Name: acute mouse brain slicing to investigate spontaneous hippocampal network activity
Uploaded: 2020-08-28T14:00:00
Description: Watch this Scientific Journal Video about Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity at JoVE.com

The author would like to thank Steve Siegelbaum for support. Funding is provided by 5R01NS106983-02 as well as 1 F31 NS113466-01.

All methods described here have been approved by the Institutional Animal Care and Use Committee at Columbia University (AC-AAAU9451).
1. Prepare solutions
<ol>
	<li>Prepare sucrose cutting solution for slicing as described in Table 1. 
	NOTE: After preparing 1 L of sucrose solution, freeze a small amount (approximately 100&#8211;200 mL) in an ice tray. These frozen sucrose ice cubes will be blended into an icy slurry (see step 4.3).</li>
	<li>Prepare a...

There are several steps in this slicing protocol designed to promote tissue health and favor the emergence of spontaneous naturalistic network activity: the mouse is transcardially perfused with chilled sucrose cutting solution; horizontal-entorhinal cortex (HEC) slices are cut at a thickness of 450 &#956;m from the intermediate or ventral hippocampus; slices recover at the interface of warmed ACSF and humidified, carbogen-rich air; during recordings slices are superfused with ACSF warmed to 32 &#176;C and delivered at a...

Electrophysiological measurement from living hippocampal slices in vitro is a powerful experimental approach with numerous advantages. The experimenter can use a microscope, micromanipulators, and a recording system to directly visualize and collect measurements from individual neurons in the tissue. Tissue slices are also very accessible to photostimulation or drug delivery for optogenetic, chemogenetic, or pharmacological experiments.
The hippocampal network generates highly synchronous population activity in vivo, visible as oscillations in the extracellular local field potential1,2,3,4,5. Brain slice methods have been leveraged to gain insight into the cellular and circuit mechanisms underlying these neuronal network oscillations. Foundational work from Maier et al. demonstrated that sharp wave-ripple complexes (SWRs) can emerge spontaneously in slices of the ventral hippocampus6,7. Subsequent studies from multiple investigators have gradually elucidated many aspects of SWRs, including the role of neuromodulators in regulating the network state of the hippocampus8,9,10 and the synaptic mechanisms that drive the in vitro reactivation of neuronal ensembles previously active during behavior in vivo11. Brain slice experiments have also provided insight into the gamma range oscillation (30&#8211;100 Hz), a distinct hippocampal network state believed to support memory encoding and recall12,13. Finally, recognizing the central role of the hippocampus and associated structures in the pathophysiology of temporal lobe epilepsy14,15, researchers have used hippocampal slice preparations to investigate the generation and propagation of epileptiform activity. Carter et al. demonstrated that combined hippocampal-entorhinal cortex slices prepared from chronically epileptic animals can spontaneously generate epileptiform discharges in vitro16. Subsequently, Karl&#243;cai et al. explored the mechanisms underlying epileptiform discharges in hippocampal slices by using modified artificial cerebrospinal fluid (ACSF) with altered ion concentrations (reduced Mg2+ or elevated K+) or added drugs (4AP or gabazine)17.
Investigators have developed numerous hippocampal slice approaches that differ in key ways: (1) the region of the hippocampus contained in the slice (dorsal, intermediate, or ventral); (2) the presence or absence of extrahippocampal tissues such as the entorhinal cortex; (3) the orientation used to cut slices (coronal, sagittal, horizontal, or oblique); and (4) the conditions under which the tissue is maintained after slicing (submerged fully in ACSF or held at the interface of ACSF and humidified, carbogen-rich air).
The choice of which slicing approach to use should be determined by the experimental objective. For example, transverse or coronal slices of the dorsal hippocampus maintained under submerged conditions have been used very effectively for the investigation of intrahippocampal circuitry and synaptic plasticity18,19,20. However, such preparations do not spontaneously generate network oscillations as readily as slices from the ventral hippocampus21,22,23. Although a state of persistent SWR activity can be induced by tetanic stimulation in transverse slices from the dorsal and ventral hippocampus24, spontaneous SWRs are more readily observed in ventral slices7,25.
An inherent physiological and anatomical distinction between the dorsal and ventral hippocampus is supported by studies performed both in vivo and in vitro26. Recordings in rats revealed strongly coherent theta rhythms throughout the dorsal and intermediate hippocampus, yet poor coherence between the ventral region and the rest of the hippocampus27. SWRs in vivo propagate readily between the dorsal and intermediate hippocampus, while SWRs that originate in the ventral hippocampus often remain local28. The associational projections originating from CA3 pyramidal neurons that reside in the dorsal and intermediate hippocampus project long distances along the longitudinal axis of the hippocampus. CA3 projections originating from ventral regions remain relatively local, and thus are less likely to be severed during the slicing process29,30. Ventral slices may, therefore, better preserve the recurrent network necessary to generate population synchrony. The propensity of ventral slices to generate spontaneous network activities in vitro may also reflect higher intrinsic excitability of pyramidal neurons or weaker GABAergic inhibition in the ventral hippocampus as compared to more dorsal regions31. Indeed, ventral hippocampal slices are more susceptible to epileptiform activity32,33. Thus, many studies of spontaneous physiological8,9,11,24 or pathological16,34,35,36 network oscillations have traditionally used a horizontal slicing approach, sometimes with a slight angle in the fronto-occipital direction, which yields tissue slices parallel to the transverse plane of the ventral hippocampus.
Network connectivity is unavoidably impacted by the slicing procedure as many cells in the slice will be severed. The angle and thickness of the slice and the tissue retained in the preparation should be considered to optimize connectivity in the circuits of interest. Many studies have utilized horizontal combined hippocampal-entorhinal cortex slices (HEC) to explore interactions between the two structures in the context of physiological or pathological network oscillations. Roth et al. performed dual recordings from the CA1 subfield of the hippocampus and layer V of the medial entorhinal cortex to demonstrate propagation of SWR activity through the HEC slice37. Many studies of epileptiform activity have used the HEC slice preparation to investigate how epileptiform discharges propagate through the corticohippocampal network16,35,36,38. It is important to note that preservation of the intact corticohippocampal loop is not a prerequisite for spontaneous SWRs, epileptiform discharges, or gamma oscillations; network oscillations can be generated in transverse slices of the dorsal or ventral hippocampus with no attached parahippocampal tissues21,22,23, 25,39,40,41. A more important factor for the spontaneous generation of network oscillations in hippocampal slices may be the thickness of each slice, as a thicker slice (400&#8211;550 &#956;m) will preserve more connectivity in the CA2/CA3 recurrent network21,22,25.
Although angled horizontal HEC slices (cut with an approximately 12&#176; angle in the fronto-occipital direction) have been used to study the functional connectivity of the corticohippocampal loop11,16,34,35,42, such angled preparations are not required for spontaneous network activity43,44,45. However, the use of an angled slicing plane does allow the investigator to selectively make slices that best preserve the transversely-oriented lamellae of either the ventral or intermediate hippocampus, depending on whether a downward or an upward angle is applied (Figure 1). This approach is conceptually similar to that used by Papatheodoropoulos et al., 2002, who dissected each hippocampus free and then used a tissue chopper to create transverse slices along the entire dorsal-ventral axis21. In the light of the aforementioned functional distinctions between the ventral and dorsal-intermediate hippocampus, investigators should consider the anatomical origin of slices when designing experiments or interpreting results. Using an agar ramp during the slicing procedure is a simple way to preferentially produce slices from either the intermediate or ventral hippocampus.
Hippocampal slices can be maintained in either a submerged chamber (with the tissue fully immersed in ACSF), or an interface-style chamber (e.g., Oslo or Haas chamber, with slices covered only by a thin film of flowing media). Interface maintenance enhances oxygenation of the tissue, which promotes neuronal survival and allows for sustained high levels of interneuronal activity. Traditionally, submerged recording conditions use a slower ACSF flow rate that does not provide adequate tissue oxygenation for stable expression of network-level oscillations. In submerged hippocampal slices carbachol-induced gamma oscillations are only observed transiently46,47, while they can be stably maintained in interface recording chambers10,48,49. As such, many studies of complex spontaneous activity in vitro have relied on interface recording chambers to investigate sharp-wave ripple complexes6,7,8,9,10,25,37, gamma oscillations10,13, and epileptiform activity16,38,45,47.
In a submerged-style recording chamber, an immersion microscope objective can be used to visualize individual cells and selectively target healthy-looking cells for recordings. The submerged preparation also allows fine control over the cellular milieu, as submersion facilitates rapid diffusion of drugs or other compounds to the tissue. Thus, a modified methodology in which stable network oscillations are maintained under submerged conditions represents a powerful experimental approach. This approach is exemplified by the work of H&#225;jos et al., in which hippocampal slices recover in a simplified interface-style holding chamber for several hours before transfer to a modified submerged recording chamber with a high flow rate of ACSF (~6 mL/min) to enhance oxygen supply to the tissue12,48,49. Under these conditions, high levels of interneuron activity and stable spontaneous network oscillations can be maintained in a submerged recording chamber. This modified approach allows the investigators to perform visually guided whole-cell patch clamp recordings and characterize the contribution of morphologically identified cell types to carbachol-induced gamma oscillations12. SWRs can also occur spontaneously in submerged hippocampal slices with a fast flow rate of ACSF11,48,49. Maier et al. demonstrated that hippocampal slices that recovered in an interface chamber before transfer to a submerged recording chamber reliably exhibited spontaneous SWRs, whereas slices that recovered submerged in a beaker before transfer to a submerged recording chamber showed smaller evoked field responses, lower levels of spontaneous synaptic currents, and only very rarely exhibited spontaneous SWRs43. Schlingloff et al. used this improved methodology to demonstrate the role of parvalbumin-expressing basket cells in the generation of spontaneous SWRs44.
The following protocol presents a slicing method through which spontaneously active neurons in horizontal hippocampal slices can be recovered under interface conditions and subsequently maintained in a submerged recording chamber suitable for pharmacological or optogenetic manipulations and visually guided recordings.

<table>
	<tbody>
		<tr>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Lulzbot</td>
			<td>LulzBot TAZ 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acute brain slice incubation holder</td>
			<td>NIH 3D Print Exchange</td>
			<td>3DPX-001623</td>
			<td>Designed by ChiaMing Lee, available at https://3dprint.nih.gov/discover/3dpx-001623</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate magnesium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9187-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag-Cl ground pellets</td>
			<td>Warner</td>
			<td>64-1309, (E205)</td>
			<td></td>
		</tr>
		<tr>
			<td>agar</td>
			<td>Becton, Dickinson</td>
			<td>214530-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Alfa Aesar</td>
			<td>36237</td>
			<td></td>
		</tr>
		<tr>
			<td>beaker (250 mL)</td>
			<td>Kimax</td>
			<td>14000-250</td>
			<td></td>
		</tr>
		<tr>
			<td>beaker (400 mL)</td>
			<td>Kimax</td>
			<td>14000-400</td>
			<td></td>
		</tr>
		<tr>
			<td>biocytin</td>
			<td>Sigma Aldrich</td>
			<td>B4261</td>
			<td></td>
		</tr>
		<tr>
			<td>blender</td>
			<td>Oster</td>
			<td>BRLY07-B00-NP0</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn scissors, small</td>
			<td>becton, Dickinson</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillaries with filament (O.D. 1.5 mm, I.D. 0.86 mm, length 10 cm)</td>
			<td>Sutter Instruments</td>
			<td>BF150-86-10HP</td>
			<td>Fire polished capillaries are preferable.</td>
		</tr>
		<tr>
			<td>calcium chloride solution (1 M)</td>
			<td>G-Biosciences</td>
			<td>R040</td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>OLY-150</td>
			<td></td>
		</tr>
		<tr>
			<td>compressed carbogen gas (95% oxygen / 5% carbon dioxide)</td>
			<td>Airgas</td>
			<td>X02OX95C2003102</td>
			<td></td>
		</tr>
		<tr>
			<td>compressed oxygen</td>
			<td>Airgas</td>
			<td>OX 200</td>
			<td></td>
		</tr>
		<tr>
			<td>constant voltage isolated stimulator</td>
			<td>Digitimer Ltd.</td>
			<td>DS2A-Mk.II</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslips (22x50 mm)</td>
			<td>VWR</td>
			<td>16004-314</td>
			<td></td>
		</tr>
		<tr>
			<td>cyanoacrylate adhesive</td>
			<td>Krazy Glue</td>
			<td>KG925</td>
			<td>Ideally use the brush-on form for precision</td>
		</tr>
		<tr>
			<td>data acquisition software</td>
			<td>Axograph</td>
			<td>N/A</td>
			<td>Any equivalent software (e.g. pClamp) would work.</td>
		</tr>
		<tr>
			<td>Dell Precision T1500 Tower Workstation Desktop</td>
			<td>Dell</td>
			<td>N/A</td>
			<td>Catalog number will depend on specific computer - any computer will work as long as it can run electrophysiology acquisition software.</td>
		</tr>
		<tr>
			<td>Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td>1-2950-0367</td>
			<td></td>
		</tr>
		<tr>
			<td>digital timer</td>
			<td>VWR</td>
			<td>62344-641</td>
			<td>4-channel Traceable timer</td>
		</tr>
		<tr>
			<td>disposable absorbant pads</td>
			<td>VWR</td>
			<td>56616-018</td>
			<td></td>
		</tr>
		<tr>
			<td>dissector scissors</td>
			<td>Fine Science Tools</td>
			<td>14082-09</td>
			<td></td>
		</tr>
		<tr>
			<td>double-edge razor blades</td>
			<td>Personna</td>
			<td>BP9020</td>
			<td></td>
		</tr>
		<tr>
			<td>dual automatic temperature controller</td>
			<td>Warner Instrument Corporation</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>dual-surface or laminar-flow optimized recording chamber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>The chamber presented in this protocol is custom made. A commercial equivalent would be the RC-27L from Warner Instruments.</td>
		</tr>
		<tr>
			<td>equipment rack</td>
			<td>Automate Scientific</td>
			<td>FR-EQ70&#34;</td>
			<td>A rack is not strictly necessary but useful for organizing electrophysiology</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethyiether)- N,N,N&#39;,N&#39;-teetraacetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>324626-25GM</td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>Whatman</td>
			<td>1004 070</td>
			<td></td>
		</tr>
		<tr>
			<td>fine scale</td>
			<td>Mettler Toledo</td>
			<td>XS204DR</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>glass petri dish (100 x 15 mm)</td>
			<td>Corning</td>
			<td>3160-101</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>G8877-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ice buckets</td>
			<td>Sigma Aldrich</td>
			<td>BAM168072002-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane vaporizer</td>
			<td>General Anesthetic Services</td>
			<td>Tec 3</td>
			<td></td>
		</tr>
		<tr>
			<td>lab tape</td>
			<td>Fisher Scientific</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>lens paper</td>
			<td>Fisher Scientific</td>
			<td>11-996</td>
			<td></td>
		</tr>
		<tr>
			<td>light source</td>
			<td>Olympus</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride solution (1 M)</td>
			<td>Quality Biological</td>
			<td>351-033-721EA</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stir bars</td>
			<td>Fisher Scientific</td>
			<td>14-513-56</td>
			<td>Catalog number will be dependent on the size of the stir bar.</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Luigs &#38; Neumann</td>
			<td>SM-5</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator (manual)</td>
			<td>Scientifica</td>
			<td>LBM-2000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>microspatula</td>
			<td>Fine Science Tools</td>
			<td>10089-11</td>
			<td></td>
		</tr>
		<tr>
			<td>monitor</td>
			<td>Dell</td>
			<td>2007FPb</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700B Microelectrode Amplifier</td>
			<td>Molecular Devices</td>
			<td>MULTICLAMP 700B</td>
			<td>The MultiClamp 700B should include headstages, pipette holders, and a model cell.</td>
		</tr>
		<tr>
			<td>N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid), (HEPES)</td>
			<td>Sigma Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>needle (20 gauge, 1.5 in length)</td>
			<td>Becton, Dickinson</td>
			<td>305176</td>
			<td></td>
		</tr>
		<tr>
			<td>nylon filament</td>
			<td>YLI Wonder Invisible Thread</td>
			<td>212-15-004</td>
			<td>size 0.004. This cat. # is from Amazon.com</td>
		</tr>
		<tr>
			<td>nylon mesh</td>
			<td>Warner Instruments Corporation</td>
			<td>64-0198</td>
			<td></td>
		</tr>
		<tr>
			<td>perstaltic pump</td>
			<td>Harvard Apparatus</td>
			<td>70-2027</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine di(tris) salt</td>
			<td>Sigma Aldrich</td>
			<td>P1937-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette holders</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td></td>
		</tr>
		<tr>
			<td>platinum wire</td>
			<td>World Precision</td>
			<td>PT0203</td>
			<td></td>
		</tr>
		<tr>
			<td>polylactic acid (PLA) filament</td>
			<td>Ultimaker</td>
			<td>RAL 9010</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P3911-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium gluconate</td>
			<td>Sigma Aldrich</td>
			<td>1550001-200MG</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>60377-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>roller clamp</td>
			<td>World Precision Instruments</td>
			<td>14041</td>
			<td></td>
		</tr>
		<tr>
			<td>scale</td>
			<td>Mettler Toledo</td>
			<td>PM2000</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>slice harp</td>
			<td>Warner</td>
			<td>SHD-26GH/2</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Fisher Chemical</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9888-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic anhydrous</td>
			<td>Fisher Chemical</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Fisher Chemical</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>spatula</td>
			<td>VWR</td>
			<td>82027-520</td>
			<td></td>
		</tr>
		<tr>
			<td>spatula/spoon, large</td>
			<td>VWR</td>
			<td>470149-442</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile scalpel blades</td>
			<td>Feather</td>
			<td>72044-10</td>
			<td></td>
		</tr>
		<tr>
			<td>stirrer / hot plate</td>
			<td>Corning</td>
			<td>6795-220</td>
			<td></td>
		</tr>
		<tr>
			<td>stopcock valves, 1-way</td>
			<td>World Precision Instruments</td>
			<td>14054</td>
			<td></td>
		</tr>
		<tr>
			<td>stopcock valves, 3-way</td>
			<td>World Precision Instruments</td>
			<td>14036</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Acros Organics</td>
			<td>AC177142500</td>
			<td></td>
		</tr>
		<tr>
			<td>support for swivel clamps</td>
			<td>Fisher Scientific</td>
			<td>14-679Q</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors, sharp/blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe (1 mL)</td>
			<td>Becton, Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe (60 mL with Luer-Lok tip)</td>
			<td>Becton, Dickinson</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>three-pronged clamp</td>
			<td>Fisher Scientific</td>
			<td>05-769-8Q</td>
			<td></td>
		</tr>
		<tr>
			<td>tissue forceps, large</td>
			<td>Fine Science Tools</td>
			<td>11021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>tissue forceps, small</td>
			<td>Fine Science Tools</td>
			<td>11023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>tubing</td>
			<td>Tygon</td>
			<td>E-3603</td>
			<td>ID 1/16 inch, OD 3/16 inch</td>
		</tr>
		<tr>
			<td>tubing</td>
			<td>Tygon</td>
			<td>R-3603</td>
			<td>ID 1/8 inch, OD 1/4 inch</td>
		</tr>
		<tr>
			<td>vacuum grease</td>
			<td>Dow Corning</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>vibrating blade microtome</td>
			<td>Leica</td>
			<td>VT 1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>vibration-dampening table with faraday cage</td>
			<td>Micro-G / TMC-ametek</td>
			<td>2536-516-4-30PE</td>
			<td></td>
		</tr>
		<tr>
			<td>volumetric flask (1 L)</td>
			<td>Kimax</td>
			<td>KIM-28014-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>volumetric flask (2 L)</td>
			<td>PYREX</td>
			<td>65640-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>warm water bath</td>
			<td>VWR</td>
			<td>1209 
			&#160;</td>
		</tr>
	</tbody>
</table>

acute mouse brain slicing to investigate spontaneous hippocampal network activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

Presented here are representative recordings from HEC slices prepared as described in this protocol. Following recovery in an interface holding chamber (Figure 1C), slices are transferred individually to a submerged recording chamber (Figure 2B). The recording chamber is supplied with carbogen-saturated ACSF using a peristaltic pump (Figure 2A). The pump first draws ACSF from a holding beaker into a heated reservoir. Carbogen lines ...

Watch this Scientific Journal Video about Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity at JoVE.com

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Using this protocol, researchers can investigate the mechanisms underlying hippocampal network oscillations in acute mouse brain slice preparations. In this preparation, recordings are performed under submerged conditions in slices generating spontaneous network oscillations, allowing pharmacological and optogenetic techniques to be performed, and single-cell recordings to be visualized. For transcardial perfusion, immediately before removing the mouse from the isoflurane chamber, fill a Petri dish lid with three to five millimeters of chilled sucrose solution, and fill the Petri dish bottom with approximately one centimeter of chilled sucrose solution.Quickly transfer the anesthetized mouse to the left absorbent pad, and use three pieces of tape to secure the forelimbs and tail. Using large tissue forceps and surgical scissors, tent the skin and make a lengthwise incision from the bottom of the sternum to the top of the chest. Use the forceps to pull up on the sternum and use the scissors to cut through the diaphragm.Use the scissors to cut through the rib cage on each side in one large motion toward the point at which the forelimb meets the body, and use the forceps to position the front of the rib cage toward the head. Use the scissors to make a horizontal cut to completely remove the ribs, and use the forceps to hold the heart in place while inserting a 20-gauge perfusion needle into the left ventricle. When the needle is in place, use small dissection scissors to make an incision in the right atrium, and allow the blood to be flushed out of the circulatory system.To extract the brain, after decapitation, use small bond scissors to make two lateral cuts through the skull toward the midline at the front of the skull near the eyes, and make two additional cuts on either side of the base of the skull. Immerse the head in the glass Petri dish, and use the scissors to cut along the midline across the entire length of the skull, while pulling up with the scissors to minimize damage to the underlying brain tissue. Use small tissue forceps to firmly grasp each side of the skull, and to lift the bone up and away from the brain to open the cranium like a book.Using the fingers of the left hand to hold the flaps of the skull open, insert the microspatula under the brain near the olfactory bulbs, and flip the brain out of the skull into the sucrose. Use the microspatula to sever the brainstem, and wash the brain to remove any residual blood, fur, or tissues. Use the large spatula to transfer the brain to the glass Petri dish lid, and use half of a double-edged razor blade to make a coronal cut in the most anterior portion of the brain, including the olfactory bulbs.Next, make a coronal cut to remove the cerebellum, and apply cyanoacrylate adhesive to an agar ramp. Use forceps to briefly dry the brain on a piece of filter paper, before placing the tissue into the adhesive on the agar ramp, ventral-side down. Place the slicing platform into the slicing chamber of a microtome, and completely cover the setup with chilled sucrose solution.Use the large spatula to stir some sucrose slurry into the chamber, melting any frozen sucrose and rapidly bringing down the mixture temperature to one to two degrees Celsius. Cut slices to a thickness of 450 microns at 0.07 millimeters per second slicing speed. As each slice is freed, use the small-tissue forceps and a sharp scalpel to separate the two hemispheres, and to cut away tissue until the slice consists primarily of the hippocampus and perihippocampal regions.Use a plastic transfer pipette to transfer the slices individually to an interface recovery chamber containing warmed aCSF. With the slices positioned at the interface of the aCSF and air, and with only a thin meniscus of aCSF covering the slices. When all of the slices have been acquired, tightly close the chamber to allow the slices to recover at 32 degrees Celsius for 30 minutes.At the end of the recovery period, place the chamber on a stirrer set to a slow speed to promote aCSF circulation within the chamber. To record local field potentials, fill a 400 milliliter beaker with carbogen-bubbled aCSF, and place one end of the pump tubing into the beaker. Turn on a peristaltic pump at 8 to 10 milliliters per minute to direct aCSF from the 400 milliliter beaker to a heated reservoir, and from the reservoir to the recording chamber at 32 degrees Celsius.Next, briefly clamp the tubing and turn off the pump to pause the flow. Using fine forceps, transfer a brain tissue slice to the recording chamber by the corner of the lens paper the tissue is resting on, slice down. Peel away the lens paper, leaving the slice emerged in the recording chamber, and use a harp to secure the slice.Using a manual micromanipulator, slowly advance the tip of a sodium chloride-filled stimulation pipette into the surface of the slice at a 30 to 45 degree angle, then use a second micromanipulator to slowly advance the tip of an aCSf-filled local field potential pipette into the region of interest at a 30 to 45 degree angle, and record the local field potential of the sample according to standard protocols. In this figure, representative recordings from hippocampal entorhinal cortex slices, prepared according to the protocol as demonstrated, can be observed. In healthy slices, electrical stimulation should produce a field post-synaptic potential with a small pre-synaptic fiber volley and a large post-synaptic potential with a rapid initial descent.Spontaneous sharp-wave ripples should also be visible as positive deflections in the local field potential in the stratum pyramidale. In suboptimal slices, evoked field post-synaptic potentials demonstrate a large fiber volley, and a relatively small post-synaptic potential, and such slices do not exhibit spontaneous sharp-wave ripples. In vitro sharp-wave ripples demonstrate a positive field potential in the stratum pyramidale layer, with an overlaid high-frequency oscillation paired with a negative field potential in the stratum radiatum layer.As illustrated, sharp-wave ripples in hippocampal entorhinal cortex slices originate within CA2/CA3 recurrent circuits, and propagate to CA1. It is essential to bubble carbogen into the solutions throughout the procedure, to ensure that the sucrose solution is chilled, and to perform each step as quickly as possible. After preparing these slices, extracellular or intracellular recordings can be performed, along with optogenetic or pharmacological experiments to determine how different cell types contribute to the function of neural networks.

Research

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Neuroscience

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Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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Horizontal Hippocampal Slices of the Mouse Brain

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