Hippocampal Stratum Oriens Somatostatin-Positive Cells Undergo CB1-Dependent Long-Term Potentiation and Express Endocannabinoid Biosynthetic Enzymes

Abstract

The hippocampus is thought to encode information by altering synaptic strength via synaptic plasticity. Some forms of synaptic plasticity are induced by lipid-based endocannabinoid signaling molecules that act on cannabinoid receptors (CB1). Endocannabinoids modulate synaptic plasticity of hippocampal pyramidal cells and stratum radiatum interneurons; however, the role of endocannabinoids in mediating synaptic plasticity of stratum oriens interneurons is unclear. These feedback inhibitory interneurons exhibit presynaptic long-term potentiation (LTP), but the exact mechanism is not entirely understood. We examined whether oriens interneurons produce endocannabinoids, and whether endocannabinoids are involved in presynaptic LTP. Using patch-clamp electrodes to extract single cells, we analyzed the expression of endocannabinoid biosynthetic enzyme mRNA by reverse transcription and then real-time PCR (RT-PCR). The cellular expression of calcium-binding proteins and neuropeptides were used to identify interneuron subtype. RT-PCR results demonstrate that stratum oriens interneurons express mRNA for both endocannabinoid biosynthetic enzymes and the type I metabotropic glutamate receptors (mGluRs), necessary for endocannabinoid production. Immunohistochemical staining further confirmed the presence of diacylglycerol lipase alpha, an endocannabinoid-synthesizing enzyme, in oriens interneurons. To test the role of endocannabinoids in synaptic plasticity, we performed whole-cell experiments using high-frequency stimulation to induce long-term potentiation in somatostatin-positive cells. This plasticity was blocked by AM-251, demonstrating CB1-dependence. In addition, in the presence of a fatty acid amide hydrolase inhibitor (URB597; 1 µM) and MAG lipase inhibitor (JZL184; 1 µM) that increase endogenous anandamide and 2-arachidonyl glycerol, respectively, excitatory current responses were potentiated. URB597-induced potentiation was blocked by CB1 antagonist AM-251 (2 µM). Collectively, this suggests somatostatin-positive oriens interneuron LTP is CB1-dependent.

Keywords: 12-lipoxygenase; DAGLα; LTP; anandamide; eCB; hippocampus; mGluR1; mGluR5.

Figure 1

High-frequency stimulus long-term potentiation (HFS-LTP) only occurs in a subset of rat hippocampal oriens interneurons and is cannabinoid receptor (CB1)-dependent. (A) Oriens interneurons were recorded in whole cell voltage-clamp mode and 100 Hz high-frequency stimulus (HFS; arrow) while in current clamp mode was used to induce LTP. A majority (67%) of interneurons exhibited significant LTP (n = 8; ANOVA, p < 0.05), while a minority did not (n = 4; ANOVA, p > 0.05). ANOVAs were used to compare baseline to post-HFS excitatory postsynaptic currents (EPSCs) in order to group a cell having significant LTP versus no plasticity. These two groups (black symbols, LTP; white symbols, no plasticity) were significantly different from each other (p < 0.05, t-test). Inset: top traces are an LTP example and bottom traces are a no plasticity example. Traces were taken from 10–12 sweeps just before HFS (black) or ~10–15 min post-HFS (light gray). (B) This LTP was significantly blocked by AM-251 (n = 6; p < 0.05 compared to control LTP), suggesting it is CB1-dependent. Scale bars 100 pA, 10 msec. Plots, mean with s.e.m.

Figure 2

Real-time (RT)-PCR confirms somatostatin-positive (SOM+) rat hippocampal cells exhibit LTP. (A) An example SOM+ cell from Figure 1A that expressed type I metabotropic glutamate receptor 5 (mGluR5), and exhibited HFS-LTP. (B) An example parvalbumin-positive (PV+; somatostatin-negative) cell from Figure 1A that expressed diacylglycerol lipase (DAGLα), mGluR5, and n-acylphosphatidylethanolamine phospholipase D (NAPE-PLD, abbreviated PLD) that did not potentiate following HFS. (C) An example SOM+ neuron from Figure 1B expressing 12-lipoxygenase (12-lipo), that likely could have potentiated, but did not exhibit plasticity in the presence of the CB1 antagonist AM-251. Inset: Gel electrophoresis of the amplicon from the somatostatin (SOM) primers of the appropriate size with accompanying ladder in base pairs (bp). As a note, amplicons from all other primers have been published previously [10,29]. Trace color key: Black, somatostatin; blue mGluR5; lime green, DAGLα; red, parvalbumin; magenta, NAPE-PLD; forest green, 12-lipoxygenase.

Figure 3

Fatty acid amide hydrolase (FAAH) inhibition potentiates rat hippocampal oriens interneurons in a CB1-dependent manner. (A) Stratum oriens interneurons were patched (n = 8) in whole cell voltage-clamp mode and recorded while the enzyme FAAH was inhibited by URB-597 (1 µM). A significant (p < 0.05; ANOVA) potentiating effect was observed, suggesting a role of anandamide in oriens synaptic plasticity. (B) CB1 antagonist AM-251 significantly blocked FAAH-induced potentiation (n = 7; p < 0.05; t test, comparing URB-597 to URB-597 + AM-251). (C) The monoacylglycerol lipase (MAG lipase) inhibitor, JZL184 (1 µM), used to prevent degradation of 2-AG also resulted in significant (p < 0.05; ANOVA; n = 5) enhancement of EPSC responses (individual cells were examined for significance in their potentiation by ANOVA and included if significant). Note that of the six significantly potentiating cells (of nine recorded from), four were confirmed SOM+ by posthoc PCR analysis while two were unclassifiable. Inset: traces were taken of 10–12 sweeps just before HFS (black) or ~15 min post-drug application (light gray).

Figure 4

DAGLα is expressed in a subset of mouse hippocampal interneurons. GAD67-green fluorescent protein (GFP) knock-in mice were probed for DAGLα expression. We observed DAGLα immunoreactivity in the cytosol of GAD67-GFP stratum oriens (SO) interneurons and pyramidal cells of the stratum pyramidale (SP). Semi-quantitative analysis of co-labeling occurred in ~64% of GAD67-positive neurons in the same region as physiological recordings (n = 31 of 48; from slices in three different mouse brains of dorsal CA1). Closed arrows illustrate an interneuron that is double labeled with GAD67-GFP and DAGLα in the oriens. Open arrow denotes a GAD67-GFP labeled, DAGLα-negative neuron in the SP. GAD67-positive cells include populations of PV+ and SOM+ cells [31]. Scale Bar: 50 µm.