The hippocampus is required for short-term memory and contains both excitatory pyramidal cells and inhibitory interneurons. These cells exhibit various forms of synaptic plasticity, the mechanism underlying learning and memory. More recently, endocannabinoids were identified to be involved in synaptic plasticity. Our goal was to describe the distribution of endocannabinoid biosynthetic enzymes within CA1 stratum radiatum interneurons and CA3/CA1 pyramidal cells. We extracted mRNA from single interneurons and pyramidal cells and used real-time quantitative PCR to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase, and type I metabotropic glutamate receptors, known to be involved in endocannabinoid production and plasticity. We observed that the expression of endocannabinoid biosynthetic enzyme mRNA does occur within interneurons and that it is coexpressed with type I metabotropic glutamate receptors, suggesting interneurons have the potential to produce endocannabinoids. We also identified that CA3 and CA1 pyramidal cells express endocannabinoid biosynthetic enzyme mRNA. Our data provide the first molecular biological evidence for putative endocannabinoid production in interneurons, suggesting their potential ability to regulate endocannabinoid-mediated processes, such as synaptic plasticity.

2.1 Preparation of Slices

All experiments were performed in accordance with Institutional Animal Care and Use Committee protocols and followed the NIH guidelines for the care and use of laboratory animals. These guidelines include minimizing animal suffering and the number of animals used to perform the required experiments. Sprague-Dawley rats (16–28 days old) were used in all experiments. Animals were anesthetized using isoflurane and decapitated using a rodent guillotine. The brain was rapidly removed, sectioned into 400 µm thick coronal slices, and stored for at least one hour submerged on a net in artificial cerebrospinal fluid containing (in mM) 119 NaCl, 26 NaHCO₃, 2.5 KCl, 1.0 NaH₂PO₄, 2.5 CaCl₂, 1.3 MgSO₄, and 11 glucose, saturated with 95% O₂/5% CO₂ (pH 7.4). Slices were then transferred to a submerged recording chamber and bathed in oxygenated artificial cerebrospinal fluid.

2.2 Electrophysiological Recordings and Extraction

Slices were continuously perfused with filtered artificial cerebrospinal fluid at a flow rate of 2–3 mL/min. Hippocampal CA1 stratum radiatum interneurons were visually selected using infrared optics, CCD camera and monitor, with an Olympus BX51WI microscope with a 40× water immersion objective. Upon selection, each cell was patched with a borosilicate glass pipette filled with filtered internal solution composed of (in mM) 117 potassium gluconate, 2.8 NaCl, 20 HEPES, 5 MgCl2, and 0.6 EGTA-K (pH 7.28, 275–285 mOsm). Spiking patterns were acquired in whole cell current clamp configuration by injecting 1000 pA positive current for 500 msec. Most cells recorded in current clamp mode were approximately −60 to −70 mV, while those cells depolarized by more than −40 mV were excluded from our spiking analysis; though these cells as analyzed by PCR were very consistent with others. Electrophysiological traces were recorded with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA). Signals were filtered at 4 kHz and digitized with an Axon 1440A digitizer (Molecular Devices) connected to a Dell personal computer with pClamp 10.2 Clampfit software (Molecular Devices). Each cell was then extracted from the slice with gentle suction. Once free of the slice, the entire cell was carefully aspirated into the pipette tip to avoid aspiration of artificial cerebrospinal fluid and transferred immediately into a chilled reverse transcription mixture and processed within two hours. The entire cell was harvested in order to attain sufficient mRNA to examine the large number of desired targets we investigated. An artificial cerebrospinal fluid control sample was extracted for every slice, where the electrode was first placed in the slice and then artificial cerebrospinal fluid was aspirated just above the slice. This was done to ensure any contaminating mRNA if seen in these artificial cerebrospinal fluid controls from the slice could be eliminated from single cell analysis to avoid false positives.

2.3 Reverse Transcription Reaction

The reverse transcription reaction was accomplished using iScript cDNA Synthesis Kit (BioRad), following the prescribed protocol, with a final reaction mixture of 10 µL. This mixture was then cycled in a C1000 Thermocycler (BioRad) under the following conditions: 25.0 °C for 8 minutes, 42.0 °C for 60 minutes, and 70 °C for 15 minutes.

For primer optimization (see below for more details) a cDNA library was created by reverse transcription of total RNA from homogenized brain tissue. Homogenization and mRNA extraction were performed using TriZol reagents (Invitrogen), according to its published protocol, followed by mRNA conversion to cDNA using iScript cDNA synthesis kit (BioRad), according to its published protocol.

2.4 Primer Design, Verification, and Optimization

Primers for selected cDNA of endocannabinoid signaling components, calcium-binding proteins, and other targets were designed using Vector NTI software (Invitrogen) and PrimerExpress software (Applied Biosystems Inc.), using identical parameters (T_m, GC content, minimum primer length) for each primer set. All primer sets were designed to cross an intron boundary and amplify from exon to exon in order to avoid nuclear DNA amplification, with the exception of CB1 because it is intron-less. For control purposes each primer was tested using a serial dilution series of cDNA from rat whole brain and SsoFast EvaGreen Supermix (BioRad), followed by melt curve analysis to verify amplification of one product. The resulting amplification mixture was tested by 4% agarose gel electrophoresis to confirm that the size of the amplified cDNA fragment matched the designed amplicon size. Once primers were verified, each primer set was optimized to 90–95% amplification efficiency using probes specific to the amplified fragment and iQ Supermix (BioRad) (Figure 2). The primers were also grouped and tested to ensure that no primer cross binding occurred during the multiplex reaction by performing the multiplex reaction using mixed primers with cDNA template of 10 ng/µL from whole brain homogenate.

Figure 2

Optimization and verification of RT-qPCR primers and probes. a) A dose-response set of fluorescent curves of the primer/probe set for CCK ranging from 100 ng to 0.3 ng cDNA. Inset: The linear fit of the dose response from (a) in log scale for CCK. Ct …

2.5 Preamplification (Multiplex) Reaction

Once the reverse transcription reaction was complete, each cell was divided into two portions of approximately 5 µL each. The primers for each target were divided into two groups, and then a mixture including iQ Supermix (BioRad), ddH₂O, and one group of 10-fold diluted primers (see Table 1) was added to each aliquot. The same two groups were used for both interneurons and pyramidal cells, except interneurons were not examined for VGlut1. Next, both aliquots were then placed in a C1000 Thermocycler (BioRad) and processed as follows: 95 °C hot start for 3 minutes, followed by 15 cycles of 95 °C for 15 seconds, 57 °C for 20 seconds, and 72 °C for 25 seconds.

Table 1

Primer and probe sequences used for RT-qPCR, and whether primers are intron spanning.

2.6 Quantitative PCR Reaction

For qPCR, cDNA from the pre-amplified multiplex reaction was used for probe-based gene detection. Each target was run individually in triplicate, with undiluted primers, the appropriate FAM-TAMRA probe (Applied BioSystems, Inc.) specific to each target (see Table 1), and iQ Supermix (BioRad). Each cell was run on a CFX96 qPCR machine (BioRad) according to the following protocol: 95 °C hot start for three minutes, followed by 50 cycles of 95 °C for 15 seconds, 57 °C for 20 seconds, and 72 °C for 25 seconds. Amplification was measured by increased relative fluorescence during each cycle and a cycle threshold (Ct) value was assigned to each target using BioRad CFX Manager software. Each target from a cell was also examined using 4% agarose gel electrophoresis to verify amplicon size (see Figure 3B). For TRPV1, we sequenced the amplification product to verify amplicon identity. In addition, because CB1 lacks introns, 5 cells were tested as controls to determine whether the amplified CB1 PCR product came from genomic DNA rather than reverse transcribed mRNA (cDNA). These cells were extracted in the same manner as all other cells, except that half the reaction mixture lacked reverse transcriptase and was processed for CB1, while the other half was run with reverse transcriptase for 18S in order to determine Ct values. These cells were then multiplexed and evaluated by qPCR. Fluorescence curves for CB1 were extremely weak and shifted back compared to CB1 positive cells. Ct values from these 5 cells were significantly different from the other interneurons (P < 0.05; T-test), indicating that detected CB1 was indeed from expressed mRNA, rather than genomic DNA.

Figure 3

Identification of CA1 stratum radiatum interneuron subtypes by their expression of endocannabinoid biosynthetic enzyme mRNA and spiking pattern. a) A representative CCK-CB cell expressing DAGL (royal), 12-LO (wine), and mGluR1 (purple). Data are displayed …

2.7 Data Analysis

Ct value data from the qPCR reaction from each cell was compared to Ct data from artificial cerebrospinal fluid samples extracted from each slice. If any target noted in artificial cerebrospinal fluid samples was either within 5 cycles of the cell Ct value or not significantly different from the Ct value of the cell, it was excluded from the cell analysis. Some artificial cerebrospinal fluid samples displayed expression of several targets; in this case, the cells corresponding to the artificial cerebrospinal fluid sample were classified as failures and not fully analyzed. Ct values for 18S were subtracted from the Ct value for each target in a cell wise manner to obtain a ΔCt value for each target. Any target with a ΔCt value greater than 20 was excluded from analysis as non-specific. To quantify mRNA expression levels, the ΔΔCt method (Livak and Schmittgen, 2001) was used. Expression data was obtained using CFX Manager software (BioRad) or by double-derivative analysis using GraphPad Prism 4 (GraphPad Software, Inc.). All expression levels were tested for significance (p < 0.05) using an unpaired two-way Student’s T-test.

4.1 Interneuron Subtypes

In undertaking this study, we first verified that the interneuron subtypes we categorized matched those of previously published studies. Our results were consistent with ratios of stratum radiatum interneurons and molecular profiles outlined previously (Freund and Buzsáki, 1996, Jinno and Kosaka, 2002, 2006,Klausberger, 2009). In addition, we noted all CCK containing cells also expressed CB1, as would be expected (Katona et al., 1999, Marsicano and Lutz, 1999). These prior reports mainly use immunocytochemistry or western blot methodologies. These current experiments using qPCR still reveal a similar pattern of calcium binding protein and neuropeptide expression, supporting our qPCR methodology as a valid technique for identification of interneuron subtypes. Indeed, using selected targets to classify only some of the many interneuron subtypes in the hippocampus, we identified four major subpopulations within stratum radiatum using this technique. Other subtypes, such as trilaminar or Schaeffer-collateral associated cells, which are present in stratum radiatum (Ferraguti et al., 2004, Boscia et al., 2008, Szilagyi et al., 2011), could possibly be among the cells we did not attempt to categorize. Many of the 14 unidentified cells also expressed these endocannabinoid-producing enzymes and type I mGluRs. While spiking is often used to help discriminate interneuron subtypes, it is difficult to employ as a clear identifier of interneuron subtypes due to variability in spiking among subtypes (Ascoli et al., 2008,Wierenga et al., 2010). However, our data suggests that general spiking patterns support our characterization of subtype groups based on calcium binding proteins and CCK as compared to others (Kawaguchi et al., 1995, Buhl et al., 1996, Pawelzik et al., 1999, Galarreta et al., 2004).

4.2 Endocannabinoid biosynthetic enzyme mRNA expression within interneurons

The major find of this study is the description of mRNA for endocannabinoid biosynthetic enzymes NAPE-PLD, 12-LO, and DAGL in hippocampal interneurons. NAPE-PLD produces anandamide in the brain (Morishita et al., 2005, Ueda et al., 2005, Placzek et al., 2008) and is reported to be present and active in the hippocampus (Morishita et al., 2005). Anandamide activates endocannabinoid receptors such as CB1 and TRPV1 (Caterina et al., 1997), and may also be produced by other enzymes (Liu et al., 2006, Simon and Cravatt, 2010). While some stratum radiatum interneurons in mice were identified to express NAPE-PLD using immunocytochemistry (Cristino et al., 2008), the expression pattern of NAPE-PLD in specific interneuron subtypes was not described. We determined that NAPE-PLD is most highly expressed in CCK-CB and CB expressing cells.

DAGL, responsible for the synthesis of 2-arachidonylglycerol (Tanimura et al., 2010, Ludanyi et al., 2011), was previously identified using in situ hybridization in CA3 and CA1 pyramidal cells, and was either absent or expressed at undetectable levels for this technique in interneurons and glia (Katona et al., 2006). Our qPCR data confirm the expression of DAGL in both CA3 and CA1 pyramidal cells and also suggest its presence in CA1 stratum radiatum interneurons. Expression was highest in CCK-CB and CB cells and somewhat lower in CCK-only cells. This is the first report of hippocampal interneurons expressing DAGL.

12-LO synthesizes 12-HPETE (Hwang et al., 2000), and has been identified in some stratum radiatum interneurons in mice (Cristino et al., 2008). The interneuron subtypes expressing 12-LO, however, were not identified. Our data indicate that 12-LO is mainly expressed in the radiatum by CCK-CB interneurons.

While others have identified protein expression to some degree of these endocannabinoid biosynthetic enzymes in the hippocampus using immunocytochemistry, which provides support for our RT-qPCR data, we now identify which cells types they are expressed in and their co-expression with type I mGluRs demonstrating their capacity to produced endocannabinoids. In short, endocannabinoid biosynthetic enzymes are indeed likely expressed by interneurons and this expression is at least partly subtype specific, where NAPE-PLD and DAGL expression is fairly broadly distributed, unlike 12-LO, and calretinin cells had very little expression of mRNA for these enzymes. While the ability of hippocampal interneurons to produce endocannabinoids has been debated (Hoffman et al., 2003), our data suggest it as a strong possibility, as has been described for cerebellar interneurons (Beierlein and Regehr, 2006). Therefore, signaling (retrograde and otherwise) and plasticity that is mediated by endocannabinoids produced within hippocampal interneurons, appears to be a possibility. Furthermore, a recent study has shown that activation of CB1 receptors on interneurons decreases gamma oscillations in the hippocampus (Holderith et al., 2011). This suggests that the coexpression of CB1 and DAGL or NAPE-PLD by CA1 stratum radiatum interneurons could lead to autoregulation of oscillatory behavior or self-inhibition as described in neocortical interneurons (Bacci et al., 2004), and further highlights the importance of our findings.

4.3 Type I mGluR expression

As type I mGluR activation often results in production of endocannabinoid precursors such as arachidonic acid, they are usually necessary for endocannabinoid production within cells that modulate plasticity (Huber et al., 2001, Edwards et al., 2006). Therefore, it was critical to identify type I mGluR coexpression with endocannabinoid biosynthetic enzymes to provide evidence for interneurons’ role in endocannabinoid production. Regarding type I mGluRs, it was previously shown that mGluR1 is expressed by non-principle cells in the radiatum (Kerner et al., 1997, van Hooft et al., 2000, Ferraguti et al., 2004), where mGluR1 expression was identified in aspiny interneurons (Wittner et al., 2006). Our data using RT-qPCR demonstrate mGluR1 expression in radiatum interneurons, particularly in CB and CCK-CB cells, which also coexpressed CB1. This supports prior immunocytochemical evidence that CB1 is coexpressed with mGluR1, particularly in CCK-CB interneurons in both rats and mice (Boscia et al., 2008). Boscia et al. also identified mGluR1 in other interneuron populations, including CCK positive and CCK negative cells. Our data suggest that some of these previously unidentified mGluR1 expressing interneuron subtypes may include CB cells as well as CCK-CB cells. While mGluR1 expression in calretinin containing cells has been described previously (Ferraguti et al., 2004), we did not observe this coexpression, possibly because of our sample size for calretinin cells.

Expression of mGluR5, which is widely present in hippocampal pyramidal cells (Kerner et al., 1997, van Hooft et al., 2000, Huber et al., 2001), was previously noted immunocytochemically in some CA1 stratum radiatum cells that appeared to be GABAergic, but were not classified as such (Romano et al., 1995). We are the first to positively identify mGluR5 expression in stratum radiatum interneurons, which appear to be mainly in CCK expressing cells. This is also the first report to specifically examine coexpression of type I mGluR and endocannabinoid biosynthetic enzyme mRNA in CA1 stratum radiatum interneurons, where our data support the potential capacity of interneurons to produce endocannabinoids. Finally, the identification or suggestion that type I mGluRs are at least present in some interneurons using immunocytochemistry or physiological, as listed above, also supports our identification of these receptors using RT-qPCR in radiatum interneurons.

4.4 TRPV1 expression in hippocampal neurons and components involved in mGluR/TRPV1-mediated interneuron plasticity

TRPV1 has been shown to be expressed in the hippocampus (Sanchez et al., 2001, Tóth et al., 2005, Cristino et al., 2006, Cristino et al., 2008, Bennion et al., 2011) with some important exceptions (Kofalvi et al., 2006, Cavanaugh et al., 2011), and has been demonstrated to be involved in hippocampal synaptic plasticity (Gibson et al., 2008, Chavez et al., 2010, Bennion et al., 2011). We recently used RT-qPCR to identify TRPV1 mRNA present in whole hippocampal homogenates (Bennion et al., 2011). Indeed, TRP conductance mediated by type I mGluRs was identified in the hippocampus previously (Gee et al., 2003), and endocannabinoid signaling via TRPV1 is known to be initiated by the activity of type I mGluRs (Gibson et al., 2008, Bennion et al., 2011). Presently, we identified weak TRPV1 mRNA expression in CA3 pyramidal cells using RT-qPCR, supporting prior reports that it is present in the hippocampus. The low levels of TRPV1 noted might suggest that high TRPV1 expression is not necessary to influence synaptic plasticity or that TRPV1 mRNA is transported toward its axonal expression site, which would reduce the amount of TRPV1 mRNA within the soma, where mRNA was harvested for our study. Because we noted TRPV1 expression in CA3 pyramidal cells as well as type I mGluRs and 12-LO in CA1 stratum radiatum interneurons, it supports physiological data of the proposed mechanism for presynaptic TRPV1-mediated long-term depression in stratum radiatum interneurons (Gibson et al., 2008). Additionally, 12-LO-expressing cells, such as CCK-CB cells, or those expressing NAPE-PLD that produce anandamide could potentially induce TRPV1-mediated interneuron long-term depression by themselves.

4.5 CA1/CA3 pyramidal cell expression of endocannabinoid biosynthetic enzymes

In addition to interneurons, endocannabinoid biosynthetic enzymes are also expressed in pyramidal cells. Prior studies using immunocytochemistry indicate that 12-LO, NAPE-PLD, and DAGL are expressed in CA3 pyramidal cells, while 12-LO and DAGL are expressed in CA1 pyramidal cells (Nishiyama et al., 1993,Cristino et al., 2008, Egertová et al., 2008, Tanimura et al., 2010). 12-LO was also shown to be physiologically active in CA3 Schaeffer collaterals (Feinmark et al., 2003). Overall, our data corroborate the published evidence for endocannabinoid biosynthetic enzyme expression in pyramidal cells, except that we did not detect 12-LO expression in CA1 pyramidal cells. Additionally, mGluR5 was noted in both CA3 and CA1 pyramidal cells, while mGluR1 was noted to be expressed solely in CA3 cells using immunocytochemistry or physiology (Lujan et al., 1996, Shigemoto et al., 1997, Chuang et al., 2002, Le Duigou et al., 2011). Our qPCR data confirm this finding. Collectively, our data support previously published data and represent the first time that the expression of these endocannabinoid biosynthetic enzymes in pyramidal cells has been described using RT-qPCR. This also provides support for the reliability of our RT-qPCR methodology, as it closely models prior physiological and immunocytochemical data, supporting RT-qPCR as a viable method to study neuronal gene expression.

4.6 Conclusion

In summary, our qPCR data indicate that CCK, CCK-CB, and CB expressing CA1 stratum radiatum interneurons have the potential to produce some endocannabinoids due to their coexpression of type I mGluRs and endocannabinoid biosynthetic enzymes. This suggests that these subtypes could be involved in some forms of endocannabinoid-mediated signaling, including synaptic plasticity and regulating oscillatory behavior. Our data also indicate that calretinin cells display very little mRNA for endocannabinoid biosynthetic enzymes or the receptors involved in their activation. Collectively, our data clearly demonstrate the capacity for hippocampal stratum radiatum interneurons to produce endocannabinoids, providing evidence that interneuron involvement in endocannabinoid signaling in the hippocampus may be greater than previously thought.

We acknowledge Andrew Martin for technical assistance. This work was supported by National Institute of Health 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. This work was also supported by institutional Brigham Young University mentoring grants.

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