. Wash three times with PBS-Tween for 15 min. each time.

7. Block with the Biotin block for 15 min.

8. Wash three times with PBS-Tween for 15 min. each time.

9. Incubate with anti-PSD-95, anti-NR1, or anti-SYP primary antibodies for 1 hr.

10. Wash three times with PBS-Tween for 15 min. each time.

11. Incubate with anti-mouse IgG biotinylated secondary antibody for PSD-95 and NR1 stainings or with anti-rabbit IgG biotinylated secondary antibody for SYP staining for 30 min.

12. Wash three times with PBS-Tween for 15 min. each time.

13. Incubate with Cy3/Streptavidin for 30 min.

14. Wash three times with PBS-Tween for 15 min. each time.

15. Stain neuronal nuclei with DAPI solution for 40 sec. (see Notes 10 and 11).

16. Wash twice with cold Methanol 80% for 2 min each time (see Note 12).

17. Place the coverslips neurons down on acid cleaned histological slides and affix them using Fluoromount g mounting medium.

3.6.2. Image acquisition

1. Image immunostained neurons using LSCM. Z-stack of images should be acquired through the entire thickness of the neuronal layer. Our verified imaging parameters include the 63 × 1.4 N.A. objective, a capture speed of 7, and a pinhole diameter of 1 Airy unit. In the z-axis, each two consecutively acquired slices have a 67% overlap between each other. The fluorescence level should be carefully adjusted to avoid saturation (see Note 13).

3.6.3. Analysis of synaptic protein expression using FIJI

1. In FIJI convert acquired Z-stacks into two-dimensional images using the maximum intensity projection function and save them into an 8-bit format.

2. Process images by enhancing the contrast to a value of 0.3 % normalized and adjust the threshold until single puncta representing synaptic proteins became visibly separated from each other (Fig 3 A, B.). Then use the sharpen tool to better define the puncta shape (see Note 13).

3. Place a rectangular test area (4 μm × 20 μm) over one of secondary or tertiary dendrites aligning the long axis of the test area with the axis of the dendrite (Fig. 3A, B).

4. Use the command analyze particles to collect the integrated density value for the analyzed test area. The integrated density is defined as a product of the area and the mean intensity value for a positive immunostaining within the test area (Fig. 3C).

5. Repeat steps 3 and 4 4 to 5 times for the same neuron and average obtained integrated density values.

Fig. 3. Analysis of synaptic protein expression.

A. Shown is a collapsed Z-stack of confocal images through a representative hippocampal neuron immunostained against PSD-95. B Shown is a threshold correction of the neuron imaged in A, which eliminates non-specific staining and highlights PSD-95 specific particles (or puncta) distributed through soma and dendritic arbor along with the mask of thresholded particles (C). Particles are routinely quantified within rectangular test areas (20 μm x 10 μm) superimposed over primary and secondary dendrites with the long axis of the test area oriented parallel to the long axis of the dendrite. Examples of test areas as shown underneath A through C. D. Shown is rendering of particle integrated density using Imaris's spot tool, where the integrated density of particles is represented as different size and color ball objects. The pseudo color bar at the bottom of the image represents size range across particle. E Imaris's spot tool rendering of particle size with highlighted outline of the neuron. Selected section of dendrites are enlarged underneath D and E. In neurons depicted in D and E a DAPI stained nucleus is also visualized. Scale bar 40 μm in the main panel and 5 μm in the magnified section in (A) and (D).

3.6.4. Analysis of synaptic protein expression in Imaris

1. Open an image Z-stack using Imaris.

2. Select the spot function for the channel and set diameter, quality, and threshold parameters needed to visualize small and large puncta. Maintain the same diameter, quality and threshold parameters across all experimental groups.

3. Generate dendritic surface to visualize dendritic outline.

4. Statistical information function will automatically display the area of all puncta per neuron.

5. For statistical visualization of synaptic protein expression use the color tool to highlight differences across the experimental groups. Select statistical parameters such as area, or volume and assign a pseudocolor (Fig. 3D, E).

6. Alternatively, to analyze five areas of interest per neuron, create a surface using the circle drawing mode, copy and paste the same area for the first and the last slice of the stack. The first and last slices were selected such as they captured the entire thickness of each neuron.

7. Create a mask with the surface selected into the channel of the puncta that you want to analyze (see Note 17).

8. Create again spots for this new region of interest using the same parameters as before.

9. The area, volume and other characteristics of the puncta in the selected test areas will be displayed under statistical information. Repeat steps 6 through 9 for up to 5 test areas and average obtained values for a given neuron.

4. Notes

1. To dilute Streptavidin/Cy3 in PBS-Tween mix it vigorously the in the shaker for 20 sec., then mix the solution by inversion in a 3D shaker for at least 30 min, before use.

2. Triturate hippocampal tissue very carefully, always putting the cell mixture back to the tube by directing the pipet tip towards the tube ’s wall and avoid making bubbles.

3. Remove the supernatant from above the cell pellet very carefully since the pellet can be easily disturbed.

4. Please notice that only the Neurobasal plating medium but not the Neurobasal growth medium contains glutamine. Continuous use of glutamine can lead to neuronal death.

5. Always add fresh medium to the neurons very carefully to avoid detachment of the neurons from the coverslip. Our preferred technique is applying the medium slowly against the wall of the well

6. Make sure to always maintain at least 1.5 mL of medium per well in the 12-well-plate. If the medium accidentally evaporates, add more than 1/3 of fresh medium during the medium exchange.

7. Exchange of liquid reagents in the well must be done carefully but quickly to avoid leaving the neurons uncovered for prolonged time. Please pay attention when adding the reagents to the well. It should be done by gentle pipetting out the liquid against the wall of the well to prevent detachment of neurons from the coverslip

8. Always check under the microscope that neurons are healthy before proceeding with the experiment.

9. Perform all steps under laminar flow biosafety hood. Minor contaminations with bacteria, can produce background during immunocytochemistry.

10. DAPI working solution can be used twice.

11. If the DAPI staining is dim, consider increasing the incubation time.

12. Leave methanol at −20°C for at least 1 hr. before its use.

13. Use the same parameters to image neurons across all experimental groups.

14. The SNT plugin is a toolbox for tracing and analyzing neuronal morphologies imaged using light or fluorescent microscopy. SNT is a recent replacement of the simple neurite tracer plugin. Like NeuronJ, SNT also can be used to quantify the combined dendritic length of the arbor. The choice of one plugin over another is a personal preference of the operator.

15. DiI staining is not compatible with any staining protocol that requires methanol or ethanol.

16. It is normal for the DPBS solution to become slightly pink on the second day, as the remaining crystals in the wells will solubilize into the PBS overnight. The solution with a very intense pink color likely means a high background due to the excess of crystals after the washes. On the other hand, if the DPBS solution appears almost transparent, some of the neurons may not be stained.

17. In the dialog window, check off the duplicate channel and set the voxels outside of the surface to 0 before applying the mask.