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Expression of type I mGluRs predicts plasticity in the hippocampal stratum radiatum interneurons
Neuroscience Letters 712 (2019) 134472
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Expression of type I mGluRs predicts plasticity in the hippocampal stratum radiatum interneurons
T
Teresa M. Nuferb,1, Collin Merrilla,1, Lindsey Friendb, Zach Hopkinsb, Zach Boyceb, ⁎ Jeffrey G. Edwardsa,b, a b
Brigham Young University, Department of Physiology and Developmental Biology, Provo, UT, 84602, USA Brigham Young University, Neuroscience, Provo, UT, 84602, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: 12-LO DAGLα NAPE-PLD mGluR1 mGluR5 Synaptic plasticity LTD Hippocampus
Changes in synaptic strength between hippocampal CA1 pyramidal cell synapses are partly responsible for memory acquisition. This plasticity is modulated by feedforward inhibitory interneurons in the stratum radiatum. While radiatum interneurons experience either long-term depression (LTD), short-term depression (STD), or lack plasticity, it is unclear whether plasticity correlates to specific interneuron subtypes. Using wholecell electrophysiology and real-time quantitative PCR, we characterized the plasticity expressed by different interneuron subtypes. We first analyzed calcium binding proteins and cholecystokinin mRNA expression patterns to determine cell subtype. We then assessed endocannabinoid (eCB) biosynthetic enzyme mRNA expression, including diacylglycerol lipase α, N-acyl-phosphatidylethanolamine phospholipase D, and 12-lipoxygenase, and metabotropic glutamate receptors that often mediate plasticity. Neurons exhibiting LTD tended to co-express mRNA for at least one eCB biosynthetic enzyme and the metabotropic glutamate receptor 5 (mGluR5). Conversely, mGluR5 was not expressed by neurons exhibiting STD or no plasticity. Neurons that exhibited STD tended to express mRNA for at least one eCB biosynthetic enzyme and mGluR1, but not mGluR5. This suggests that plasticity of stratum radiatum interneurons could be predicted based on type I mGluR expression.
1. Introduction Acquisition and retention of short-term memory in the hippocampus is mediated by synaptic plasticity, which is defined as changes in synaptic strength between neurons [1]. Traditionally, increased synaptic activity strengthens the synaptic connection [1], via long-term potentiation (LTP) [1], while decreased activity weakens the synapse [2], via long-term depression (LTD) [2]. Hippocampal LTP and LTD occur at excitatory synapses onto pyramidal cells and inhibitory interneurons. Excitatory pyramidal cells comprise a relatively homogenous neuron population. However, inhibitory interneurons, which modulate pyramidal cell activity, vary widely in morphology [3], expression of calcium-binding proteins, neuropeptides [4], and stimulus-induced plasticity [5]. The plasticity of CA1 pyramidal cells is also modulated by feedforward inhibitory interneurons in the stratum radiatum, including parvalbumin (PV)-containing axo-axonic cells, calretinin (CR)-containing interneuron-selective cells, cholecystokinin (CCK)/calbindin (CB)-positive basket cells, among others [4]. While plasticity of interneurons within the stratum radiatum varies dramatically, ranging from
LTD to short-term depression (STD) to lack of plasticity [6], little is known regarding whether plasticity correlates to specific interneuron subtypes. Interneuron variety in the hippocampus is essential for their unique functional roles, but also makes them difficult to study [7]. Understanding the differences in form and function among hippocampal interneurons will paint a more complete picture of how plasticity and memory formation occur on a cellular and molecular level. Defining the roles of specific interneuron subtypes requires an understanding of the molecular mechanisms driving synaptic change. Endocannabinoid (eCB) lipid signaling molecules are important and widely-used modulators of synaptic plasticity [8]. Produced from membrane lipids predominately in postsynaptic neurons, eCB signaling molecules diffuse retrogradely across synapses to bind ionotropic or Gprotein-coupled receptors (GPCRs) and influence presynaptic neurotransmitter release. These lipid messengers are synthesized “on-demand” by several enzymes. For example, the eCB anadamide is made by n-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) and binds to presynaptic receptors such as cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) [9]. Similarly, 2-
⁎
Corresponding author at: Physiology and Developmental Biology, 4005 LSB, Provo, UT 84602, USA. E-mail address: Jeff[email protected] (J.G. Edwards). 1 Co-first authors. https://doi.org/10.1016/j.neulet.2019.134472 Received 30 July 2019; Received in revised form 27 August 2019; Accepted 29 August 2019 Available online 06 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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a Multiclamp 700B amplifier and digitized with an Axon 1440A digitizer (Molecular Devices). Signals were filtered at 4 kHz and recorded using Clampex 10.4 (Molecular Devices) on a personal computer. Electrophysiological data was analyzed using Clampfit software (Molecular Devices), Microsoft Excel, and Origin 10.8 (OriginLab Corporation). Current amplitude 5 min before conditioning was compared to 5–10 and 15–20 min post-conditioning using an ANOVA.
arachidonylglycerol (2-AG) is produced by diacylglycerol lipase alpha (DAGLα) and usually activates GPCRs, in particular CB1 [10]. Finally, 12-lipoxygenase (12-LO) synthesizes the eicosanoid 12-HPETE to modulate synaptic plasticity by acting on the presynaptic cell [11]. Postsynaptic activation of type I metabotropic glutamate receptors (mGluRs) often initiates eCB synthesis [12,13]. Type I mGluRs are widely expressed in the hippocampus, and could be used to predict plasticity expression [14,15]. Several groups have thoroughly explored the roles of eCB and type I mGluR-mediated plasticity at excitatory synapses onto interneurons in the hippocampal stratum radiatum layer, where type I mGluR activation induces LTD via TRPV1 receptors, among others [6,14,15]. The interneurons themselves express the enzymes needed to produce eCBs [16] and are directly involved in physiologically self-induced LTD [17]. Type I mGluRs (mGluR1 and mGluR5) are clearly involved in the postsynaptic induction of presynaptic depression that excitatory synapses onto inhibitory interneurons exhibit. Specifically, for LTD of CA1 stratum radiatum interneurons type I mGluR activation followed by eCB production was required, which exhibited presynaptic depression of glutamate release, as indicated by paired pulse ratio and failure rate analysis [14]. However, it is unclear if and how type I mGluRs specifically contribute to the induction of different plasticity subtypes (i.e. LTD, STD, or no plasticity) in CA1 stratum radiatum interneurons. Our goals were to 1) probe the relationship between hippocampal CA1 stratum radiatum interneuron subtypes and synaptic plasticity, and 2) correlate the relationship between synaptic plasticity and either eCB biosynthetic enzyme or type I mGluR mRNA expression. While eCBmediated synaptic plasticity within the hippocampus is well documented, there is little evidence for the involvement of specific interneuron subtypes in eCB-synaptic plasticity. Using a combination of whole-cell patch-clamp electrophysiology and quantitative real time PCR (qPCR), we identified a relationship between expression of type I mGluRs and plasticity type exhibited by CA1 radiatum interneurons. These data provide further evidence for the mechanism of interneuron synaptic plasticity within the hippocampus and the importance that eCB-mediated signaling could have in modulation of pyramidal cell activity.
2.2. PCR We quantified eCB biosynthetic enzyme mRNA using RT-qPCR [16]. Cells used for PCR analysis were extracted from the slice using gentle suction, aspirated into the recording electrode, and immediately mixed with ice-cold iScript cDNA synthesis kit reagents (BioRad). All samples were processed within 2 h. One ACSF control sample was obtained for each slice and used to identify potential background contamination from extracellular mRNA. Extracted neuronal mRNA was converted to cDNA with an iScript cDNA synthesis kit and incubated in a C1000 Thermocycler (BioRad) at 25 °C for 8 min, 42 °C for 60 min, and 70 °C for 15 min. Following reverse transcription, each cell was divided into three 5 μl aliquots, which each received a separate group of 10-fold diluted primers, iQ Supermix (BioRad), and ddH20. Additional notemplate-control samples ensured that there were no primer-dimer or hairpin interactions. Extracted mRNA was amplified in a C1000 Thermocycler (BioRad) starting at 95 °C for 3 min, followed by 15 cycles of 95 °C for 15 min, 57 °C for 20 s, and 72 °C for 25 s. Final qPCR reactions were run in triplicate with individual primers for each target and specific FAM-TAMRA© probes (ThermoFischer Scientific). Primer and probe sequences were published previously [16]. Each pre-amplified sample was then assayed for every target individually using a CFX96 qPCR machine (BioRad) with a 95 °C for 3 min, followed by 60 cycles of 95 °C for 15 s, 57 °C for 25 s, and 72 °C for 25 s. Cycle threshold (Ct) values were determined by CFX Manager 3.1 software (BioRad). Amplification curves with Ct values > 20 cycles higher than 18S Ct values that infrequently occurred were rejected as non-specific false positives. 3. Results
2. Methods
To understand how cell subtype and eCB biosynthetic enzyme or type I mGluR expression correlate with the induction of specific plasticity types in individual neurons, we used a combination of whole-cell patch-clamp electrophysiology and real-time qPCR. After inducing plasticity with high frequency stimulation (HFS) and recording postsynaptic currents, each neuron was aspirated into the pipette tip. Gene expression was assayed by qPCR (described in the Methods) for the targets listed in Table 1. This methodological combination allowed us to describe specific relationships between plasticity and mRNA expression patterns in individual interneurons. We initially hypothesized that CA1 stratum radiatum interneuron plasticity would correlate with specific, previously described interneuron subtypes or with expression qPCR products examined [3,7]. Excitatory synapses onto CA1 stratum radiatum interneurons
2.1. Electrophysiology All experiments were performed in accordance with Institutional Animal Care and Use Committee protocols and NIH guidelines for the care and use of laboratory animals. Male Sprague-Dawley rats P15-30 days old were anesthetized with isoflurane (1.5–2%) and decapitated. Brains were removed and sectioned coronally on a vibratome at 400 μm using oxygenated artificial cerebral spinal fluid (ACSF) composed of (in mM) 119 NaCl, 26 NaHCO3, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4, 11 glucose, and 400 μM ascorbic acid. Slices were stored in oxygenated ACSF at room temperature for at least 1 h before experiments began. Inhibitory GABAA currents were blocked with 100 μM picrotoxin (Abcam) throughout the entire experiment. Hippocampal CA1 stratum radiatum neurons were visualized with a BX51WI microscope with a 40x water immersion objective (Olympus). Interneurons were patched with a borosilicate glass pipette (3–6 MΩ) filled with internal solution composed of (in mM) 117 cesium gluconate, 2.8 NaCl, 20 HEPES, 5 MgCl2, 1 QX-314 (Tocris), 0.6 EGTA, 2 ATP (Sigma or Calbiochem), and 0.3 GTP (Sigma-Aldrich) (pH 7.28, 275–285 mOsm). Recordings were performed in voltage clamp mode, with cells held at −65 mV throughout the experiment. Excitatory postsynaptic currents (EPSCs) were evoked using a stainless steel bipolar stimulating electrode placed in the stratum radiatum, activated at 0.1 Hz (100 μsec duration) during baseline and post-conditioning phases. Plasticity was induced using a conditioning stimulus of two, 1-second 100 Hz stimulations, 20 s apart, while holding the cell at −65 mV. Evoked currents were recorded using
Table 1 Genes tested by qPCR. CA1 stratum radiatum interneurons were assayed for classic interneuron markers, eCB biosynthetic enzymes, and type I mGluRs. Gene targets
2
Interneuron markers
eCB enzymes
Type I mGluRs
GAD65 GAD67 Parvalbumin (PV) Calbindin (CB) Calretinin (CR) CCK
12-Lipoxygenase NAPE-PLD DAGL⍺
mGluR1 mGluR5
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Fig. 1. Plasticity Profiles of Stratum Radiatum Interneurons. A. 50% of cells displayed longterm depression (LTD) of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in whole cell voltage clamp mode (n = 15, p < 0.05 between baseline and 10–20 min post-conditioning). B. In addition, 37% of interneurons expressed short-term depression (STD) which returned to baseline approximately 10–15 min post-conditioning (n = 11; p < 0.05 between 0–10 min post-conditioning compared to baseline; p > 0.05 at 15+ minutes post-conditioning). C. Lastly, approximately 13% of cells studied displayed no plasticity (n = 4; p > 0.05 at all time points). ANOVA was used for quantification and to categorize types. Scale bars: 10 ms, 100pA. Data represent means ± SEM.
positive cell noted in this study, we did not included it in the Table 2 analysis, though we thought it important to mention. Finally, interneurons that do not exhibit plasticity following HFS expressed eCB biosynthetic enzymes less often and did not express DAGLα ever. Interestingly, these neurons were most often CCK + basket cells that did not express mGluRs (Fig. 2C, Table 2). As a note, many cells that exhibited STD or LTD were labeled in Table 2 as “uncategorized,” because we were not able to unequivocally categorize them into classically-recognized interneuron subtypes, and thus they were grouped together. These likely include either cell subtypes we did not examine and/or false negatives from our qPCR data, which we address in the discussion section. In summary, we determined that plasticity is not necessarily an indicator of interneuron subtype, nor does subtype predict plasticity. Instead, the key commonality between LTD-expressing neurons is mGluR5 expression. Though not all neurons exhibiting LTD expressed mGluR5 (possibly due to false negatives for mGluR5), every neuron that expressed mGluR5 exhibited LTD (Table 2). Therefore, mGluR5 activation could be necessary to produce the eCBs needed to induce LTD at excitatory synapses onto radiatum interneurons. Additionally, mGluR1
exhibit distinct types of plasticity, including LTD (depression maintained) or STD (depression returning to baseline within 10–20 min). They may also lack plasticity following HFS [6]. We recorded EPSCs from 30 interneurons. Specifically, we identified following HFS 15 cells exhibiting LTD (Fig. 1A; 50%), 11 with STD (Fig. 1B; 36.7%), and 4 with no plasticity (Fig. 1C; 13.3%). This ratio is very similar to that noted by McMachon and Kauer [6]. Next, we examined whether neurons exhibiting different plasticity correlate to specific interneuron subtypes or expression of either mGluRs or eCB biosynthetic enzymes. Stratum radiatum interneurons exhibiting LTD in response to HFS typically expressed eCB biosynthetic enzymes, but also expressed mGluR5 and often mGluR1 (Fig. 2A, Table 2). Surprisingly, neurons exhibiting LTD did not represent particular subtypes but included all interneuron subtypes we examined. They also expressed all varieties of eCB biosynthetic enzymes. Interneurons exhibiting STD also expressed one or more varieties of eCB biosynthetic enzymes and furthermore represented various subtypes, including CCK + basket cells. Some STD-exhibiting cells also express mGluR1, but not mGluR5 (Fig. 2B, Table 2). In the STD group there was also a CR + cell that expressed mGluR1. As this was the only CR3
Neuroscience Letters 712 (2019) 134472
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Table 2 Expression of eCB Biosynthetic enzymes and type I mGluRs as a function of plasticity and interneuron subtype. Percentages indicate neuron subtype proportions within each plasticity group. An ‘X’ indicates that a target came up within that given cell subtype population. Plasticity
Cell Type
None
CCK (75%) PV (25%) CCK (36%) CCK-CB (18%) Undifferentiated (46%) CCK (20%) CCK-CB (13%) CB (13%) PV (7%) Undifferentiated (47%)
STD
LTD
DAGLα
NAPE-PLD
12-LO
mGluR5
mGluR1
X X
X X
X X
X
X
X X
X
X X
X
X X
X X
was only expressed in neurons with one form of plasticity, either LTD or STD (Table 2). Nearly all stratum radiatum interneuron subtypes exhibiting plasticity appear to express at least one eCB biosynthetic enzyme, regardless of plasticity type. However, these enzymes were rarely present in neurons without plasticity (Table 2). 4. Discussion Our data illustrate a potential relationship between mGluR5 expression and LTD in CA1 stratum radiatum interneurons. Neurons expressing mGluR5, and often mGluR1, exhibit LTD preferentially over other plasticity types in radiatum interneurons. Furthermore, nearly all the neurons in our study expressed eCB biosynthetic enzyme mRNA as demonstrated previously [16], confirming that eCB synthesis is likely a common feature of CA1 stratum radiatum interneurons. We recently demonstrated differential distribution of eCB biosynthetic enzyme mRNA, suggesting that eCB-mediated processes do not occur equally at CA1 stratum radiatum interneuron synapses [16]. In fact, in eCB-mediated LTD studies, all interneurons tested did not respond equally to HFS [6,14]. McMahon & Kauer (1997) were the first to describe synaptic plasticity of excitatory inputs to hippocampal stratum radiatum interneurons. They reported bistratified and basket cell morphology among other interneurons, and concluded that plasticity did not be correlated with interneuron subtype [6]. Later, CA1 stratum radiatum interneuron LTD dependence on TRPV1, 12-HPETE, and mGluRs with a presynaptic loci of action was illustrated [14,18]. Our data also support that interneuron plasticity is not cell-type specific, but rather determined by mGluR5/1and potentially eCB-biosynthetic enzyme expression. Our results also indicate a qualitative effect on plasticity: all interneurons that express mGluR5 exhibit LTD. There were a few neurons exhibiting LTD without mGluR5 expression, though we cannot confirm whether they actually did not express mGluR5 or whether our qPCR assay did not detect mGluR5. These hippocampal interneurons also express eCB biosynthetic enzyme mRNA coding for DAGLα, which others also noted is expressed in interneurons, but more highly expressed in pyramidal cells [17]. In contrast, Péterfi and colleagues demonstrated a quantitative effect where higher stimulation frequencies and higher DHPG concentrations were required to induce plasticity in CA1 hippocampal interneurons than in pyramidal cells [17]. We did not test whether increasing the number of stimulus trains also increased LTD induction, however, it is possible that increased stimulation could induce plasticity in neurons we recorded from that lacked plasticity or neurons with STD, uncovering a quantitative effect. Collectively, the high correlation between mGluR5 and LTD in our study suggests that plasticity induction is more likely in mGluR5-expressing neurons.
Fig. 2. Gene expression profiles of stratum radiatum interneurons. A. The quantitative PCR (qPCR) of an example cell from Fig. 1A that demonstrated LTD. These cells always expressed mRNA for at least one eCB biosynthetic enzyme and most often for type I metabotropic glutamate receptors (mGluRs), especially mGluR5. B. An example cell from Fig. 1B that demonstrated STD. These cells often expressed mRNA for eCB biosynthetic enzymes and also for mGluR1. C. An example of one cell that did not exhibit plasticity from Fig. 1C. While a few of these neurons express mRNA for eCB biosynthetic enzymes (see Table 2), none expressed type I mGluRs. Data are represented as log-scaled relative fluorescence curves from FAM-TAMRA probe-based qPCR. 4
Neuroscience Letters 712 (2019) 134472
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study of DHPG-induced LTD of stratum radiatum interneurons noted that mGluR5 activation alone caused true LTD, while mGluR1 activation alone induced a short-term transient depression with a very similar time course to our HFS-induced STD [5], supporting the interpretation of mGluR5/1 in this study. As a note, the STD we report could also possibly correspond to depolarization-induced suppression of excitation or DSE, however DSE most often lasts 1–2 min and STD in this report lasts much longer. Our data confirm that LTD is inducible at excitatory synapses onto interneurons in the CA1 stratum radiatum and highlight the importance of mGluR5 in this LTD induction. In addition, mGluR1 could play a role in STD and possibly LTD. Furthermore, eCB synthesis likely plays an important role in hippocampal interneuron plasticity, and eCB enzymes appear to be dispersed fairly ubiquitously among certain CA1 radiatum interneuron subtypes [16]. While it was somewhat surprising that all interneurons of each subtype do not express the same plasticity type, this finding does fit with our data as a whole. Collectively, these data demonstrate the potential of distinct CA1 interneuron populations of various subtypes to differentially modulate learning and memory processing within the hippocampus through differing forms of plasticity.
We acknowledge that a quantitative effect as demonstrated by Péterfi et al. could still be present and that other eCB biosynthetic enzymes, even produced from other neurons, may contribute to plasticity, either synergistically or in a non-cell-autonomous manner. Lastly, our data do not suggest that any particular eCB biosynthetic enzyme expression correlates with a certain type of plasticity. Indeed, NAPE-PLD and 12-LO were expressed in neurons exhibiting LTD, STD, or no plasticity, and DAGLα was present in STD- or LTD-expressing neurons. In addition, it is conceivable that while most all interneurons express some form of eCB producing enzyme, the reason some lack or have different plasticity types is tied to their expression profile of type I mGluRs, which could be a way for the cell to regulate its ability to induce plasticity or not. The highly heterogeneous nature of hippocampal interneurons has historically made them difficult to study. Therefore, effective interneuron studies require careful definition of the target population, and as techniques differ this often leads to challenges when comparing findings between studies. For example, one limitation of the present study is that we were unable to examine cell morphology and correlate projection patterns, to plasticity and type I mGluR expression. To reduce false negative qPCR results, we extracted the entire neuron following the plasticity experiment, making it impossible to also image neuron morphology. We therefore limited our study to well-described CA1 stratum radiatum interneurons. Our data describe interneurons that are likely classically-described subtypes including parvalbuminpositive axo-axonic cells, calretinin-positive interneuron-selective cells, and CCK/CB-positive basket cells [4]. That being said, interneuron subtype in our study did not necessarily correlate to interneuron plasticity, nor did GAD65 and/or GAD67 expression. Lastly, many neurons were classified as “uncategorized” in our study, because their interneuron marker expression patterns did not match classical descriptions. We believe that the high rate of uncategorized cells is due to the technical limitations of single-cell RTqPCR. We recognize that qPCR often has ∼35% false negative results. Therefore, it is possible that many uncategorized cells actually expressed more interneuron markers than we detected, or expressed different markers that we did not examine. Additionally, negative qPCR results are difficult to interpret, because lack of detection of a target does not positively indicate its absence. We are aware of only one other study examining and finding a correlation between interneuron subtype and plasticity. This study determined that CA1 interneurons in or near the stratum pyramidale layer could be categorized into three main groups based on HFS (100 Hz)induced plasticity: PV bistratified cells that exhibit LTD, PV perisomatic targeting/axo-axonic cells that exhibit LTP and CB1-containing cells that lacked plasticity. A key determinant in plasticity was that PV cells contained calcium-permeable AMPA receptors while CB1-containing cells lacked them. In contrast, we noted no differences in plasticity based on cell subtype in stratum radiatum interneurons stimulated at 100 Hz, though AMPA receptor subtypes were not examined for comparison and we did not use perforated patch. Péterfi et al. (2012) also examined different interneuron subtypes and plasticity type, but in the stratum oriens as well as stratum pyramidale interneurons. They noted that both PV and somatostatin-positive cells both display 10-Hz-induced and DHPG-induced LTD, and thus no difference in plasticity of interneurons subtypes was noted. This LTD required mGluR5 and DAG lipase activity [17]. This study found that eCB-producing interneurons can mediate their own plasticity, which supports our previous study [16] and the current study. Our results, the first to focus on stratum radiatum interneurons subtypes only, corroborate the mGluR5 requirement for LTD in a non-specific interneuron subtype fashion. Though differences in methodology should be considered between these studies, it is of interest to note that cell-determined expression of type I mGluRs, particularly mGluR5, is likely a key factor in an interneuron’s ability to produce plasticity and its duration, or perhaps in the quantitative stimulus needed to induce the plasticity. Indeed, another
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Expression of type I mGluRs predicts plasticity in the hippocampal stratum radiatum interneurons
T
Teresa M. Nuferb,1, Collin Merrilla,1, Lindsey Friendb, Zach Hopkinsb, Zach Boyceb, ⁎ Jeffrey G. Edwardsa,b, a b
Brigham Young University, Department of Physiology and Developmental Biology, Provo, UT, 84602, USA Brigham Young University, Neuroscience, Provo, UT, 84602, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: 12-LO DAGLα NAPE-PLD mGluR1 mGluR5 Synaptic plasticity LTD Hippocampus
Changes in synaptic strength between hippocampal CA1 pyramidal cell synapses are partly responsible for memory acquisition. This plasticity is modulated by feedforward inhibitory interneurons in the stratum radiatum. While radiatum interneurons experience either long-term depression (LTD), short-term depression (STD), or lack plasticity, it is unclear whether plasticity correlates to specific interneuron subtypes. Using wholecell electrophysiology and real-time quantitative PCR, we characterized the plasticity expressed by different interneuron subtypes. We first analyzed calcium binding proteins and cholecystokinin mRNA expression patterns to determine cell subtype. We then assessed endocannabinoid (eCB) biosynthetic enzyme mRNA expression, including diacylglycerol lipase α, N-acyl-phosphatidylethanolamine phospholipase D, and 12-lipoxygenase, and metabotropic glutamate receptors that often mediate plasticity. Neurons exhibiting LTD tended to co-express mRNA for at least one eCB biosynthetic enzyme and the metabotropic glutamate receptor 5 (mGluR5). Conversely, mGluR5 was not expressed by neurons exhibiting STD or no plasticity. Neurons that exhibited STD tended to express mRNA for at least one eCB biosynthetic enzyme and mGluR1, but not mGluR5. This suggests that plasticity of stratum radiatum interneurons could be predicted based on type I mGluR expression.
1. Introduction Acquisition and retention of short-term memory in the hippocampus is mediated by synaptic plasticity, which is defined as changes in synaptic strength between neurons [1]. Traditionally, increased synaptic activity strengthens the synaptic connection [1], via long-term potentiation (LTP) [1], while decreased activity weakens the synapse [2], via long-term depression (LTD) [2]. Hippocampal LTP and LTD occur at excitatory synapses onto pyramidal cells and inhibitory interneurons. Excitatory pyramidal cells comprise a relatively homogenous neuron population. However, inhibitory interneurons, which modulate pyramidal cell activity, vary widely in morphology [3], expression of calcium-binding proteins, neuropeptides [4], and stimulus-induced plasticity [5]. The plasticity of CA1 pyramidal cells is also modulated by feedforward inhibitory interneurons in the stratum radiatum, including parvalbumin (PV)-containing axo-axonic cells, calretinin (CR)-containing interneuron-selective cells, cholecystokinin (CCK)/calbindin (CB)-positive basket cells, among others [4]. While plasticity of interneurons within the stratum radiatum varies dramatically, ranging from
LTD to short-term depression (STD) to lack of plasticity [6], little is known regarding whether plasticity correlates to specific interneuron subtypes. Interneuron variety in the hippocampus is essential for their unique functional roles, but also makes them difficult to study [7]. Understanding the differences in form and function among hippocampal interneurons will paint a more complete picture of how plasticity and memory formation occur on a cellular and molecular level. Defining the roles of specific interneuron subtypes requires an understanding of the molecular mechanisms driving synaptic change. Endocannabinoid (eCB) lipid signaling molecules are important and widely-used modulators of synaptic plasticity [8]. Produced from membrane lipids predominately in postsynaptic neurons, eCB signaling molecules diffuse retrogradely across synapses to bind ionotropic or Gprotein-coupled receptors (GPCRs) and influence presynaptic neurotransmitter release. These lipid messengers are synthesized “on-demand” by several enzymes. For example, the eCB anadamide is made by n-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) and binds to presynaptic receptors such as cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) [9]. Similarly, 2-
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Corresponding author at: Physiology and Developmental Biology, 4005 LSB, Provo, UT 84602, USA. E-mail address: Jeff[email protected] (J.G. Edwards). 1 Co-first authors. https://doi.org/10.1016/j.neulet.2019.134472 Received 30 July 2019; Received in revised form 27 August 2019; Accepted 29 August 2019 Available online 06 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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a Multiclamp 700B amplifier and digitized with an Axon 1440A digitizer (Molecular Devices). Signals were filtered at 4 kHz and recorded using Clampex 10.4 (Molecular Devices) on a personal computer. Electrophysiological data was analyzed using Clampfit software (Molecular Devices), Microsoft Excel, and Origin 10.8 (OriginLab Corporation). Current amplitude 5 min before conditioning was compared to 5–10 and 15–20 min post-conditioning using an ANOVA.
arachidonylglycerol (2-AG) is produced by diacylglycerol lipase alpha (DAGLα) and usually activates GPCRs, in particular CB1 [10]. Finally, 12-lipoxygenase (12-LO) synthesizes the eicosanoid 12-HPETE to modulate synaptic plasticity by acting on the presynaptic cell [11]. Postsynaptic activation of type I metabotropic glutamate receptors (mGluRs) often initiates eCB synthesis [12,13]. Type I mGluRs are widely expressed in the hippocampus, and could be used to predict plasticity expression [14,15]. Several groups have thoroughly explored the roles of eCB and type I mGluR-mediated plasticity at excitatory synapses onto interneurons in the hippocampal stratum radiatum layer, where type I mGluR activation induces LTD via TRPV1 receptors, among others [6,14,15]. The interneurons themselves express the enzymes needed to produce eCBs [16] and are directly involved in physiologically self-induced LTD [17]. Type I mGluRs (mGluR1 and mGluR5) are clearly involved in the postsynaptic induction of presynaptic depression that excitatory synapses onto inhibitory interneurons exhibit. Specifically, for LTD of CA1 stratum radiatum interneurons type I mGluR activation followed by eCB production was required, which exhibited presynaptic depression of glutamate release, as indicated by paired pulse ratio and failure rate analysis [14]. However, it is unclear if and how type I mGluRs specifically contribute to the induction of different plasticity subtypes (i.e. LTD, STD, or no plasticity) in CA1 stratum radiatum interneurons. Our goals were to 1) probe the relationship between hippocampal CA1 stratum radiatum interneuron subtypes and synaptic plasticity, and 2) correlate the relationship between synaptic plasticity and either eCB biosynthetic enzyme or type I mGluR mRNA expression. While eCBmediated synaptic plasticity within the hippocampus is well documented, there is little evidence for the involvement of specific interneuron subtypes in eCB-synaptic plasticity. Using a combination of whole-cell patch-clamp electrophysiology and quantitative real time PCR (qPCR), we identified a relationship between expression of type I mGluRs and plasticity type exhibited by CA1 radiatum interneurons. These data provide further evidence for the mechanism of interneuron synaptic plasticity within the hippocampus and the importance that eCB-mediated signaling could have in modulation of pyramidal cell activity.
2.2. PCR We quantified eCB biosynthetic enzyme mRNA using RT-qPCR [16]. Cells used for PCR analysis were extracted from the slice using gentle suction, aspirated into the recording electrode, and immediately mixed with ice-cold iScript cDNA synthesis kit reagents (BioRad). All samples were processed within 2 h. One ACSF control sample was obtained for each slice and used to identify potential background contamination from extracellular mRNA. Extracted neuronal mRNA was converted to cDNA with an iScript cDNA synthesis kit and incubated in a C1000 Thermocycler (BioRad) at 25 °C for 8 min, 42 °C for 60 min, and 70 °C for 15 min. Following reverse transcription, each cell was divided into three 5 μl aliquots, which each received a separate group of 10-fold diluted primers, iQ Supermix (BioRad), and ddH20. Additional notemplate-control samples ensured that there were no primer-dimer or hairpin interactions. Extracted mRNA was amplified in a C1000 Thermocycler (BioRad) starting at 95 °C for 3 min, followed by 15 cycles of 95 °C for 15 min, 57 °C for 20 s, and 72 °C for 25 s. Final qPCR reactions were run in triplicate with individual primers for each target and specific FAM-TAMRA© probes (ThermoFischer Scientific). Primer and probe sequences were published previously [16]. Each pre-amplified sample was then assayed for every target individually using a CFX96 qPCR machine (BioRad) with a 95 °C for 3 min, followed by 60 cycles of 95 °C for 15 s, 57 °C for 25 s, and 72 °C for 25 s. Cycle threshold (Ct) values were determined by CFX Manager 3.1 software (BioRad). Amplification curves with Ct values > 20 cycles higher than 18S Ct values that infrequently occurred were rejected as non-specific false positives. 3. Results
2. Methods
To understand how cell subtype and eCB biosynthetic enzyme or type I mGluR expression correlate with the induction of specific plasticity types in individual neurons, we used a combination of whole-cell patch-clamp electrophysiology and real-time qPCR. After inducing plasticity with high frequency stimulation (HFS) and recording postsynaptic currents, each neuron was aspirated into the pipette tip. Gene expression was assayed by qPCR (described in the Methods) for the targets listed in Table 1. This methodological combination allowed us to describe specific relationships between plasticity and mRNA expression patterns in individual interneurons. We initially hypothesized that CA1 stratum radiatum interneuron plasticity would correlate with specific, previously described interneuron subtypes or with expression qPCR products examined [3,7]. Excitatory synapses onto CA1 stratum radiatum interneurons
2.1. Electrophysiology All experiments were performed in accordance with Institutional Animal Care and Use Committee protocols and NIH guidelines for the care and use of laboratory animals. Male Sprague-Dawley rats P15-30 days old were anesthetized with isoflurane (1.5–2%) and decapitated. Brains were removed and sectioned coronally on a vibratome at 400 μm using oxygenated artificial cerebral spinal fluid (ACSF) composed of (in mM) 119 NaCl, 26 NaHCO3, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4, 11 glucose, and 400 μM ascorbic acid. Slices were stored in oxygenated ACSF at room temperature for at least 1 h before experiments began. Inhibitory GABAA currents were blocked with 100 μM picrotoxin (Abcam) throughout the entire experiment. Hippocampal CA1 stratum radiatum neurons were visualized with a BX51WI microscope with a 40x water immersion objective (Olympus). Interneurons were patched with a borosilicate glass pipette (3–6 MΩ) filled with internal solution composed of (in mM) 117 cesium gluconate, 2.8 NaCl, 20 HEPES, 5 MgCl2, 1 QX-314 (Tocris), 0.6 EGTA, 2 ATP (Sigma or Calbiochem), and 0.3 GTP (Sigma-Aldrich) (pH 7.28, 275–285 mOsm). Recordings were performed in voltage clamp mode, with cells held at −65 mV throughout the experiment. Excitatory postsynaptic currents (EPSCs) were evoked using a stainless steel bipolar stimulating electrode placed in the stratum radiatum, activated at 0.1 Hz (100 μsec duration) during baseline and post-conditioning phases. Plasticity was induced using a conditioning stimulus of two, 1-second 100 Hz stimulations, 20 s apart, while holding the cell at −65 mV. Evoked currents were recorded using
Table 1 Genes tested by qPCR. CA1 stratum radiatum interneurons were assayed for classic interneuron markers, eCB biosynthetic enzymes, and type I mGluRs. Gene targets
2
Interneuron markers
eCB enzymes
Type I mGluRs
GAD65 GAD67 Parvalbumin (PV) Calbindin (CB) Calretinin (CR) CCK
12-Lipoxygenase NAPE-PLD DAGL⍺
mGluR1 mGluR5
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Fig. 1. Plasticity Profiles of Stratum Radiatum Interneurons. A. 50% of cells displayed longterm depression (LTD) of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in whole cell voltage clamp mode (n = 15, p < 0.05 between baseline and 10–20 min post-conditioning). B. In addition, 37% of interneurons expressed short-term depression (STD) which returned to baseline approximately 10–15 min post-conditioning (n = 11; p < 0.05 between 0–10 min post-conditioning compared to baseline; p > 0.05 at 15+ minutes post-conditioning). C. Lastly, approximately 13% of cells studied displayed no plasticity (n = 4; p > 0.05 at all time points). ANOVA was used for quantification and to categorize types. Scale bars: 10 ms, 100pA. Data represent means ± SEM.
positive cell noted in this study, we did not included it in the Table 2 analysis, though we thought it important to mention. Finally, interneurons that do not exhibit plasticity following HFS expressed eCB biosynthetic enzymes less often and did not express DAGLα ever. Interestingly, these neurons were most often CCK + basket cells that did not express mGluRs (Fig. 2C, Table 2). As a note, many cells that exhibited STD or LTD were labeled in Table 2 as “uncategorized,” because we were not able to unequivocally categorize them into classically-recognized interneuron subtypes, and thus they were grouped together. These likely include either cell subtypes we did not examine and/or false negatives from our qPCR data, which we address in the discussion section. In summary, we determined that plasticity is not necessarily an indicator of interneuron subtype, nor does subtype predict plasticity. Instead, the key commonality between LTD-expressing neurons is mGluR5 expression. Though not all neurons exhibiting LTD expressed mGluR5 (possibly due to false negatives for mGluR5), every neuron that expressed mGluR5 exhibited LTD (Table 2). Therefore, mGluR5 activation could be necessary to produce the eCBs needed to induce LTD at excitatory synapses onto radiatum interneurons. Additionally, mGluR1
exhibit distinct types of plasticity, including LTD (depression maintained) or STD (depression returning to baseline within 10–20 min). They may also lack plasticity following HFS [6]. We recorded EPSCs from 30 interneurons. Specifically, we identified following HFS 15 cells exhibiting LTD (Fig. 1A; 50%), 11 with STD (Fig. 1B; 36.7%), and 4 with no plasticity (Fig. 1C; 13.3%). This ratio is very similar to that noted by McMachon and Kauer [6]. Next, we examined whether neurons exhibiting different plasticity correlate to specific interneuron subtypes or expression of either mGluRs or eCB biosynthetic enzymes. Stratum radiatum interneurons exhibiting LTD in response to HFS typically expressed eCB biosynthetic enzymes, but also expressed mGluR5 and often mGluR1 (Fig. 2A, Table 2). Surprisingly, neurons exhibiting LTD did not represent particular subtypes but included all interneuron subtypes we examined. They also expressed all varieties of eCB biosynthetic enzymes. Interneurons exhibiting STD also expressed one or more varieties of eCB biosynthetic enzymes and furthermore represented various subtypes, including CCK + basket cells. Some STD-exhibiting cells also express mGluR1, but not mGluR5 (Fig. 2B, Table 2). In the STD group there was also a CR + cell that expressed mGluR1. As this was the only CR3
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Table 2 Expression of eCB Biosynthetic enzymes and type I mGluRs as a function of plasticity and interneuron subtype. Percentages indicate neuron subtype proportions within each plasticity group. An ‘X’ indicates that a target came up within that given cell subtype population. Plasticity
Cell Type
None
CCK (75%) PV (25%) CCK (36%) CCK-CB (18%) Undifferentiated (46%) CCK (20%) CCK-CB (13%) CB (13%) PV (7%) Undifferentiated (47%)
STD
LTD
DAGLα
NAPE-PLD
12-LO
mGluR5
mGluR1
X X
X X
X X
X
X
X X
X
X X
X
X X
X X
was only expressed in neurons with one form of plasticity, either LTD or STD (Table 2). Nearly all stratum radiatum interneuron subtypes exhibiting plasticity appear to express at least one eCB biosynthetic enzyme, regardless of plasticity type. However, these enzymes were rarely present in neurons without plasticity (Table 2). 4. Discussion Our data illustrate a potential relationship between mGluR5 expression and LTD in CA1 stratum radiatum interneurons. Neurons expressing mGluR5, and often mGluR1, exhibit LTD preferentially over other plasticity types in radiatum interneurons. Furthermore, nearly all the neurons in our study expressed eCB biosynthetic enzyme mRNA as demonstrated previously [16], confirming that eCB synthesis is likely a common feature of CA1 stratum radiatum interneurons. We recently demonstrated differential distribution of eCB biosynthetic enzyme mRNA, suggesting that eCB-mediated processes do not occur equally at CA1 stratum radiatum interneuron synapses [16]. In fact, in eCB-mediated LTD studies, all interneurons tested did not respond equally to HFS [6,14]. McMahon & Kauer (1997) were the first to describe synaptic plasticity of excitatory inputs to hippocampal stratum radiatum interneurons. They reported bistratified and basket cell morphology among other interneurons, and concluded that plasticity did not be correlated with interneuron subtype [6]. Later, CA1 stratum radiatum interneuron LTD dependence on TRPV1, 12-HPETE, and mGluRs with a presynaptic loci of action was illustrated [14,18]. Our data also support that interneuron plasticity is not cell-type specific, but rather determined by mGluR5/1and potentially eCB-biosynthetic enzyme expression. Our results also indicate a qualitative effect on plasticity: all interneurons that express mGluR5 exhibit LTD. There were a few neurons exhibiting LTD without mGluR5 expression, though we cannot confirm whether they actually did not express mGluR5 or whether our qPCR assay did not detect mGluR5. These hippocampal interneurons also express eCB biosynthetic enzyme mRNA coding for DAGLα, which others also noted is expressed in interneurons, but more highly expressed in pyramidal cells [17]. In contrast, Péterfi and colleagues demonstrated a quantitative effect where higher stimulation frequencies and higher DHPG concentrations were required to induce plasticity in CA1 hippocampal interneurons than in pyramidal cells [17]. We did not test whether increasing the number of stimulus trains also increased LTD induction, however, it is possible that increased stimulation could induce plasticity in neurons we recorded from that lacked plasticity or neurons with STD, uncovering a quantitative effect. Collectively, the high correlation between mGluR5 and LTD in our study suggests that plasticity induction is more likely in mGluR5-expressing neurons.
Fig. 2. Gene expression profiles of stratum radiatum interneurons. A. The quantitative PCR (qPCR) of an example cell from Fig. 1A that demonstrated LTD. These cells always expressed mRNA for at least one eCB biosynthetic enzyme and most often for type I metabotropic glutamate receptors (mGluRs), especially mGluR5. B. An example cell from Fig. 1B that demonstrated STD. These cells often expressed mRNA for eCB biosynthetic enzymes and also for mGluR1. C. An example of one cell that did not exhibit plasticity from Fig. 1C. While a few of these neurons express mRNA for eCB biosynthetic enzymes (see Table 2), none expressed type I mGluRs. Data are represented as log-scaled relative fluorescence curves from FAM-TAMRA probe-based qPCR. 4
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study of DHPG-induced LTD of stratum radiatum interneurons noted that mGluR5 activation alone caused true LTD, while mGluR1 activation alone induced a short-term transient depression with a very similar time course to our HFS-induced STD [5], supporting the interpretation of mGluR5/1 in this study. As a note, the STD we report could also possibly correspond to depolarization-induced suppression of excitation or DSE, however DSE most often lasts 1–2 min and STD in this report lasts much longer. Our data confirm that LTD is inducible at excitatory synapses onto interneurons in the CA1 stratum radiatum and highlight the importance of mGluR5 in this LTD induction. In addition, mGluR1 could play a role in STD and possibly LTD. Furthermore, eCB synthesis likely plays an important role in hippocampal interneuron plasticity, and eCB enzymes appear to be dispersed fairly ubiquitously among certain CA1 radiatum interneuron subtypes [16]. While it was somewhat surprising that all interneurons of each subtype do not express the same plasticity type, this finding does fit with our data as a whole. Collectively, these data demonstrate the potential of distinct CA1 interneuron populations of various subtypes to differentially modulate learning and memory processing within the hippocampus through differing forms of plasticity.
We acknowledge that a quantitative effect as demonstrated by Péterfi et al. could still be present and that other eCB biosynthetic enzymes, even produced from other neurons, may contribute to plasticity, either synergistically or in a non-cell-autonomous manner. Lastly, our data do not suggest that any particular eCB biosynthetic enzyme expression correlates with a certain type of plasticity. Indeed, NAPE-PLD and 12-LO were expressed in neurons exhibiting LTD, STD, or no plasticity, and DAGLα was present in STD- or LTD-expressing neurons. In addition, it is conceivable that while most all interneurons express some form of eCB producing enzyme, the reason some lack or have different plasticity types is tied to their expression profile of type I mGluRs, which could be a way for the cell to regulate its ability to induce plasticity or not. The highly heterogeneous nature of hippocampal interneurons has historically made them difficult to study. Therefore, effective interneuron studies require careful definition of the target population, and as techniques differ this often leads to challenges when comparing findings between studies. For example, one limitation of the present study is that we were unable to examine cell morphology and correlate projection patterns, to plasticity and type I mGluR expression. To reduce false negative qPCR results, we extracted the entire neuron following the plasticity experiment, making it impossible to also image neuron morphology. We therefore limited our study to well-described CA1 stratum radiatum interneurons. Our data describe interneurons that are likely classically-described subtypes including parvalbuminpositive axo-axonic cells, calretinin-positive interneuron-selective cells, and CCK/CB-positive basket cells [4]. That being said, interneuron subtype in our study did not necessarily correlate to interneuron plasticity, nor did GAD65 and/or GAD67 expression. Lastly, many neurons were classified as “uncategorized” in our study, because their interneuron marker expression patterns did not match classical descriptions. We believe that the high rate of uncategorized cells is due to the technical limitations of single-cell RTqPCR. We recognize that qPCR often has ∼35% false negative results. Therefore, it is possible that many uncategorized cells actually expressed more interneuron markers than we detected, or expressed different markers that we did not examine. Additionally, negative qPCR results are difficult to interpret, because lack of detection of a target does not positively indicate its absence. We are aware of only one other study examining and finding a correlation between interneuron subtype and plasticity. This study determined that CA1 interneurons in or near the stratum pyramidale layer could be categorized into three main groups based on HFS (100 Hz)induced plasticity: PV bistratified cells that exhibit LTD, PV perisomatic targeting/axo-axonic cells that exhibit LTP and CB1-containing cells that lacked plasticity. A key determinant in plasticity was that PV cells contained calcium-permeable AMPA receptors while CB1-containing cells lacked them. In contrast, we noted no differences in plasticity based on cell subtype in stratum radiatum interneurons stimulated at 100 Hz, though AMPA receptor subtypes were not examined for comparison and we did not use perforated patch. Péterfi et al. (2012) also examined different interneuron subtypes and plasticity type, but in the stratum oriens as well as stratum pyramidale interneurons. They noted that both PV and somatostatin-positive cells both display 10-Hz-induced and DHPG-induced LTD, and thus no difference in plasticity of interneurons subtypes was noted. This LTD required mGluR5 and DAG lipase activity [17]. This study found that eCB-producing interneurons can mediate their own plasticity, which supports our previous study [16] and the current study. Our results, the first to focus on stratum radiatum interneurons subtypes only, corroborate the mGluR5 requirement for LTD in a non-specific interneuron subtype fashion. Though differences in methodology should be considered between these studies, it is of interest to note that cell-determined expression of type I mGluRs, particularly mGluR5, is likely a key factor in an interneuron’s ability to produce plasticity and its duration, or perhaps in the quantitative stimulus needed to induce the plasticity. Indeed, another
Author contributions CM, TMN and JGE designed experiments, acquired and analyzed data and wrote the paper. LF, ZB, and ZH acquired and analyzed data, and edited the paper. Declaration of Competing Interest There is no conflict of interest for any of the authors. Acknowledgements This work was supported by a National Institute of Neurological Diseases and Stroke grant (R15NS078645). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. Institutional Mentoring Environment Grants (JGE) and Brigham Young University Graduate Fellowship Awards (TMN) also supported this work. References [1] T.V.P. Bliss, T. Lømo, Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol. 232 (1973) 331–356, https://doi.org/10.1113/jphysiol.1973.sp010273. [2] S.M. Dudek, M.F. Bear, Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 4363–4367, https://doi.org/10.1073/pnas.89.10.4363. [3] W. Nissen, A. Szabo, J. Somogyi, P. Somogyi, K.P. Lamsa, Cell type-specific longterm plasticity at glutamatergic synapses onto hippocampal interneurons expressing either parvalbumin or CB1 cannabinoid receptor, J. Neurosci. 30 (2010) 1337–1347, https://doi.org/10.1523/JNEUROSCI.3481-09.2010. [4] T.F. Freund, G. Buzsáki, Interneurons of the hippocampus, Hippocampus 6 (1998) 347–470, https://doi.org/10.1002/(SICI)1098-1063(1996)6:4<347::AIDHIPO1>3.0.CO;2-I. [5] C. Le Duigou, T. Holden, D.M. Kullmann, Short- and long-term depression at glutamatergic synapses on hippocampal interneurons by group I mGluR activation, Neuropharmacology 60 (2011) 748–756, https://doi.org/10.1016/J. NEUROPHARM.2010.12.015. [6] L.L. McMahon, J.A. Kauer, Hippocampal interneurons express a novel form of synaptic plasticity, Neuron 18 (1997) 295–305, https://doi.org/10.1016/S08966273(00)80269-X. [7] D.M. Kullmann, K.P. Lamsa, LTP and LTD in cortical GABAergic interneurons: emerging rules and roles, Neuropharmacology 60 (2011) 712–719, https://doi.org/ 10.1016/J.NEUROPHARM.2010.12.020. [8] V. Chevaleyre, K.A. Takahashi, P.E. Castillo, Endocannabinoid-mediated synaptic plasticity in the CNS, Annu. Rev. Neurosci. 29 (2006) 37–76, https://doi.org/10. 1146/annurev.neuro.29.051605.112834. [9] J. Liu, L. Wang, J. Harvey-White, D. Osei-Hyiaman, R. Razdan, Q. Gong, A.C. Chan, Z. Zhou, B.X. Huang, H.-Y. Kim, G. Kunos, A biosynthetic pathway for anandamide, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 13345–13350, https://doi.org/10.1073/
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