Name: dual electrophysiological recordings of synaptically-evoked astroglial and neuronal responses in acute hippocampal slices
Uploaded: 2012-11-26T12:00:00
Duration: 16 min 38 s
Description: Watch this Scientific Journal Video about Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices at JoVE.com

The authors would like to thank Dana Kamalidenova, Morgan Autexier, and Roch Chopier, who made the video and animations, as well as Florian Beck for the photographs and the post-production of the video voice-over. This work was sponsored by Olympus and supported by grants from the HFSPO (Career Development Award), ANR (Programme Jeunes chercheurs and Programme Blanc Neurosciences), FRC (F&eacute;d&eacute;ration pour la Recherche sur le Cerveau), INSERM and La Piti&eacute; Salp&ecirc;tri&egrave;re hospital (Translational research contract) to N.R., from French Research Ministry and Deutsche Forschungsgemeinschaft postdoc fellowships to U.P., and from the doctoral school "Frontiers in Life Science", Paris Diderot University, Bettencourt Schuller foundation, and FRM (Fondation pour la Recherche M&eacute;dicale) doctoral fellowship to J.S.

1. Preparation of Artificial Cerebrospinal Fluid and Intracellular Solution
<ol>
 <li>Before starting the experiment, one needs to prepare the internal solution for the patch clamp recordings, as well as the artificial cerebrospinal fluid (ACSF) for the hippocampus preparation. You will furthermore need a dissection kit consisting of surgical scissor and fine iris scissor, two spatulas and forceps (Fine science tools); a glass gassing device (micro-filter candle, ROBU Germany) and tissue grid (mosquito net or nylon tight), as well as superglue (Uhu Dent). The configuration of the electrophysiology slice patch setup was described by Finkel &amp; Bookman, 20011. </li>
 <li>For the internal solution, dissolve (in mM): 105 K-gluconate, 30 KCl, 10 HEPES and 0.3 EGTA in deionized water (in 70-80% of the final volume). Cool the solution to 4 &deg;C and add (in mM): 4 ATP-Mg, 0.3 GTP-Tris and 10 phosphocreatine. Adjust the pH to 7.4 with KOH and fill up with deionized water to the final volume. (Osmolaritiy: ~ 280 mOsm). Filter this solution (pore size 0.2 &mu;m). Aliquoted solution is stable for 3-4 weeks at - 20 &deg;C. For one experimental day, ~ 1 ml of internal solution is needed. </li>
 <li>Unless otherwise stated, the ACSF used for hippocampus preparation and recordings of cells in the CA1 region, contains (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3 and 11 glucose. Dissolve these salts in deionized water (Osmolarity ~ 320 mOsm) and oxygenate this solution for at least 10 min (pH ~ 7.3 - 7.4) with carbogen (95 % O2 and 5 % CO2). Prepare at least 1 liter of solution per experiment. Particular care should be taken for preparation of hippocampal tissue that will be used to perform experiments in the CA3 region of the hippocampus. Indeed, this region is prone to epileptiform activity and subsequent neuronal death. Thus synaptic activity should be strongly reduced during slice preparation, and this is achieved by performing the hippocampus dissection in ice-cold sucrose solution containing (in mM): 87 NaCl, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 1 NaH2PO4, 25 NaHCO3, 10 Glucose and 75 Sucrose. In this solution, the combination of low sodium, low calcium and high magnesium concentrations massively reduce presynaptic firing and release probability, as well as postsynaptic NMDA receptor activity, thus minimizing spontaneous activity and cell death. Once prepared, hippocampal slices used for recordings of cells from the CA3 region are perfused with modified ACSF, containing 4 mM CaCl2 and 4 MgSO4, to minimize polysynaptic activity. </li>
</ol>
2. Acute Hippocampal Slice Preparation
<ol>
 <li>Cool down ~ 300 ml of ACSF for the slicing chamber, as well as for the preparation at 4 &deg;C, while constantly oxygenating with carbogen. Prepare a small beaker with ACSF at room temperature (RT) for slice storage, which is also oxygenated with carbogen (Scheme Figure 1). </li>
 <li>Anesthetize the mouse under a hood with a small paper towel soaked with 1 ml isoflurane that is added into the cage. </li>
 <li>After the mouse is deeply anesthetized, cut the head off and add it directly into a small dish with ice-cold oxygenated ACSF. Remove the scalp with a small scissor and transfer the head onto tissue for the subsequent steps. Start to dissect the hippocampus, as illustrated and described in Figure 1. </li>
 <li>Cut 300-400 &mu;m thick transverse slices at low speed (3 &mu;m/sec) and vibration frequency of 70 Hz in ice cold oxygenated ACSF, and transfer them into a storage chamber. Let the slices rest at RT for at least 1 hr prior recording. Slices for CA3 experiments are stored 25 min at 34 &deg;C and at least 30 min at RT, to recover from the slicing process. </li>
</ol>
3. Dual Recording of Evoked Astroglial Currents and Neuronal Field Potentials
We here describe how to record synaptically-evoked astroglial and neuronal responses, i.e. responses induced by synapse activation through afference stimulation using an extracellular electrode. 
<ol>
 <li>Constantly perfuse the recording chamber with oxygenated ACSF (1.5-2 ml/min, RT), containing 100 &mu;M picrotoxin (GABAA antagonist) to isolate excitatory responses. Transfer a slice onto a poly-L-lysine (1.5 to 3 mg/ml) coated coverslip, soak the liquid to achieve a good slice adhesion and add a drop of ASCF on top of the slice. Place the coverslip into the recording chamber. Blockade of inhibitory transmission by picrotoxin can result in epileptiform activity, i.e. spontaneous, synchronous firing of neuronal populations, which will distort the measurement of evoked events. Thus, to prevent epileptiform activity, make a flat cut (only the surface) between the CA1 and CA3 regions to prevent the propagation via the Schaffer collaterals (as indicated in Figure 2a). </li>
 <li>Stratum radiatum astrocytes can be identified by their small soma size (~ 10 &mu;m) and stellate process assembly. Choose a cell at least 20-30 &mu;m below the slice surface. Mount a glass stimulation electrode (tip resistance ~ 1 M&Omega;) on the silver wire that is connected to the stimulus isolation box and grounded to the bath (simply by wrapping the second silver wire around the glass pipette). Place the stimulation electrode into the Schaffer collateral region, as indicated in Figure 2A at a distance of 200-300 &mu;m away from the chosen astrocyte. Mount the field recording electrode (~2-5 M&Omega;) onto a chlorinated silver wire connected to the headstage of the amplifier. Both electrodes are filled before with ACSF. Choose in multiclamp the mode I=0, which disables external command input and place the electrode ~ 50 &mu;m away from the astrocyte into the stratum radiatum region (Figure 2A). Record the responses with Gain 2 to 10 and filter with a 2 kHz Bessel filter. Electric current injection into the brain slice triggers action potentials in the surrounding Schaffer collateral axons and subsequent transmitter release at the presynaptic terminals that project to postsynaptic CA1 pyramidal neurons. The released transmitters will trigger a positive charge flow into the cells through postsynaptic ionotropic receptors, which is measurable extracellularly as a small negative potential. This field excitatory postsynaptic potential (fEPSP) integrates the activity of a group of simultaneously active neurons, while inhibitory transmission is blocked pharmacologically. Apply some test pulses (0.1 msec duration) to evoke a fEPSP; some repositioning of the stimulation electrode might help to increase the fEPSP. A typical CA1 stratum radiatum fEPSP is illustrated in Figure 2B. Further details on positioning and response waveforms can be found in Yuan et al. 20032. The fEPSP amplitude in a healthy hippocampal slice should usually be more than twice as big as the amplitude of the fiber volley. For accurate quantification of fEPSP amplitude or slope, the evoked reponse should be monosynaptic, as polysynaptic activity (detectable as a multi-peak response) indicates synaptic activity independent of the electrical stimulation, which could be a sign for hyperexcitability. For experiments performed in the CA3 region of the hippocampus, stimulation and recording pipettes are positioned as illustrated in Figure 3C. To clearly identify mossy fiber inputs, which are strongly facilitating, paired-pulse stimulation (50 msec interpulse interval) and 1 Hz stimulation for a few seconds are applied to massively enhance the initially low amplitude evoked fEPSP responses.At the end of the experiment, DCGIV, a mGluR2/3 receptor antagonist, can be washed in to further verify that indeed mossy fiber inputs were stimulated. Application of this antagonist should reduce the fEPSP by ~ 90% due to the high expression of mGluR2/3 receptors, inhibiting presynaptic release from mossy fiber boutons. </li>
 <li>Fill a patch pipette (~ 2-5 M&Omega;) with filtered internal solution and mount it onto a chlorinated silver wire connected to the second headstage, apply positive pressure with a syringe, which is connected via tubing to your pipette holder. Constantly apply a 20 msec test pulse of 10 mV and move the pipette into the tissue until you reach the cell surface and a deflection in the membrane becomes visible. Zero the pipette offset, remove the positive pressure, and clamp the membrane to - 80 mV. Wait until a gigaseal (at least 1 G&Omega;) is reached (it should not take longer than several seconds). Gentle application of some negative pressure might help to reach a gigaseal. Break into the cell is achieved by a short application of negative pressure or using the zap function in multiclamp. Start the simultaneous recording of the Schaffer collateral evoked fEPSP and the astroglial response in voltage clamp (Vhold - 80 mV; frequency 0.1 Hz, bessel filter 2 kHz, gain 10). The astroglial current response is biphasic: first you will see a fast transient outward current, reflecting fEPSPs generated by adjacent pyramidal cells. This is followed by a slowly rising and decaying inward current (persisting several seconds (&gt; 10 sec) after termination of neuronal responses). This current is mainly due to potassium entry into astrocytes, following release by surrounding depolarized postsynaptic terminals. A simultaneously activated fast transient glutamate transporter current (GLT), triggered by presynaptic glutamate release is masked by the potassium current. The holding potential of the astrocyte, as well as the access resistance of the patch should be monitored throughout the experiment and should not vary more than ~ 20%, to avoid inaccurate monitoring of astroglial reponses due to changes in the recording conditions. Only astrocytes with an initial holding potential &gt; -70 mV should be investigated to study healthy cells. </li>
 <li>Switch from voltage to current-clamp in order to record the evoked astrocytic membrane depolarization. Isolate the glial glutamate transporter (GLT) current by perfusion of the ionotropic glutamate receptor blocker kynurenic acid (5 mM), until the fEPSP is fully blocked and the GLT amplitude has reached a plateau. To clearly identify the GLT current, apply the specific antagonist DL-threo-&beta;-Benzyloxyaspartatic acid (DL-TBOA, 200 &mu;M). Increase the stimulation strength by 2-fold to 5-fold to record neuronal and astroglial responses at different synaptic strength, and apply two closely spaced stimuli (50 msec interval) to investigate responses to paired pulse stimulation. Stability of series resistance and membrane potential of the glial cell should be monitored throughout the recording. </li>
 <li>To visualize the extent of the gap-junction mediated astroglial networks, dye-coupling experiments should be performed in current clamp mode, without any current injection, to enable passive diffusion of low molecular dyes (&lt; 1.5 kDa), such as sulforhodamine-B, through gap junction channels. To minimize dye spillover into the surrounding tissue, positive pressure should be applied through the patch pipette just when entering the tissue and the patch should be reached as soon as possible. </li>
</ol>

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No conflicts of interest declared.

Dual recording of synaptically-induced neuronal and glial responses is a useful method to study online alterations in pre- and postsynaptic activities associated to changes in astroglial properties. The synaptically-evoked glial membrane depolarization is a direct measure of the extracellular potassium rise8, due in part to presynaptic action potential firing, but mostly to postsynaptic depolarization 7. Therefore recordings of glial membrane potential dynamics can be used to investigate modifications in presynaptic excitability, postsynaptic activity, extracellular space volume and potassium buffering capacities6, 8. The astroglial glutamate transporter current is a sensitive measure of presynaptic glutamate release, able to monitor short-term changes in release probability3, 5, 9. It can furthermore be used to characterize the functional synapse-glia interactions at different synapses or at different developmental stages10. It should be highlighted that GLTs are highly temperature sensitive11 and are driven by the electrochemical gradient of Na+, K+ and H+12. Thus the amplitude and kinetics of the GLT current highly depend on the chosen experimental conditions. Furthermore, the actual time course of astroglial glutamate clearance derived from the recorded GLT current is known to be partially obscured. This is due to the filtering of GLT currents by factors such as the electrotonic properties of astrocytes or the asynchronous transmitter release, which distort their kinetics13. Methods extracting the temporal features of the filtering mechanisms have been developed and can be used to derive the actual glutamate clearance time course in physiological or pathological situations, as recenly performed6,13,14. Additionally, the simultaneous recording of the astroglial membrane depolarization, in current clamp, can provide insights into possible alterations of extracellular potassium transients. Single astrocytes contact up to 100,000 synapses of ~ 100 different neurons, and do therefore integrate and modulate the activity of local neuronal networks. 
When using the technique presented here, i.e. recording electrophysiological whole-cell responses from astrocytes to gain insights into basal synaptic activity, one should keep in mind that in astrocytes, patch-clamp recordings at the soma level allow detecting currents mostly originating from the cell soma or proximal processes. Indeed, currents detected at the soma only partially originate from fine distal processes when a strong activation of receptors and channels occuring in multiple fine processes can generate currents propagating to the cell soma. Thus basal receptor and channel activity in individual small astroglial processes covering synaptic compartments is hardly detectable. This is due in part to the limited spatial and temporal control of membrane currents and voltages by whole-cell patch-clamp recordings from astrocytes in situ. However, it should be noted that the surface of the abundant tiny astrocytic processes exceeds by far the membrane area of the soma and main processes. In addition, these perisynaptic astroglial microdomains contain the functionally relevant receptors and channels, which likely play an important role in neuroglial communication and synaptic regulation. The technique we presented here is therefore mostly useful to study the astrocytic integration of synchronous activity from neuronal ensembles, occurring in particular during afference stimulation. It should not be used to study the dialogue between individual synapses and adjacent fine astrocytic processes occurring during basal spontaneous activity. An alternative method to study local astroglial responses induced by basal synaptic activity would be to perform patch-clamp recordings from fine processes, as done in dendrites15. Although patching these fine astroglial processes is likely challenging due to their small size, it is probably an avenue to pursue to unravel more intimate dialog between astroglial microdomains and individual synapses. However, the likely small electrophysiological astroglial responses resulting from individual fine astroglial processes may be below threshold detection, since electrical noise reaches in average 3-5 pA in patch-clamp recordings. Another method to study astroglial responses to synaptic activity is calcium imaging, since activation of astrocytic membrane receptors or transporters by neuroactive substances can trigger intracellular calcium transients. However, bulk loading of astrocytes with calcium indicators may also mainly reflect somatic activity16. The combination of electrophysiology and calcium imaging also enables detecting small calcium signals from fine astroglial processes, either occurring spontaneously or triggered by minimal synaptic stimulation17, 18. However, one should keep in mind that high-affinity calcium indicators might act like calcium buffers, inhibiting important calcium signaling pathways, whereas low-affinity indicators might work below detection level. Finally, an elegant and non-invasive technique to study calcium events in fine astrocytic processes, which also circumvents the washout of intracellular signaling molecules during whole-cell patch-clamp, consists in using a membrane targeted calcium sensor, which can be expressed in astrocytes in situ, as well as in vivo19. However, calcium imaging can only provide information about one signaling molecule, which is involved in many, but not all cellular activities, whereas whole-cell patch-clamp provides quantitative information about all the different ionic currents triggered upon channel and receptor activation. Therefore simultaneous electrophysiological recordings from neurons and astrocytes are a unique and powerful method to unravel online the dynamics of neuroglial ionic signalling and its role brain information processing.

<table border="1">
	<tbody>
 	<tr>
 	<td>Name of the reagent</td>
			<td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 	<td>Picrotoxin</td>
 <td>Sigma</td>
 <td>P1675</td>
 <td>dissolve in DMSO</td>
 </tr>
 <tr>
 	<td>Kynurenic Acid</td>
 <td>Tocris</td>
 <td>0223</td>
 <td>dissolve at 34 &deg;C stirring or sonication</td>
 </tr>
 <tr>
 	<td>DL-TBOA</td>
 <td>Tocris</td>
 <td>1223	</td>
 <td>DCG IV	Tocris	0975	</td>
 </tr>
 </tbody>
</table>

dual electrophysiological recordings of synaptically-evoked astroglial and neuronal responses in acute hippocampal slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

A representative simultaneous recording of synaptically-evoked astroglial and neuronal responses (fEPSPs) in the CA1 area of the hippocampus is shown in Figure 2 A-B. The evoked astroglial current is biphasic, i.e. it consists of a transient outward current and a slowly decaying inward current (&gt; 10 sec) (Figure 2B). The outward current reflects the evoked fEPSP, and is blocked after inhibition of ionotropic glutamate receptors by kynurenic acid (dark grey trace, Figure 2B)3. The majority of the slow inward current reflects potassium entry into the astrocyte following postsynaptic depolarization, since it is also abolished by kynurenic acid, which inhibits postsynaptic ionotropic glutamate receptor activity (Figure 2B and Figure 2C1)3-6, known to represent the main source (80 %) of potassium release7. The remaining rapidly rising and decaying inward current is inhibited by the GLT antagonist DL-TBOA (light grey trace, Figure 2B and Figure 2C2). Post-hoc subtraction of the remaining slow current in TBOA (light grey trace) from the current in kynurenic acid (dark grey trace) allows the isolation of the pure astroglial glutamate transporter current (black trace), as illustrated in Figure 2C2. The persistent slowly decaying current in kynurenic acid and TBOA (light grey trace, Figure 2B and Figure 2C2) can be blocked by TTX (data not shown), and reflects most likely the accumulation of extracellular K+ released during presynaptic afferent firing3. Moderate single stimulation of Schaffer collaterals induces a relatively large synaptically-evoked astroglial current compared to the small evoked depolarization recorded in the same cell (Figure 2D). This is due to the low membrane resistance of astrocytes. Recording of the synaptically-evoked astroglial membrane potential dynamics, as illustrated in Figure 2D, is a direct measure of local extracellular potassium levels8. Normalization of the evoked astroglial responses to the underlying neuronal activity allows the direct comparison of different experiments, as recently shown6. Astroglial currents can furthermore monitor very reliably alterations in excitatory transmission, as the total synaptically-evoked astroglial current follows linearly the increase in the fEPSP (Figure 2E). Astroglial currents also reflect short-term synaptic plasticity, since they show, as neurons, paired-pulse facilitation (Figure 2F). Paired whole-cell recording of a CA1 pyramidal cell and an astrocyte reveal very different electrophysiological behavior in both cell types, since the neuron display action potentials in response to a depolarizing pulse, while the neighboring astrocyte is silent (Figure 3A-B). However, moderate stimulation of the Schaffer collaterals can evoke simultaneously a fast excitatory posynaptic potential in the CA1 pyramidal cell and a fast outward and slow inward currents in the adjacent astrocyte (Figure 3B2). Dual recordings of synaptically-evoked neuronal and astroglial responses can also be recorded in the CA3 area of the hippocampus, as shown in Figure 3C. Indeed, single stimulation of CA3 mossy fibers evokes in basal conditions very small neuronal responses, recorded as local fEPSPs, associated to small fast outward and slow inward currents in astrocytes (Figure 3D1). In contrast, 1 Hz stimulation of CA3 mossy fibers for a few seconds strongly potentiates the fEPSP, while it only moderately increases the astroglial response (Figure 3D2). 
<img src="/files/ftp_upload/4418/4418fig1.jpg" alt="Figure 1" /> 
Figure 1. Hippocampus isolation to prepare transversal slices. To dissect the brain, cut the scull along the midline (a). Make a coronal cut at the level of the olfactory bulb (b) and subsequently at the level of the cerebellum (c). Carefully remove the scull with the help of a forceps (d), separate the two hemispheres with a blade (e), and transfer them on a small spoon into cold oxygenated ACSF (f). After ~ 5 min equilibration, place one hemisphere on dry tissue with the medial surface up (g). With the help of two spoons remove the diencephalon (h-j). The hippocampus is now visible, as illustrated by the dashed lines (k). Dissect the hippocampus with a spoon out, starting from the fimbria, visible as white structure (l-m). Transfer the hippocampus back into the cold ACSF. Prepare a small agarose-block, position the two hippocampi with the alveus side up and the ventral hippocampus facing the edge of the agar block, and soak carefully all liquid away to allow a good attachment to the agar (n). Glue the hippocampus attached to the agar block onto the ventral part (o).
<img src="/files/ftp_upload/4418/4418fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4418/4418fig2highres.jpg"/> 
Figure 2. Simultaneous neuronal and astroglial responses evoked-synaptically in the CA1 area of the hippocampus. A) Scheme of the hippocampal slice illustrating the arrangement of the stimulating electrode, to activate the Schaffer collaterals (SC), the patch pipette electrode, to record astrocytic currents, and the extracellular electrode, to record fEPSP, evoked by SC stimulation in the hippocampal CA1 area. B) Representative traces of simultaneous recordings of fEPSPs (upper panel) and astrocytic currents (lower panel) evoked-synaptically by SC stimulation in the presence of pharmacological drugs. The responses are first recorded in the presence of a GABAA receptor blocker (picrotoxin, 100 &mu;M, black traces) to isolate excitatory responses. Subsequent application of an ionotropic glutamate receptor blocker (kynurenic acid, 5 mM, dark grey traces), inhibits the fEPSP and the major part of the long-lasting astroglial current, unmasking a small and fast transient component of the astrocytic current response, which is sensitive to a glutamate transporter blocker (TBOA, 200 &mu;M, light grey traces). Scale bar, fEPSP 0.1 mV, astrocytic current 15 pA, 10 msec. C1). Sample trace of the astroglial potassium current (1-2), which can be isolated from the evoked response, shown in B (lower panel, black trace), by subtracting the current component remaining in kynurenic acid (2) from the total current (1). Scale bar, 20 pA, 1 sec. C2) Sample trace of the glutamate transporter current (2-3), obtained by subtraction of the TBOA insensitive slow component (3) from the current in kynurenic acid (2). Scale bar, 2.5 pA, 25 msec. D) Sample traces of an inward current recorded in voltage-clamp (lower panel) and the corresponding membrane depolarization recorded in current-clamp (upper panel) induced in an astrocyte by SC stimulation. Scale bar, current-clamp 1.5 mV, voltage-clamp 5 pA, 1 sec. E) Input-output curves illustrating the relationship between the presynaptic fiber volleys (input) and the total astroglial current (output) recorded simultaneously in response to SC stimulation (n = 6). The astroglial current increases linearly with the increased fiber volleys, as the neuronal fEPSP. F) Sample traces of the neuronal response (fEPSP) and the astrocytic current are shown for paired-pulse stimulation at a 40 msec interpulse interval. The synaptically-evoked astroglial current exhibits, like neurons, paired-pulse facilitation. Scale bar, 0.1 mV, 5 pA, 20 msec.
<img src="/files/ftp_upload/4418/4418fig3.jpg" alt="Figure 3" fo:content-width="3.75in" fo:src="/files/ftp_upload/4418/4418fig3highres.jpg"/> 
Figure 3. Dual recordings of synaptically-induced neuronal and astroglial responses in the CA1 and CA3 areas of the hippocampus. A) Reconstruction of a CA1 pyramidal cell injected with sulforhodamine-B (red, 0.1 %) and an astrocyte filled with fluorescein dextran (green, 0.1 %), using the whole-cell patch-clamp technique. Scale bar, 10 &mu;m. B1) Representative traces of simultaneous whole-cell recordings of membrane potentials recorded in current-clamp from a CA1 pyramidal cell and an adjacent astrocyte. Neuronal action potential firing (black trace), evoked by injection of a 20 pA depolarizing current pulse, evokes no response in the adjacent astrocyte (grey trace). Scale bar, 20 mV, 10 pA, 100 msec. B2) Sample traces of dual whole-cell recordings of a CA1 pyramidal cell in current-clamp and a neighboring astrocyte in voltage-clamp after SC stimulation in the presence of picrotoxin (100 &mu;M). SC stimulation evokes an excitatory postsynaptic potential (EPSP, black trace), associated to a small and long-lasting astrocytic current (grey trace). Scale bar, 5 mV, 10 pA, 100 msec. C) Scheme of the hippocampal slice depicting in the dentate gyrus (DG) and CA3 areas the arrangement of the stimulating electrode, to activate the mossy fibers, the patch pipette electrode (grey), to record astrocytic currents, and the extracellular electrode, to record fEPSP, evoked by CA3 mossy fibers stimulation. D, E) Representative traces of paired recordings of CA3 fEPSPs (upper panels, black traces in D1 and D2) and astrocytic whole-cell responses (lower panels, grey traces in D1 and D2) to single stimulation of CA3 mossy fibers at 0.02 Hz (zoom in the inset) (D1) or at 1 Hz stimulation frequency (D2). Scale bar for D1 and D2, 0.2 mV, 15 pA, time: D1 1 sec; D2 100 msec. Inset scale bar, 0.1 mV, 100 msec.

Watch this Scientific Journal Video about Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices at JoVE.com

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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JoVE Journal

Neuroscience

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.