Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Podsumowanie

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Streszczenie

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg²⁺ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Wprowadzenie

Acute seizure models can successfully reproduce electrographic signatures reminiscent of ictal events observed in the electroencephalogram (EEG) of individuals experiencing a seizure. Researchers use these ictal-like events (herein referred to as 'ictal events') as surrogates for the seizure event¹. Clinically, ictal events serve as a reliable proxy for seizure events since seizures are a neurological disorder which originates from the brain. In the epilepsy monitoring unit, neurologists rely upon the detection of ictal events to confirm the brain's epileptogenic region and isolate it for resection². In the intensive care unit, physicians monitor ictal activity to assess if any seizure activity persists in sedated patients³. Controlling seizures remains to be a challenging issue for the medical community, as 30% of epilepsy patients are drug resistant to the available medication^4,5, and 10% of medical cases involving drug-induced seizures are unresponsive to the standard treatment³. This presents a serious concern for the society, as 10% of the American population is prospected to experience one seizure event in their lifetime and 3% are expected to develop epilepsy⁶.

Studying seizures in chronic epilepsy models is expensive, laborious, and often take months to prepare⁷. It is also difficult to perform electrophysiological recordings in freely moving animals. Human clinical trials face similar issues, as well as additional complexities related to patient consent, variability in participants' backgrounds, and the moral and ethical considerations involved⁸. Acute seizure models, on the other hand, are favorable because they are relatively convenient to prepare, cost-efficient, and capable of generating large volumes of ictal events for study⁹. Additionally, the tissue is fixed in a stable position, so the conditions are ideal for performing the electrophysiological recordings necessary to study seizure dynamics and the related underlying pathophysiology. Acute seizure models remain favorable over in silico (computer) models because they are based on biological material comprised of the brain's constituent neuronal network with all its inherent factors and synaptic connectivity, that may not be captured by even the most detailed computer models¹⁰. These features make acute seizure models poised to be efficient at screening for potential anti-seizure therapies and making preliminary findings before advancing them for further investigation in chronic epilepsy models and clinical trials.

Typically, acute seizure models are derived from the normal brain tissue that has been subjected to hyper-excitable conditions. To induce clinically relevant ictal events in healthy brain tissue, it is important to understand that the brain functions optimally in a critical state¹¹ where excitation (E) and inhibition (I) are balanced¹². A disruption of the E-I balance can lead to the hyper-excitable seizure state in which ictal events precipitate. Accordingly, within this conceptual framework, there are two major strategies to generate ictal events in brain slices (in vitro) or in whole-brain (in vivo) preparations: either decreased inhibition ("disinhibition") or increased excitation ("non-disinhibition"). However, ictal events are highly ordered and synchronized events that require the influence of GABAergic interneurons to orchestrate the neural network activity^13,14. For this reason, non-disinhibition models are the most effective for generating ictal events in isolated neural networks, such as in an in vitro brain slice¹⁵, whereas in vitro disinhibition models commonly lead to spiking activity reminiscent of interictal-like spiking. Furthermore, within this conceptual framework, a momentary synchronizing event can also reliably trigger an ictal event¹⁶. In fact, an ictal event can be triggered by any minor perturbation applied to the neural system¹⁷ when it is at a critical state transition ("bifurcation") point¹⁸. Traditionally, these perturbations were induced by electrical stimulation. The recent development of optogenetics in neuroscience, however, now offers a more elegant strategy to induce critical state transitions¹⁶.

The methods described in this paper demonstrate how to generate ictal events on-demand in acute seizure models for both in vitro (step 1 of the Protocol) and in vivo studies (step 2 of the Protocol). They involve the choice of brain region, seizure induction method, study type, and species; however, the focus will be on the recommended choice of an acute 4-AP cortical seizure model because of its versatility in a wide variety of study types. The acute in vitro 4-AP seizure model is based on the standard protocol to prepare high-quality brain slices for electrophysiological recordings and imaging studies¹⁹. These protocols have already been used to make in vitro coronal brain slices from the somatosensory-motor cortex of mice^16,20 and humans²¹. Modifications to generate ictal events in these types of brain slices have been previously demonstrated¹⁶ and the full details are described in the Protocol below. The acute in vivo 4-AP cortical seizure model is based on the standard protocol to prepare a craniotomy for imaging studies²². The modification is that no (glass slide) window is installed following the craniotomy. Instead, proconvulsant agents (4-AP) are topically applied to the exposed cortex to induce ictal events while the animal is under general anesthesia. To our knowledge, our group was the first to develop this acute in vivo cortical seizure model in mice^16,23. The acute in vivo 4-AP cortical seizure model prepared from adult mice was developed to complement the in vitro slice model from juvenile tissue. The replication of findings in the adult in vivo seizure model helps to generalize the findings from slice models by addressing the inherent concerns regarding the non-physiological conditions of a 2D brain slice (versus a 3-D whole-brain structure) and the physiological differences between juvenile and adult tissue.

The method of on-demand ictal event initiation is demonstrated using either puffs of neurotransmitters with a picospritzer or optogenetic strategies. To the best of our knowledge, our group is the first to initiate ictal events in human tissue using neurotransmitters via a picospritzer¹⁶. For optogenetic strategies, the C57BL/6 mice strain is the conventional strain used for expressing transgenes. The expression of channelrhodopsin-2 (ChR2) in either GABAergic interneurons or glutamatergic pyramidal cells will provide the optional ability to generate ictal events on-demand with brief light pulses. Suitable optogenetic mice strains include the commercially available C57BL/6 variant that expresses ChR2 in either interneurons, using the mouse vesicular GABA transporter promotor (VGAT)²⁴, or pyramidal cells, using the mouse thymus cell antigen 1 promotor (Thy1)²⁵. These commercially available VGAT-ChR2 and Thy1-ChR2 mice offer the opportunity to activate GABAergic neurons or glutamatergic neurons, respectively, in the neocortex with blue (470 nm) light. The ability to generate ictal events on demand in acute seizure models can offer novel opportunities to study seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Protokół

All research involving patients was performed under a protocol approved by the University Health Network Research Ethics Board in accordance with the Declaration of Helsinki. Procedures involving animals were in accordance with guidelines by the Canadian Council on Animal Care and approved by the Krembil Research Institute Animal Care Committee.

1. Protocol I: Acute In vitro Seizure Model

1. Preparation of dissection solutions and artificial cerebrospinal fluid
   1. Carbogenate ultrapure water (which is water filtered with a resistivity of 18.2 MΩ·cm) with carbogen (95% O₂/5% CO₂) for 5 min using air stone bubble diffusers (aerators for aquariums).
      Note: Air stones can be connected to the carbogen tank’s regulator via standard silicone tubing. If air stones are not available, seal shut the end of the silicone tubing and poke small holes in the seal to allow the carbogen gas to bubble out and carbogenate the solution.
   2. Prepare a dissection solution by dissolving the solutes from Table 1 in ultrapure water. Add CaCl₂ to a solution that has been carbogenated for at least 5 min to help it dissolve.
      Note: Alternatively, add CaCl₂ first, so that it can dissolve easily in an unsaturated solution. The solution, in liquid form, should be 300 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen.
      1. Chill the dissection solution to 0 - 4 °C. Leave the solution in the fridge overnight or place the solution in the freezer for 1 h and continually monitor the temperature.
         Note: The dissection solution can be stored for up to 3 nights; discard afterward.
      2. Carbogenate the dissection solution for 5 - 10 min before use.
         Note: Ideally, the solution should either appear as slush or be transitioning to slush prior to use.
   3. Prepare rodent artificial cerebrospinal fluid (ACSF) by dissolving the solutes from Table 2 (or Table 3 for human ACSF) in ultrapure water. Carbogenate the ACSF solution while it is in a water bath heated to 35 °C until use. Add CaCl₂ to a solution that has been carbogenated for at least 5 min to help it dissolve.
      Note: Alternatively, add CaCl₂ first so that it can dissolve easier in an unsaturated solution. The solution, in liquid form, should be 290 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen. Solutions should be made in 1, 2, or 4 L volumes depending on the duration of the experiments. Make 4 L for a full day (8 h) of experiments. The ACSF should be made fresh each day; do not store it for longer than 1 d.
2. Mouse dissection to collect brain tissue
   Note: For human tissue, proceed to the next step (step 1.3) on making brain slices with the vibratome. The cube-shaped blocks (1 cm³) of brain tissue should be obtained from the neurosurgeon using procedures previously described^26,27.
   1. Collect all the tools and materials necessary for animal dissections. Set up the workspace to prepare for animal dissections.
      1. Check the carbogen tank (95%O₂/5%CO₂) to ensure it is not empty. Replace the gas cylinder prior to experiments if there is less than 500 psi of pressure remaining.
      2. Calibrate the vibratome (vibrating blade tissue slicer) according to the manufacturer’s instruction manual. Adjust the vibratome setting to have a cutting amplitude of 1 mm and a cutting speed of 0.12 mm/s.
         Note: The optimal settings may differ for each individual vibrating tissue blade cutter; however, in general, the amplitude should be high and the cutting speed should be low.
      3. Fill the brain slice incubation chamber (i.e., the brain slice keeper) with ACSF and bubble it with carbogen. Then place the incubation chamber in a water bath set at 35 °C.
         Note: Use rodent ACSF for rodent brain slices and human ACSF for human brain slices.
   2. Obtain a juvenile mouse of either sex, between the age of 13 d (p13) to 21 d (p21, where p0 is the date of birth).
   3. Anesthetize the mouse with an intraperitoneal injection of sodium pentobarbital (55 mg/kg of body weight). Once the mouse is deeply anesthetized, as indicated by the absence of the toe pinch reflex, promptly use a vet-approved guillotine instrument to decapitate the mouse in one swift movement.
      Note: Prepare the sodium pentobarbital injection according to the procedures recommended by institutional guidelines.
   4. Hold the mouse’s nose to stabilize the decapitated head and gently make a midline incision starting between the mouse’s eyes and along the entire length of the scalp to expose the skull. Ensure the skull is not compressed by the pressure applied by the razor. Use the corner of the razor’s edge to increase cut precision.
   5. Spread apart the flaps of the mouse’s scalp using fingers to expose the skull. Then, use the fresh corner of a razor’s edge to make an incision along the midline of the skull; pay careful attention to avoid any contact with the cerebral cortex.
   6. Insert splinter forceps into the mouse’s eye sockets to stabilize the decapitated head and place it in a Petri dish filled with cold (0 - 4 °C) dissection solution. Ensure that the head is fully submerged in the dissection solution.
   7. Use a second set of splinter forceps to gently peel away the mouse’s scalp, remove the nose bone by peeling it, and remove the back (caudal side) of the skull.
      Note: The nose bone of juvenile mice is very soft and easily broken.
   8. Use a micro spatula to cut the optic nerves, trigeminal nerves, and spinal cord. Then, use the micro spatula to gently separate the brain from the cranium. Leave the brain fully submerged in the Petri dish filled with dissection solution.
3. Preparation of cortical slices from the somatosensory-motor cortex
   1. Fill the vibratome’s buffer tray with ~150 mL of cold (0 - 4°C) dissection solution.
      Note: Optionally, keep the buffer tray in the freezer until needed to maintain a low temperature during the slicing procedure.
   2. Glue the brain tissue from mouse or human onto the vibratome stage (specimen holder/tray) using instant adhesive glue. For mice, cut off a small caudal portion of the brain (i.e., the cerebellum) so that it can be easily glued flat onto the specimen holder (allowing the rostral side of the brain to face the ceiling).
      Note: Alternatively, horizontal mouse brain slices can be prepared by gluing the dorsal side of the brain onto the specimen holder (the ventral side of the brain should face the ceiling)²⁸.
   3. Gently place the specimen holder (with brain tissue) into the buffer tray. Ensure that the dorsal portion of the brain is facing the vibratome’s blade.
      Note: It is critical to minimize the amount of time the brain is exposed to air.
4. Brain tissue sectioning and collection
   1. Slice the brain into 450 μm thick slices using the vibratome in the dorsal to ventral direction. Ensure that the brain remains completely anchored to the specimen tray while slicing it.
      1. Make the first cut in the mouse brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory-motor area is observed (located approximately halfway between the olfactory bulb and bregma).
         Note: Optionally, slice the brain in thinner (200 μm) slices until the corpus callosum appears in the coronal view of the brain, which indicates that the somatosensory-motor area is close.
   2. Use a wide-bore transfer pipette to collect coronal slices (450 μm) that contain the somatosensory-motor area and submerge them in a Petri dish containing cold (0 - 4 °C) dissection solution.
   3. Use a new razor blade and cut off any excess tissue from the slices. For coronal slices from mice, perform a transverse cut just below the neocortical commissure (i.e., corpus callosum). Do not cut in a sawing motion; simply apply pressure on the blade into the tissue and use a detailing brush to gently separate the tissue. Make sure to minimize the movement of the coronal slice.
   4. Use a wide-bore transfer pipette to transfer the dorsal portion of the coronal slices that contain the neocortex (layer 1 - 6) to a second Petri dish filled with warm (35 °C) ACSF for a moment (~1 s). Then, promptly transfer the slices to an incubation chamber containing warm (35 °C) carbogenated ACSF.
      Note: The purpose of the transfer into the Petri dish with ACSF is to minimize the transfer of dissection solution to the incubation chamber while transferring brain slices with the wide-bore pipette. Utilize the incubation chamber’s multiple wells to keep different brain slices organized.
   5. Discard the rest of the brain and animal carcass according to the institutional guidelines.
5. Incubation and maintenance
   1. Leave the brain slices slightly submerged in the incubation chamber at 35 °C for 30 min. Then, remove the incubation chamber from the water bath and allow it to return to room temperature (20 - 25 °C). Wait 1 h for the brain slices to recover before performing electrophysiological recordings.
      Note: Ensure there are no air bubbles that collect underneath the brain slices in the incubation chamber. Air bubbles are air interfaces that cause tissue damage. The mouse brain slices can be maintained for 6 - 8 h, while human brain tissue can be stored for up to 24 h in a well-carbogenated incubation chamber with the appropriate ACSF.
6. Electrophysiological recordings of the superficial cortical layer
   Note: Ictal events are observed in the extracellular local field potential (LFP) recordings from brain slices. The LFP of the brain slice can be observed and recorded with either an interface type chamber²⁹ or a submerged multi-electrode array (MEA) system¹⁶. The procedure has been previously described¹⁶ and additional details are described below.
   1. Use a wide-bore transfer pipette or a detailing brush to move a brain slice onto slightly larger pre-cut lens paper that is held in place using a dental tweezer. Transfer the lens paper (that the brain slice is resting upon) to the recording chamber and secure it in position with a harp screen.
   2. Run warm (35 °C), carbogenated ACSF perfusate through the recording chamber over the brain slice at a rate of 3 mL/min (~1 drip/s). Use a digital thermometer to ensure the recording chamber is 33 - 36°C.
   3. Pull glass electrodes with an impedance of 1 - 3 M‎Ω from borosilicate glass tubing (with an outer diameter of 1.5 mm) using a puller. Backfill the glass electrodes with ACSF (~10 µL) using a Hamilton syringe. Immediately discard the electrode if the tip is damaged.
      Note: Bleach the silver wire (5 min) and only allow a minimal portion (i.e., the tip) of the silver wire to be submerged in the ACSF back-filled glass electrodes to minimize noise and drift during the recordings. Remove any excess ACSF from the glass electrode with a Hamilton syringe if necessary.
   4. Use a 20X stereo microscope to accurately guide the recording glass electrode into the superficial cortical layer (2/3) using manual manipulators. Record/view the electrical activity of the brain slice on a computer with standard software.
      Note: Viable (high-quality) brain slices will exhibit a robust evoked potential in response to the applied electrical stimuli (100 µs, 30 - 300 μA) or light pulses (30 ms, 10 mW/mm²) for optogenetic tissue.
7. Induction of seizure-like activities
   1. Perfuse ACSF containing 100 µM 4-aminopyrimidine (4-AP) over the brain slice. Dissolve 80 mg of 4-AP in 8.5 mL of water to make a stock solution of 100 mM 4-AP. Add 100 µL of 100 mM 4-AP stock solution to 100 mL of ACSF to achieve an ACSF perfusate with 100 µM 4-AP.
      Note: Alternatively, use Zero-Mg²⁺ ACSF (Table 4), a modified ACSF solution containing no added Mg²⁺, to perfuse the brain slice. For human tissue, use a combination of 100 µM 4-AP in Zero-Mg²⁺ Human ACSF (Table 5) to achieve optimal results. The average time for ictal events to appear is 15 min; however, it may take up to 40 min for some brain slices.
8. On-demand seizure generation: an optogenetic strategy for optogenetic mice
   1. Apply a brief (30 ms) pulse of blue (470 nm) light (with a minimum 1 mW/mm² output intensity) to initiate an ictal event. Use a manual manipulator to position a 1,000 µm core diameter optical fiber (0.39 NA) directly above the recording region.
      Note: Set the rate of the photo-stimulation to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 50 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.
9. On-demand seizure generation: a non-optogenetic strategy for human tissue
   1. Position the brain slice so that the superficial layers are upstream, and the deep layers are downstream to the flow of the perfusate through the recording chamber.
   2. Pull a glass electrode (the same type as used for the LFP recordings) and gently touch its tip with a delicate task wiper to create a small opening. Backfill the electrode with ~25 µL of 100 mM GABA (dissolved in water) and attach it to the picospritzer. Alternatively, backfill the electrode with 200 µM glutamate (dissolved in water).
      Note: To estimate the volume being puffed from the picospritzer (~50 µL), apply a test puff onto a plastic plate (preferably gridded). Then, apply a droplet of known volumes (i.e., 10 µL, 20 µL, 50 µL, or 100 µL) with a pipette for a comparison with the test puff.
   3. Apply puffs of the neurotransmitter (GABA or glutamate) to the human tissue with a picospritzer (10 - 20 psi for 30 - 100 ms) to initiate an ictal event. Apply a single puff of 100 mM GABA onto the deep layer (5) of the brain tissue to generate ictal events in the superficial layer (2/3). Alternatively, apply a single puff of 200 µM glutamate directly onto the superficial layer to generate ictal events in the superficial layer.
      Note: Set the rate of the picospritzer puff to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 50 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.

2. Protocol II: Acute In vivo Seizure Model

1. Surgical preparation and surgery
   1. Obtain an adult mouse (p35 - p60) of either sex.
   2. Deeply anesthetize mouse with Ketamine (95 mg/kg) and Xylazine (5 mg/kg). Perform the toe-pinch reflex test to ensure the mouse is sedated. Alternatively, use isoflurane (4% for induction, 1.5 - 2% for surgery) to anesthetize the mouse.
      Note: The choice of anesthetics can impact the seizure activity^30,31.
   3. Mount the mouse into a stereotaxic frame by securing its head with ear bars. Place a heated pad underneath the mouse for the duration of the surgery.
   4. Shave the top of the mouse’s head using a rodent trimmer to expose the scalp. Inject 0.3 mL of lidocaine with a 25 G 5/8 in needle into the periosteum to avoid excessive bleeding or pain. Apply eye ointment to the mouse’s eyes to prevent them from drying out during the experiment.
   5. Pinch and lift the center of the mouse’s scalp to assess if its reflexes are gone. Subsequently, perform a horizontal cut to expose the bregma to lambda region of the skull. Gently scrape the entire exposed area of the skull with a cotton swab to create a dry surface.
   6. Perform a craniotomy of 4 mm in diameter at coordinates 2.0 mm lateromedial and -2.0 mm rostrocaudal to expose the somatosensory cortex. Mark the location with a circle (4 mm in diameter) using a permanent marker. Drill around the circle with a pneumatic dental drill until the remaining layer of the craniotomy bone easily gives way when pushed down. Apply saline solution onto the layer of bone before removing the craniotomy with splinter forceps. Subsequently, apply warm saline solution to the exposed region.
2. Recording methods and chemical induction of seizures
   1. Pull glass electrodes with an impedance of 1 - 3 M‎Ω from borosilicate glass tubing (with an outer diameter of 1.5 mm) using a puller. Backfill the glass electrodes with ~10 µL of ACSF or saline using a Hamilton syringe. Immediately discard the electrode if the tip is damaged.
      Note: Bleach the silver wire (5 min) and only allow a minimal portion (i.e., the tip) of the silver wire to be submerged in the ACSF back-filled glass electrodes to minimize noise and drift during the recordings. Remove any excess ACSF from the glass electrode with a Hamilton syringe if necessary.
   2. Use a manual manipulator to guide the glass electrode into the superficial cortical layer (2/3) in the somatosensory-motor area. Record/view the electrical activity of the brain slice on a computer with standard software.
      Note: Layer 2/3 is approximately 0.3 mm deep from the surface³².
   3. Topically apply ~0.5 mL 1.5 mM 4-AP saline onto the exposed cortex of the mouse with a syringe until it completely covers the craniotomy. Dissolve 14 mg of 4-AP in 100 mL of isotonic saline to make a stock solution of 1.5 mM 4-AP saline.
      Note: Prepare the 4-AP saline before the experiment begins. On average, ictal events appear 15 - 30 min after topical application.
3. On-demand generation of seizures in optogenetic mice
   1. Apply a brief (30 ms) pulse of blue (470 nm) light (with a minimum 10 mW/mm² output intensity) to initiate an ictal event. Use a manual manipulator to position a 1,000 µm core diameter optical fiber (0.39 NA) directly above the recording region.
      Note: Set the rate of the photo-stimulation to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 300 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.

Wyniki

The application of 100 µM 4-AP to good-quality (undamaged) 450 µm-sized cortical brain slices from a juvenile VGAT-ChR2 mouse reliably induced recurrent ictal events (> 5 s) within 15 min (Figure 1Ai). The application of 100 µM 4-AP to slices of poor-quality resulted in bursting events or spiking activity (Figure 1Aii). On average, 40% of the slices from each dissected mouse brain successfully generated ictal e...

Dyskusje

The brain slices are treated with a proconvulsant drug or an altered ACSF perfusate to increase the neural network's excitability and promote a precipitation of ictal events (electrographic seizure-like events). For mice, the preferred coronal slices of the somatosensory-motor area should contain the cingulate cortex, area 2 (CG), but not the retrosplenial area (RS); these anatomical markers help identify the range of coronal slices that are best for inducing ictal events. An optional modification for mice tissue is ...

Materiały

Name	Company	Catalog Number	Comments
Sodium pentobarbital	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
1 mm syringe	N/A	N/A	Purchased through UT Med Store
25G 5/8” sterile needle	N/A	N/A	Purchased through UT Med Store
Single edge razor blade (2x)	N/A	N/A	Purchased through UT Med Store
Instant adhesive glue	N/A	N/A	Purchased through UT Med Store
Lens paper	N/A	N/A	Purchased through UT Med Store
Glass petri dish (2x)	N/A	N/A	Purchased through UT Med Store
Splinter forceps (2x)	N/A	N/A	Purchased through UT Med Store
PVC handle micro spatula	N/A	N/A	Purchased through UT Med Store
Micro spoon with flat end	N/A	N/A	Purchased through UT Med Store
Detailing brush 5/0	N/A	N/A	Purcahsed from a boutique art store
Wide bore transfer pipette	N/A	N/A	Purchased through UT Med Store
Dental Tweezer	N/A	N/A	Purchased through UT Med Store
Thermometer (digital)	N/A	N/A	Purchased on Amazon.ca
Check carbogen tank (95%O2/5%CO2)	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
Vibratome	Leica	N/A	Purchased through the Toronto Western Hospital's Suppliers
brain slice incubation chamber (a.k.a. brain slice keeper)	Scientific Systems Design Inc	N/A
Sodium Chloride (NaCl)	N/A	N/A	Purchased through UT Med Store
Sodium Bicarbonate	N/A	N/A	Purchased through UT Med Store
Dextrose	N/A	N/A	Purchased through UT Med Store
Potassium Chloride (KCl)	N/A	N/A	Purchased through UT Med Store
Magnesium Sulfate (MgSO4 H2O)	N/A	N/A	Purchased through UT Med Store
Sodium phosphate monobasic monohydrate (HNaPO4·H2O)	N/A	N/A	Purchased through UT Med Store
Calcium Chloride (CaCl2·2H2O)	N/A	N/A	Purchased through UT Med Store
Sucrose	N/A	N/A	Purchased through UT Med Store