Name: inducing plasticity of astrocytic receptors by manipulation of neuronal firing rates
Uploaded: 2014-03-20T19:30:00
Duration: 12 min 47 s
Description: Watch this Scientific Journal Video about Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates at JoVE.com

The authors would like to acknowledge UC Riverside&#8217;s Center for Glial-Neuronal Interactions for valuable discussion of the scaling protocols and data. The authors would also like to give a sincere thank you to Abcam for sponsoring the publication of their&#160;work.

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The authors wish to disclose that the tetrodotoxin used in these studies was purchased from Abcam. Abcam had no involvement in the hypotheses, design, or collection of data. All communication regarding sponsorship of the work by Abcam occurred after the peer review process was complete.

The described scaling models represent practical methods for researching long-term plasticity of astrocytic group I mGluRs. Imaging spontaneous and evoked Ca2+ events provides a sensitive assay for measuring changes in astrocytic Gq GPCR activity, as firm evidence has been established that astrocyte Ca2+ elevations occur following release from IP3R-sensitive stores downstream of Gq GPCR activation10,12,17,18. The percentage of astrocytes in the population responding to group I mGluR agonist and the pattern of such Ca2+ responses report changes in group I mGluRs by astrocytes.
The specific technique used to load astrocytes with Ca2+ indicator is an important consideration in the design of experiments to look for changes in astrocytic Gq GPCR activity. Bolus-loading or bulk-loading multiple astrocytes, or patch-clamp loading of individual astrocytes can be used to image Ca2+ transients in astrocytes. Each approach offers certain advantages and disadvantages. Directly filling astrocytes with Ca2+ indicator via patch clamp allows unequivocal identification of the cell as an astrocyte without need for a secondary marker such as SR-101. Patch-clamp delivery of indicator also enables recording of Ca2+ activity from small astrocytic compartments including the bushy fine processes, potentially deeper in the slice where cells are healthier and with more intact interactions with synapses (depending on the laser power available). However, patch-clamp loading suffers from low throughput as data are collected one cell at a time. Bulk-loading, by contrast, allows a large number of astrocytes to be loaded with Ca2+ indicator and imaged simultaneously. However, only astrocytes near the surface (&#60;20 &#181;m) of the slice are loaded, with associated concerns about cell health and intact synapses.
The backpressure bolus-loading protocol presented here offers a middle ground, with relatively high throughput and the ability to monitor Ca2+ activity deeper within the slice (40-75 &#181;m). A significant increase in the percentage of spontaneously active astrocytes using the bolus-loading technique is observed compared to bulk-loading, suggesting that the connections among neuronal synapses and the astrocytic processes are more complete15. With good loading, one can often monitor Ca2+ activity in main processes of astrocytes (data not shown) or potentially even smaller compartments using 2-photon microscopy. However, care would need to be exercised in assigning the smaller processes to a particular astrocyte, as the boundaries blend into the nonspecific background staining. An additional concern with the use of bulk-loading or bolus-loading procedures is the need for a secondary marker for astrocyte identification. While it has been known for many years that astrocytes preferentially take up AM ester Ca2+ indicators, the secondary marker SR-101 is often used to verify the loaded cells as astrocytes. SR-101 may in itself alter the intrinsic excitability of neurons29. Use of SR-101 corroborates the need to perform all astrocyte Ca2+ measurements in TTX to limit possible SR-101 effects on neuronal excitability. Assuming that both control and experimental groups include SR-101, the marker in itself should not account for the effects observed in astrocyte Ca2+ signaling following long-term manipulation of neuronal action potentials. SR-101 may be more of a concern in high K+ experiments, however, as it may reduce the difference between 2.5 mM K+ vs. 5.0 mM K+ if the basal firing rate is not altered proportionally.
A very promising approach to deliver Ca2+ indicator to astrocytes has emerged recently which offers an attractive alternative to the more traditional approaches using Ca2+ dyes. Significant advances have been made over the past few years with genetically encoded calcium indicators (GECIs) targeted to astrocytes30-32. GECIs can be delivered to astrocytes by in vivo microinjection of adeno-associated viral vectors into a brain region of interest such as the hippocampus. Expression of GECIs is achieved after approximately two weeks following viral infection32. There are numerous advantages presented by the use of GECIs in astrocytes. First, the vectors are targeted to astrocytes using an astrocyte-specific promoter, so the labeled cells are astrocytes32. Second, the signal-to-noise now seems comparable to what can be obtained using patch-clamp delivery of dye, but without the invasiveness of having had a patch pipette on the cell32. Third, the indicators can be delivered and expressed in adult tissue, which is problematic using bulk-loading delivery methods. Furthermore, the expression is mosaic, offering the ability to differentiate among multiple astrocytes. Thus, several astrocytes can potentially be imaged simultaneously, while also recording in the soma and fine branchlets at the same time. Therefore, potentially one single technique could be used in place of three separate techniques (bulk-loading, bolus-loading, and patch-clamp loading) to record scaling activity of astrocytic Gq GPCRs, greatly increasing efficiency.
One potential drawback of using viral-mediated delivery of Ca2+ indicators to astrocytes is the possible effect on slice health32. The adeno-associated viral vectors used to deliver the GECIs have been shown previously to cause reactive gliosis of astrocytes33. Preparation of brain slices in general likely initiates early stages of pathology including release of inflammatory molecules10. Therefore, combined with the long incubation times required to induce scaling of astrocytic receptors, use of GECIs delivered using viral vectors would need to receive additional consideration in the context of slice health in these types of experiments.
When employing this protocol, it is important to keep in mind that the application time for agonist to produce a response will vary as a function of receptor availability. For a given concentration of agonist, the application time will have to be longer if receptors have scaled down, and shorter if receptors have scaled up, for the drug to reach an adequate concentration in the tissue to activate receptors sufficiently to produce a Ca2+ response. Therefore, drug application times, and potentially their concentrations, may have to be adjusted depending upon the intended direction of the scaling. For example, the agonist concentration may need to be lowered in the case of TTX to avoid saturating responses, and increased after incubating slices in high K+ to even see a response. Specifically, the DHPG concentration was shifted from 5-15 &#181;M after TTX treatment to 30-50 &#181;M after 5.0 mM K+ treatment in order to study Ca2+ response patterns, as 5-15 &#181;M was often too low to produce reliable responses in astrocytes after scaling down of group I mGluRs.
Recording of astrocyte Ca2+ activity provides no direct evidence of receptor insertion or internalization to or from the plasma membrane. However, based on the remarkable similarity of the data with data from previous studies in vitro which examined the direct relationship between Gq GPCR expression levels and spontaneous and evoked Ca2+ transients21-24, the most logical interpretation of the changes in Ca2+ signaling is that the astrocyte surface receptor expression levels have changed. A complementary approach may be an important consideration if one wants to provide additional evidence about the locus of the effect on Ca2+ activity. A strategy we used was to examine the effect of TTX incubation on hippocampal slices from astrocytic MrgA1R mice. These transgenic mice express a foreign Gq GPCR (the MrgA1R) only in astrocytes. Because this receptor is not native to the brain, there is no endogenous neurotransmitter present to change its activity levels. Previous work suggested that this receptor engages the same intracellular signaling molecules as endogenous group I mGluRs in the same astrocytes34. After long-term incubation of slices from MrgA1R mice in TTX, no differences in agonist-evoked MrgA1R responses compared to littermate control incubated slices would provide evidence that the effect on astrocyte Ca2+ activity is due to changes localized to the surface receptor, especially if group I mGluR responses are still significantly enhanced in the same astrocytes. An alternative, though perhaps more involved strategy would be to isolate astrocytes from the slices for Western blot analysis, as long as a membrane fraction could be analyzed for changes in surface receptor expression levels. Fluorescence Activated Cell Sorting (FACS) or flow cytometry may be helpful here.
The possible applications of this technique to the study of neurons, astrocytes and astrocyte-neuronal interactions are many. In our experiments, only DHPG-evoked group I mGluR astrocytic Ca2+ responses were studied, in isolated acute hippocampal slices from juvenile mice. This preparation not only has the intact afferents (Schaffer collaterals), but also the neurons that give rise to them (CA3 pyramidal cells), making it possible to manipulate the firing rates of these glutamatergic neurons onto the postsynaptic cells (CA1 pyramidal cells) and the astrocytes in stratum radiatum whose processes associate with these synapses. The acute hippocampal slice may not be the best preparation for manipulating firing rates of other types of neurons, however, as many afferents are severed from the neurons that give rise to them. Nevertheless, it may be possible in certain slice preparations to observe plasticity of other astrocytic Gq GPCR subtypes. For example, slices could be prepared with basal forebrain cholinergic neurons and their projections to hippocampus intact. Incubation of these slices in TTX or elevated K+ would affect basal firing rates of cholinergic neurons, leading to scaling of mAchRs in astrocytes of stratum oriens, which receive a significant portion of the cholinergic input1. An alternative yet untested approach to study astrocytic receptor scaling within a specific area of the brain, with all of the connections intact while scaling occurs, could be to use an in vivo model where a sustained release of TTX is achieved by implantation of a plastic polymer Elvax 40W above the region of interest35. This approach has been used previously in a study of neuronal scaling but should also be applicable to astrocytic scaling. Finally, with the proper readout, future studies could examine other GPCR families, including changes in Gs or Gi GPCRs. One might predict astrocytic GABAB Gi GPCRs to be affected following inhibition of firing in locally projecting GABA interneurons within any slice preparation. Development of new indicators targeting other signaling molecules, such as a real-time indicator of the second messenger cAMP, would open up a whole new area of research on neuron-to-astrocyte receptor communication.
Bidirectional scaling of astrocytic mGluRs by manipulation of basal neuron firing rates provides a measure of the sensitivity of astrocytes to AP-mediated release of neurotransmitter. Astrocytes can apparently sense spontaneous APs and glutamate release at Schaffer collateral-CA1 pyramidal cell synapses even when extracellular K+ is within a physiological range. While acute application of TTX does not reduce the frequency of spontaneous astrocyte Ca2+ activity18,36,37, the Ca2+ activity among the astrocytes in the population becomes decorrelated36, providing evidence that astrocytic receptors are AP detectors. This suggests that astrocytes sense spontaneous neuronal APs with no affect on their overall Ca2+ activity. It is widely accepted that intracellular concentrations of IP3 need to reach a threshold level to stimulate IP3Rs sufficiently to lead to a detectable Ca2+ elevation. Could spontaneous neuronal APs activate astrocytic GPCRs without producing measurable Ca2+ elevations? Future studies could utilize Fluorescence Resonance Energy Transfer (FRET) or a similar technique (such as BRET) to examine the relationship between G protein coupling to the receptor (a measure of receptor activation) and Ca2+ release from internal stores. BRET has been used extensively in vitro to detect G protein-to-GPCR coupling38, although it may be some time before this technology becomes available for use in intact tissue preparations. It is possible that astrocytic Gq GPCRs are being activated much more frequently than can be recorded using the currently available Ca2+ imaging tools. In addition to sensing action potentials, astrocytic Gq GPCRs may also be able to detect miniature quantal release of neurotransmitter as reported in a recent study39. The bidirectional scaling method described here may be used in future studies to provide a measure of the extent to which astrocytic Gq GPCRs detect quantal vesicular release of neurotransmitter, by including bafilomycin A1 in the incubation protocol.
Thus far, the scaling protocols have only been used in hippocampal slices from juvenile mice (p12-p18). Therefore, it is currently unknown if astrocytic receptor scaling could also be induced in tissue obtained from adult mice. A compelling recent study suggests that group I mGluR expression in astrocytes diminishes considerably after the first week of age and continues to decline until adulthood, with very low levels of receptor expression in adult astrocytes40. It would therefore be interesting to determine if astrocytic mGluRs scale up following long-term inhibition of neuronal firing in adult mouse hippocampal slices to levels approaching those seen in astrocytes from juvenile mice. This finding would suggest that astrocytic receptor expression is not static at a given age but can change rapidly depending upon levels of neuronal activity. In contrast to reduced expression of group I mGluRs in adult mice, evidence is emerging that adrenergic receptors, including &#945;1A, &#945;2A, and &#946;1 subtypes, are predominantly expressed by astrocytes in the adult brain3,4. The &#945;1A adrenergic Gq GPCR may be an attractive target for future studies of neuron-to-astrocyte&#160; communication, including whether these receptors are sensitive to changes in adrenergic neuron firing rates.&#160;

Astrocytes respond within seconds to stimulation of neurons or neuronal axons with increases in cytoplasmic Ca2+ resulting almost exclusively from activation of astrocytic Gq GPCRs. For example, muscarinic acetylcholine receptors1, cannabinoid receptors2, &#945;1A adrenergic receptors3,4, and group I mGluRs (see below) are all astrocytic Gq GPCR subtypes that acutely respond to neuronal activity. Activation of astrocytic group I mGluRs has been demonstrated most extensively, following stimulation of neuronal glutamatergic afferents in situ (such as acute hippocampal slices)5-7, as well as in adult mouse cortex in vivo following sensory stimulation8. The outcome of activation of astrocytic Gq GPCR signaling on the biology and physiology of astrocytes, neurons, or astrocyte-neuron interactions has been a matter of debate9-12. It will be some time before the function of neuron-to-astrocyte receptor signaling is fully appreciated.&#160;
While it is clear that neurons can activate astrocytic receptors using experimental protocols, there are aspects of neuron-to-astrocyte receptor communication that remain poorly understood. First, the actual amount of neuronal activity required to activate astrocytic Gq GPCRs is not well-defined, and second, the ability of astrocytic receptors to exhibit use-dependent plasticity has received little attention. To begin to address these questions, we recently developed a protocol to induce bidirectional scaling of astrocytic group I mGluRs in acute juvenile hippocampal slices in response to long-term changes in neuronal action potential (AP)-dependent synaptic activity. Similar to what has been discovered for bidirectional homeostatic plasticity of neuronal ionotropic glutamate receptors13,14, astrocytic group I mGluRs scale up following blockade of neuronal action potentials and scale down when neuronal action potential frequency is increased15. These compensatory changes in astrocytic receptors can be measured by recording spontaneous and evoked astrocyte Ca2+ transients and comparing the properties of these events to those from astrocytes in control conditions. In this manuscript, we describe the complete methodology for use of this protocol, including preparation of acute hippocampal slices, incubation conditions to induce astrocyte receptor scaling, astrocyte Ca2+ indicator dye loading, Ca2+ imaging techniques using confocal microscopy, and expected effects on astrocyte Gq GPCR activity. Predictable effects on astrocyte Ca2+ signaling properties - which match those recorded previously in cultured cells transfected with different expression levels of Gq GPCRs - provide a &#8220;roadmap&#8221; that can be used in future studies to assay for changes in astrocytic GPCR expression. The ramifications and potential applications for use of this technique will contribute to our understanding of astrocyte-neuronal interactions in the healthy and diseased brain.

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="32">
			<td height="32" style="height:32px;width:163px;">Chamber Supplies</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:163px;">Brinkmann pipette storage container</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:131px;">03-491</td>
			<td style="width:159px;">Use the drawer portion as the incubation chamber</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:163px;">Electrical drill</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:163px;">Flexible tubing</td>
			<td style="width:135px;">Tygon</td>
			<td style="width:131px;">R-3603</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:163px;">Silicone seam sealant&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;">Also called aquarium seam sealer</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:163px;">Gas tank</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;">95% oxygen, 5% carbon dioxide</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:163px;">Natural beveled pipette tip</td>
			<td style="width:135px;">USA Scientific</td>
			<td style="width:131px;">1111-1000</td>
			<td style="width:159px;">Cut-to-fit to connect oxygenate lines</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:163px;">One-to-six lines manifold</td>
			<td style="width:135px;">Warner Instruments</td>
			<td style="width:131px;">64-0210 &#160;(MP-6)</td>
			<td style="width:159px;">For the microloader-manifold apparatus</td>
		</tr>
		<tr height="59">
			<td height="65" rowspan="2" style="height:65px;width:163px;">Eppendorf microloader</td>
			<td rowspan="2" style="width:135px;">Eppendorf</td>
			<td rowspan="2" style="width:131px;">5242 956.003</td>
			<td rowspan="2" style="width:159px;">For the microloader-manifold apparatus, cut-to-fit</td>
		</tr>
		<tr height="6">
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:163px;">Floating Bubble Rack</td>
			<td style="width:135px;">Bel Art Scienceware</td>
			<td style="width:131px;">F18875-0400</td>
			<td style="width:159px;">For slice holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">600 micron Sefar Nitex nylon mesh</td>
			<td style="width:135px;">ELKO filtering Co.</td>
			<td style="width:131px;">&#160;06-600/51</td>
			<td style="width:159px;">For slice holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Krazy Glue</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;">For slice holder</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:163px;">Reagent</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:163px;">Isoflurane</td>
			<td style="width:135px;">Baxter</td>
			<td style="width:131px;">1001936060</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">NaCl</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">S271-3</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:163px;">KCl</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">P333-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:163px;">CaCl2</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">C79-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:163px;">MgCl2</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">M33-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:163px;">NaH2PO4</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">S369-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:163px;">NaHCO3</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">S233-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Glucose</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">(&#177;)-6-Hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid (Trolox)</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:131px;">53188-07-1</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Ascorbic acid</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:131px;">401471000</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Tetrodotoxin citrate (TTX)</td>
			<td style="width:135px;">Ascent Scientific</td>
			<td style="width:131px;">Asc-055</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Sulforhodamine 101 (SR-101)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:131px;">284912</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Dimethyl Sulfoxide (DMSO)&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:131px;">D128-500</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Pluronic Acid F-127</td>
			<td style="width:135px;">Invitrogen, Molecular Probes</td>
			<td style="width:131px;">P6867</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:163px;">Oregon Green 488 BAPTA-1 AM *cell permeant (special packaging)</td>
			<td style="width:135px;">Invitrogen, Molecular Probes</td>
			<td style="width:131px;">O6807</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">(RS)-3,5-DHPG</td>
			<td style="width:135px;">Ascent</td>
			<td style="width:131px;">Asc-020</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Histamine</td>
			<td style="width:135px;">Sigma-Aldrich&#160;</td>
			<td style="width:131px;">H7125</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:163px;">Carbamoylcholine chloride (Carbachol)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:131px;">C4382</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:163px;">Adenosine 5&#8217;-ATP disodium (Na-ATP)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:131px;">A7699</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:163px;">Dissection Tools</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:163px;">Single-edge razor blade&#160;</td>
			<td style="width:135px;">GEM</td>
			<td style="width:131px;">62-0161</td>
			<td style="width:159px;">For bisection&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Double edge razor blade</td>
			<td style="width:135px;">TED PELLA, INC.</td>
			<td style="width:131px;">121-6</td>
			<td style="width:159px;">For cutting slices</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Mayo Scissors, supercut</td>
			<td style="width:135px;">WPI</td>
			<td style="width:131px;">14010-15</td>
			<td style="width:159px;">For decapitation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Fine iris scissors, straight</td>
			<td style="width:135px;">Fine Science Tools</td>
			<td style="width:131px;">14094-11</td>
			<td style="width:159px;">For cutting the skull</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Iris forceps, curved</td>
			<td style="width:135px;">WPI</td>
			<td style="width:131px;">15915</td>
			<td style="width:159px;">To remove the skin and skull</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:163px;">Small spatula</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;">To remove/transfer the brain</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:163px;">Dumostar Dumont #5 Biologie Tip forceps</td>
			<td style="width:135px;">Fine Science Tools</td>
			<td style="width:131px;">11295-10</td>
			<td style="width:159px;">For hippocampus dissection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Glass Pasteur pipette</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">13-678-20B</td>
			<td style="width:159px;">For transferring brain slices</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Pasteur pipette rubber bulb</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:131px;">03-448-22</td>
			<td style="width:159px;">For transferring brain slices</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Polystyrene 100-mm tissue culture dishes</td>
			<td style="width:135px;">Corning</td>
			<td style="width:131px;">25020</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:163px;">Equipment</td>
			<td style="width:135px;"></td>
			<td style="width:131px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Vibratome</td>
			<td style="width:135px;">Leica</td>
			<td style="width:131px;">VT 1200S</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="23" rowspan="2" style="height:23px;width:163px;">Water bath</td>
			<td rowspan="2" style="width:135px;">Fisher</td>
			<td rowspan="2" style="width:131px;">ISOTEMP 210</td>
			<td rowspan="2" style="width:159px;">For warm incubation</td>
		</tr>
		<tr height="3">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:163px;">Micropipette puller</td>
			<td style="width:135px;">Narishige</td>
			<td style="width:131px;">PC-10</td>
			<td style="width:159px;">For bolus-loading pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Confocal microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:131px;">Olympus Fluoview 1000</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:163px;">Low Profile Open Diamond Bath Imaging Chamber with PM-1 platform</td>
			<td style="width:135px;">Warner Instruments</td>
			<td style="width:131px;">RC-26GLP</td>
			<td style="width:159px;">diamond bath with low profile</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:163px;">Borosilicate glass pipette</td>
			<td style="width:135px;">World Precision Instruments</td>
			<td style="width:131px;">TW150F-4</td>
			<td style="width:159px;">For bolus-loading pipette</td>
		</tr>
		<tr height="22">
			<td height="25" rowspan="2" style="height:25px;width:163px;">Micromanipulator&#160;</td>
			<td rowspan="2" style="width:135px;">Sutter instrument</td>
			<td rowspan="2" style="width:131px;">ROE-200</td>
			<td rowspan="2" style="width:159px;">For bolus-loading pipette</td>
		</tr>
		<tr height="3">
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:163px;">Spin-X centrifuge tube filter with 0.22 &#181;m cellulose acetate&#160;</td>
			<td style="width:135px;">Costar</td>
			<td style="width:131px;">8161</td>
			<td style="width:159px;"></td>
		</tr>
	</tbody>
</table>

inducing plasticity of astrocytic receptors by manipulation of neuronal firing rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Representative results in Figure 3 show the effect of incubation of acute mouse hippocampal slices in TTX for 4-6 hr on s.r. astrocyte Ca2+ activity. Data include both spontaneous Ca2+ transients and DHPG-evoked group I mGluR Ca2+ responses, from slices incubated in control ACSF vs. ACSF plus TTX. Other than basic characteristic morphological features, stellate process assembly and small soma size (~10 &#181;m), astrocytes are identified in the s.r. by overlay of the Ca2+ indicator OGB-1 AM with the selective astrocyte marker SR-10119,20 (Figure 3A). The numbered boxes over astrocyte cell bodies correspond to the numbered fluorescence over time traces shown in Figure 3B. The group 1 mGluR agonist (RS)-3.5-DHPG is applied to determine the specific scaling effects on group 1 mGluRs in astrocytes. To discriminate between&#160;physiology&#160;in the specificity of the scaling effect to group 1 mGluRs versus other Gq GPCRs, a cocktail of agonists is applied at the end of each experiment. Here we used 10 &#181;M each of the Gq GPCR agonists histamine, carbamylcholine chloride (carbachol) and adenosine 5&#8217;-ATP disodium (Na-ATP). The agonist cocktail also serves as a positive control to identify viable, responsive astrocytes in cases where cells do not respond to DHPG, presumably because those particular astrocytes do not express sufficient amounts of the receptor to elicit a response to DHPG.
We used different concentrations of DHPG, including 5 &#181;M and 15 &#181;M (Figure 3B) as well as 30 &#181;M and 50 &#181;M (Figure 4A) to assist in revealing changes in astrocytic group I mGluRs. The relationship between cellular Ca2+ responses and Gq GPCR expression levels has been examined previously in vitro21-24. First, the threshold to respond to a particular agonist concentration depends on the density of receptors expressed by each cell. In a population of cells, more cells respond with a Ca2+ elevation to a given concentration of agonist when the cells are transfected with higher densities of receptors. After incubating slices in TTX, the percentage of astrocytes in the population responding to a fixed concentration of agonist increases (Figures 3B and 3C). We have found that 5 &#181;M and 15 &#181;M DHPG reveal obvious differences in the percentage of responsive astrocytes between the control and TTX treated cells, whereas 30 and 50 &#181;M DHPG are required to compare group I mGluR responses in 5.0 mM K+ treated vs. control cells (Figure 4A).
The relationship between astrocytic Ca2+ response patterns and agonist concentration has also been examined in situ. Increasing the agonist concentration shifts the pattern of the Ca2+ response in astrocytes from single peak Ca2+ elevations to multipeak and plateau Ca2+ elevations25-27. Based on these previous findings, we predicted that the response pattern to a single concentration of agonist will shift if there are changes in the level of receptor expression. Thus, depending on which of the two scaling methods is being utilized (inhibiting neuronal firing or increasing it), the concentration of agonist necessary to produce a particular response pattern will increase or decrease. For example, astrocytes incubated in TTX shift their DHPG-evoked Ca2+ response pattern to more plateau-type Ca2+ elevations and respond to lower agonist concentrations compared to control astrocytes (Figure 3C). Taking into account the earlier studies, these observations suggest that the group I mGluR receptor expression levels in astrocytes has increased. &#160;
Rise time and onset (latency) of the Ca2+ elevations have also been shown to directly correlate to changes in Gq GPCR expression levels in cultured cells22-24. Higher receptor expression levels result in shorter response latencies and faster rise times while reduction in receptor density produces the opposite effect. For astrocytes incubated in TTX, Ca2+ transients evoked by application of DHPG have significantly faster rise times compared to astrocytes incubated in control ACSF (Figure 3D). As previously mentioned, amplitudes of agonist-evoked Ca2+ responses remain unchanged regardless of the agonist concentration or the scaling model15 (Figure 3D).
In addition to the changes observed by directly activating group I mGluRs with DHPG, spontaneous astrocyte Ca2+ activity is also significantly affected by this manipulation. We observed a 2.26 fold increase in the percentage of spontaneously active astrocytes incubated in TTX versus control. This is an increase from only 12.9% of control astrocytes exhibiting spontaneous activity in the soma to 42.1% in the TTX incubated astrocytes (Figure 3E). Because it is known that GPCRs exhibit &#8220;intrinsic&#8221; or constitutive activity in the absence of agonist21,26,28, and that the level of this intrinsic activity increases with increasing receptor expression levels, these data suggest that the density of astrocytic Gq GPCRs increases following long-term reduction in neuronal action potential firing. Similar to agonist-evoked responses, the rise times of the spontaneous Ca2+ elevations are also increased (Figure 3E).
Representative data using the second protocol, incubation in elevated extracellular potassium (5.0 mM), is depicted in Figure 4. An increase in extracellular K+ from 2.5-5.0 mM results in a significant increase in basal CA3 neuron action potential frequency15. Higher concentrations of DHPG (30 &#181;M and 50 &#181;M) are necessary in order to evoke group I mGluR Ca2+ responses from the astrocytes incubated in high potassium (Figures 4A and&#160;4B). This is consistent with a reduced level of group I mGluR responsiveness in astrocytes following a long-term increase in neuronal action potentials. In addition, the evoked response pattern to a fixed concentration of DHPG shifts from plateau-like responses to weaker single-peak responses (Figure 4B). Examining the percentage of spontaneously active astrocytes in the two potassium conditions reveals that fewer astrocytes incubated in high K+ are spontaneously active compared to the control condition (Figure 4C). This effect is in the opposite direction of the TTX condition in which the percentage of astrocytes exhibiting spontaneous Ca2+ elevations is increased. Last, both evoked and spontaneous Ca2+ elevations have slower rise times in astrocytes incubated in high K+ versus the control condition (Figures&#160;4C and 4D). Overall these data suggest that astrocytic Gq GPCR expression levels scale bidirectionally depending upon the level of neuronal action potential activity over an extended time period.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51458/51458fig1highres.jpg" width="500" /> 
Figure 1.&#160;Slice incubation chamber fabrication and set up. (A) The drawer portion of a Brinkmann pipette storage container along with its air-tight lid is used to construct the slice incubation chamber. (B) Drill a hole in the side of the container approximately 1&#188; in from the top and &#188; in from the side. Fit in a piece of flexible tubing. (C) Connect the microloader-manifold apparatus to the inside end of the flexible tubing. Note cut-to-fit natural 200 &#181;l pipette tip (white arrow). (D) Six 20 &#181;l Eppendorf microloaders are cut-to-fit to a one-to-six line manifold to create a microloader-manifold apparatus. (E) Drill two small holes on the lid. (F) A Floating Bubble Rack for making the slice holder. (G) The bottom &#8220;legs&#8221; of the floating bubble rack are removed so that a piece of Nylon mesh material can be glued to the bottom. (H) Fill the incubation chamber with sufficient amount of ACSF so that the slice holder floats. Adjust the tubing length so the tips of the microloaders can rest at one corner of the chamber floor. When placing the incubation chamber in the water bath, the water level in the bath should be at the same level as the ACSF in the chamber. <a href="https://www.jove.com/files/ftp_upload/51458/51458fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51458/51458fig2highres.jpg" width="500" /> 
Figure 2.&#160;Estimation of slice health. (A) A healthy-looking hippocampal slice using differential interference contrast (DIC) optics. Healthy slices have a smooth, velvety appearance and a high percentage of healthy CA1 pyramidal neurons. Note the apical dendrites projecting into stratum radiatum. Patch-clamp of neurons that look like those shown here reveal a low resting membrane potential (-61 to -62 mV) in standard 2.5 mM K+ ACSF with few spontaneous action potentials. Membrane potential and firing rates will vary as a function of extracellular K+ (Xie et al.15). Arrows point to putative astrocytes. Abbreviations: s.r., stratum radiatum; s.py., stratum pyramidale. Scale bar, 10 &#181;m. (B) Unhealthy slices will have a high percentage of dead CA1 pyramidal neurons, which have the appearance of fried eggs (white arrows point to nuclei of dead neurons - the &#8220;yolk&#8221; of the fried egg). <a href="https://www.jove.com/files/ftp_upload/51458/51458fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="4.5in" src="/files/ftp_upload/51458/51458fig3highres.jpg" width="500" /> 
Figure 3.&#160;Recording amplified Gq GPCR activity and evoked group I mGluR Ca2+ responses after long-term inhibition of neuronal APs by incubation in TTX. (A) Representative images of cells in the recording field incubated in control conditions (upper panels) or in TTX (lower panels) that have taken up Oregon Green BAPTA-1 AM Ca2+ indicator dye (left panels) and SR-101 (middle panels). Scale bar is 10 &#181;m. Overlay of both signals (&#8220;merge&#8221;) indicates that astrocytes load with Ca2+ indicator. Boxes are drawn over individual astrocyte soma to record fluorescence intensity over time in the green channel to monitor Ca2+ activity in astrocytes. (B) Sample traces from the recording boxes in A) of Ca2+ activity in the astrocytes. Astrocytes incubated in TTX show increased spontaneous activity and more robust evoked group I mGluR Ca2+ responses as evidenced by changes in the pattern of response. Examples of single peak (circle), multipeak (rectangle), and plateau (dotted rectangle) Ca2+ transients are shown. (C) Changes in response patterns are especially evident using different concentrations of the group I mGluR agonist DHPG. More multipeak and plateau responses are evident after incubation in TTX compared to control at a given agonist concentration. (D) Rise times of DHPG-evoked Ca2+ responses are faster in astrocytes incubated in TTX compared to control (upper panel), while amplitudes do not change (lower panel), indicative of &#8220;all-or-none&#8221; response amplitudes once the threshold to respond has been reached. (E) Rise times of spontaneous astrocyte Ca2+ transients are also faster in TTX incubated vs. control incubated astrocytes (upper panel), while the percentage of astrocytes in the population exhibiting spontaneous Gq GPCR Ca2+ activity increases (lower panel). <a href="https://www.jove.com/files/ftp_upload/51458/51458fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51458/51458fig4highres.jpg" width="500" /> 
Figure 4.&#160;Recording diminished astrocytic Gq GPCR activity and evoked group I mGluR Ca2+ responses following long-term increase in neuronal APs by incubation in elevated extracellular potassium. (A) Representative traces of astrocyte Ca2+ recordings from slices incubated in 5.0 mM K+ ACSF to depolarize neurons and increase their basal firing rate compared to control ACSF (2.5 mM K+ ACSF). Astrocytes incubated with 5.0 mM K+ ACSF exhibit fewer spontaneous somatic Ca2+ transients and weaker DHPG evoked responses compared to astrocytes incubated in control ACSF. (B) A comparison of patterns of evoked responses to multiple concentrations of DHPG reveals weaker response types after long-term increase in neuronal APs. (C) A reduction in the percentage of astrocytes in the population exhibiting spontaneous Ca2+ activity is observed after long-term incubation in elevated K+ compared to control ACSF (upper panel), while rise times of the spontaneous activity become slower (lower panel). (D) Rise times of evoked astrocyte Ca2+ responses to different concentrations of DHPG become slower following 5.0 mM K+ treatment&#160; compared to astrocytes incubated in control ACSF. <a href="https://www.jove.com/files/ftp_upload/51458/51458fig4highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates at JoVE.com

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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