Identification of mRNA for endocannabinoid biosynthetic enzymes within hippocampal pyramidal cells and CA1 stratum radiatum interneuron subtypes using quantitative real-time polymerase chain reaction

Abstract

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 polymerase chain reaction (RT-PCR) to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase α, and type I metabotropic glutamate receptors, all 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.

Figure 1

Endocannabinoid biosynthetic pathways and receptor targets. Postsynaptic type I metabotropic glutamate receptor activation commonly produces metabolites used in endocannabinoid and eicosanoid synthesis. Endocannabinoid biosynthetic enzymes such as diacylglycerol lipase (DAGL), N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and 12-lipoxygenase (12-LO) produce the endocannabinoids 2-arachidonylglycerol (2-AG), anandamide (AEA), and 12-(S)-Hydroperoxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid (12-HPETE), respectively. Endocannabinoids are lipophilic substances that can act retrogradely on presynaptic terminals to modulate neurotransmitter release via 2-AG or AEA activating cannabinoid receptor 1 (CB1), or 12-HPETE or AEA activating transient receptor potential vanilloid 1 (TRPV1).

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 values for all triplicates from each concentration are included. b) Melt curve peaks of CCK from the same cDNA dose response in (a). Inset: Electrophoresis gel of CCK, showing, from left to right, 50 base pair (bp) ladder, 18S amplification product (133 bp), and CCK amplification product (111 bp). Curves produced for dose response and melt curves in (a) and (b) were triplicate averages.

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 as fluorescence from a FAM-TAMRA hydrolysis probe from a RT-qPCR reaction. Inset: A representative irregular spiking CA1 stratum radiatum interneuron. The membrane potential of this cell (not visible here) was −70 mV. Scale bar: 150 ms, 15 mV. b) A 4% agarose gel of the cell presented in (a) showing, from left to right, 50 bp ladder, mGluR1, CB1, 12-LO, DAGL, CCK, 18S, and CB, with their respective amplicon sizes.

Figure 4

Endocannabinoid biosynthetic enzyme mRNA expression in CA1 stratum radiatum interneuron subtypes. a) A representative CCK-CB cell demonstrates the presence of mGluR1 (purple), mGluR5 (dark cyan) and NAPE-PLD (olive). Note that the scaling of this figure did not allow for an accurate depiction of mGluR5, which under different scaling demonstrates a sigmoid curve, and the correct amplicon size is noted using gel electrophoresis. b) A representative CB-expressing cell demonstrates the presence of NAPE-PLD (olive) and mGluR1 (purple). c) A representative calretinin-expressing cell lacking expression of endocannabinoid components or type I mGluRs. Data are displayed as fluorescence from a FAM-TAMRA hydrolysis probe in the RT-qPCR reaction.

Figure 5

Expression of endocannabinoid biosynthetic enzyme mRNA in hippocampal pyramidal cells. a) A representative CA3 pyramidal cell demonstrates the presence of DAGL (royal), NAPE-PLD (olive), and mGluR5 (dark cyan). Note the mGluR5 no-template control (mGluR5 NTC). b) A second CA3 pyramidal cell demonstrates the presence of DAGL (royal) and12-LO (wine). c) A representative CA1 pyramidal cell demonstrates the presence of DAGL (royal) and mGluR5 (dark cyan). Data are displayed as fluorescence from a RT-qPCR reaction.

Figure 6

TRPV1 mRNA expression in a CA3 pyramidal cell. a) A representative CA3 pyramidal cell expressing VGluT1 (light gray) and TRPV1 (dark gray). b) A 4% agarose gel of the cell in a), showing, from left to right, 50 bp ladder (50 bp and 100 bp shown), TRPV1 (86 bp, arrow), TRPV1 no-template control, and VGluT1 (74 bp).