Delta-subunit-containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

The intercalated paracapsular cells (pcs) are small GABAergic interneurons that form densely populated clusters surrounding the basolateral (BLA) complex of the amygdala. Their main task in the amygdala circuitry appears to be the control of information flow, as they act as an inhibitory interface between input and output nuclei. Modulation of their activity is thus thought to affect amygdala output and the generation of fear and anxiety. Recent evidence indicates that pcs express benzodiazepine (BZ)-sensitive GABA(A) receptor (GABA(A)R) variants containing the α2- and α3-subunit for transmission of post-synaptic currents, yet little is known about the expression of extrasynaptic GABA(A)Rs, mediating tonic inhibition and regulating neuronal excitability. Here, we show that pcs from the lateral and medial intercalated cell cluster (l- and mITC, respectively) express a tonic GABAergic conductance that could be significantly increased in a concentration-dependent manner by the δ-preferring GABA(A)R agonist THIP (0.5–10 μM), but not by the BZ diazepam (1 μM). The neurosteroid THDOC (300 nM) also increased tonic currents in pcs significantly, but only in the presence of additional GABA (5 μM). Immunohistochemical stainings revealed that both the δ-GABA(A)R and the α4-GABA(A)R subunit are expressed throughout all ITCs, while no staining for the α5-GABA(A)R subunit could be detected. Moreover, 1 μM THIP dampened excitability in pcs most likely by increasing shunting inhibition. In line with this, THIP significantly decreased lITC-generated inhibition in target cells residing in the BLA nucleus by 30%. Taken together these results demonstrate for the first time that pcs express a tonic inhibitory conductance mediated most likely by α4/δ-containing GABA(A)Rs. This data also suggest that δ-GABA(A)R targeting compounds might possibly interfere with pcs-related neuronal processes such as fear extinction

Heterogeneity of GABAA Receptors and interneurons in the Amygdala

Zusammenfassung Die Amygdala spielt eine wichtige Rolle bei negativen Emotionen wie Angst und den damit verbundenen Gedächtnisvorgängen. Wesentlich für den korrekten Ablauf dieser Prozesse ist das GABAerge inhibitorische System, dessen Schlüsselkomponenten Interneurone und GABAA-Rezeptoren sind. Im ersten Teil der Arbeit wurden die Verteilung verschiedener alpha-Untereinheiten von GABAA-Rezeptoren und ihr relativer Anteil an inhibitorischen Strömen immunohistochemisch und elektrophysiologisch in der Maus-Amygdala untersucht. Der Grossteil inhibitorischer Ströme im basolateralen und im zentralen Kern wird von alpha2- GABAA-Rezeptoren getragen. Alpha1-vermittelte Ströme fehlen gänzlich im zentralen Kern, während die alpha3-Untereinheit nur minimal in beiden Kernen zur Inhibition beiträgt. Im Hippocampus formen Interneurone, die den Cannabinoid Rezeptor 1 (CB1) tragen, bevorzugt Synapsen mit alpha2-GABAA-Rezeptoren. Anders in der Amygdala: Zumindest im basolateralen Kern trägt der grössere Teil der CB1-Synapsen alpha1-GABAA-Rezeptoren. Der zweite Teil fokussiert auf die Charakterisierung eines Subtyps von Interneuronen, den sogenannten parakapsulären Zellen. Diese GABAergen Zellen ummanteln in grossen Clustern den basolateralen Kern und werden cortical aktiviert. Sie hemmen basolaterale Pyramidenzellen und Zellen des zentralen Kerns. Moduliert werden diese Interneurone über Dopamin. Aktivierung von D1-Rezeptoren öffnet G-Protein-abhängige Kalium-Kanäle und führt zur Hyperpolarisierung. Pyramidenzellen und andere Interneuronen hingegen werden D1-Rezeptor-vermittelt depolarisiert. Das etwa in Stress-Situationen ausgeschüttete Dopamin vermindert also die über die parakapsulären Zellen vermittelte corticale Kontrolle über die Amygdala und führt zu einer weniger gehemmten affektiven Reaktion. Summary The amygdala plays a crucial role in emotions, particular negative ones such as anxiety, and in related memory processes. To ensure appropriate emotional responses, an intact inhibitory system is essential, yet little is known about inhibitory GABAergic neurons and GABAA receptors in the amygdala. We therefore investigated electrophysiologically the contribution of different alpha-subunits of the GABAA receptor to inhibitory currents and mapped these subunits also immunohistochemically in mouse amygdala. We found that alpha2-GABAA receptors carry the bulk of inhibitory currents in the basolateral and in the central nucleus. While the alpha1-subunit was completely absent from the central nucleus, the alpha3-subunit contributes only modestly to inhibition in both nuclei. In addition, we detected that inhibitory terminals carrying cannabinoid receptor 1 (CB1) targeted predominantly alpha1-subunit containing GABAA receptors, which is in contrast to the situation in the hippocampus. The second part focussed on a specific type of amygdala interneurons, the so-called paracapsular cells. These small cells form distinct clusters around the basolateral complex. They are cortically activated and inhibit projection cells of the basolateral and the central nucleus. Dopamine modulates this inhibitory gate: Activation of D1 receptors opens a G- protein-dependent potassium conductance and thus hyperpolarizes these interneurons. In contrast, projection cells and other interneurons within the basolateral nucleus depolarize in response to dopamine and D1 receptor agonists. These findings complement recent in vivo studies, in which dopamine (via D1 receptor activation) was shown to attenuate cortical suppression of the amygdala, thereby facilitating sensory-driven affective responses

Microsomal epoxide hydrolase is not a 2-arachidonyl glycerol hydrolase

The endocannabinoid 2-arachidonyl glycerol (2-AG) is substantially hydrolysed by at least two enzymes, fatty acid amide hydrolase (FAAH) and monoarachidonyl glycerol lipase (MAGL), which thereby terminate its biological activity. In a recent report it has been claimed that microsomal epoxide hydrolase (mEH), hitherto known as a xenobiotic detoxifying enzyme, also rapidly catalyses the breakdown of 2-AG. However, the catalytic site architecture of mEH argues against an esterase activity. We therefore analyzed the capacity of recombinant purified human, mouse and rat mEH to hydrolyze 2-AG. In contrast to the previous finding, we find only marginal 2-AG esterase activity ( ≤ 50 nmol/mg protein/min) associated with the purified enzymes that was resistant to inhibition by the potent mechanism-based mEH inhibitor 1,1,1-trichloropropene 2,3-oxide (TCPO). Likewise, 2-AG hydrolysis in mouse liver microsomes was resistant to TCPO inhibition while being efficiently blocked by methyl arachidonyl fluorophosphonate (MAFP). MAFP, on the other hand, failed to inhibit epoxide hydrolase activity of both, purified mEH and mouse liver microsomes. We therefore conclude that mEH lacks any appreciable 2-AG hydrolase activity

Mammalian Epoxide Hydrolases

Epoxide hydrolases (EHs) metabolize highly reactive epoxides with mutagenic and carcinogenic potential to the less reactive corresponding diols and are therefore traditionally viewed as detoxicating enzymes. In few instances, however, the diols themselves can be precursors of further reactive metabolites, and EH may thereby contribute to metabolic toxification. The most important xenobiotic-metabolizing EH is the microsomal EH (mEH). Furthermore, evidence is emerging that mammalian EHs fulfill roles other than detoxication. Meanwhile, the role of soluble EH (sEH), whose physiological substrates are epoxyeicosanoids (epoxyeicosatrienoic acids, EETs), signaling molecules involved in a broad variety of regulatory pathways, is well documented. sEH is therefore considered a new drug target as respective inhibitors promise therapeutic potential in the treatment of hypertension, pain, and possibly other diseases. Two new human EHs, EH3 and EH4, have been recently identified. Their expression pattern and substrate specificity strongly suggest a role in physiological processes, similar to sEH, rather than in metabolizing xenobiotic compounds

Beyond detoxification: a role for mouse mEH in the hepatic metabolism of endogenous lipids

Microsomal and soluble epoxide hydrolase (mEH and sEH) fulfill apparently distinct roles: Whereas mEH detoxifies xenobiotics, sEH hydrolyzes fatty acid (FA) signaling molecules and is thus implicated in a variety of physiological functions. These epoxy FAs comprise epoxyeicosatrienoic acids (EETs) and epoxy-octadecenoic acids (EpOMEs), which are formed by CYP epoxygenases from arachidonic acid (AA) and linoleic acid, respectively, and then are hydrolyzed to their respective diols, the so-called DHETs and DiHOMEs. Although EETs and EpOMEs are also substrates for mEH, its role in lipid signaling is considered minor due to lower abundance and activity relative to sEH. Surprisingly, we found that in plasma from mEH KO mice, hydrolysis rates for 8,9-EET and 9,10-EpOME were reduced by 50% compared to WT plasma. This strongly suggests that mEH contributes substantially to the turnover of these FA epoxides-despite kinetic parameters being in favor of sEH. Given the crucial role of liver in controlling plasma diol levels, we next studied the capacity of sEH and mEH KO liver microsomes to synthesize DHETs with varying concentrations of AA (1-30 μM) and NADPH. mEH-generated DHET levels were similar to the ones generated by sEH, when AA concentrations were low (1 μM) or epoxygenase activity was curbed by modulating NADPH. With increasing AA concentrations sEH became more dominant and with 30 μM AA produced twice the level of DHETs compared to mEH. Immunohistochemistry of C57BL/6 liver slices further revealed that mEH expression was more widespread than sEH expression. mEH immunoreactivity was detected in hepatocytes, Kupffer cells, endothelial cells, and bile duct epithelial cells, while sEH immunoreactivity was confined to hepatocytes and bile duct epithelial cells. Finally, transcriptome analysis of WT, mEH KO, and sEH KO liver was carried out to discern transcriptional changes associated with the loss of EH genes along the CYP-epoxygenase-EH axis. We found several prominent dysregulations occurring in a parallel manner in both KO livers: (a) gene expression of Ephx1 (encoding for mEH protein) was increased 1.35-fold in sEH KO, while expression of Ephx2 (encoding for sEH protein) was increased 1.4-fold in mEH KO liver; (b) Cyp2c genes, encoding for the predominant epoxygenases in mouse liver, were mostly dysregulated in the same manner in both sEH and mEH KO mice, showing that loss of either EH has a similar impact. Taken together, mEH appears to play a leading role in the hydrolysis of 8,9-EET and 9,10-EpOME and also contributes to the hydrolysis of other FA epoxides. It probably profits from its high affinity for FA epoxides under non-saturating conditions and its close physical proximity to CYP epoxygenases, and compensates its lower abundance by a more widespread expression, being the only EH present in several sEH-lacking cell types

In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells

11,12 -Epoxyeicosatrienoic acid (11,12 EET) reduces excitability and excitatory transmission in the hippocampus

Recent studies suggest a role for the arachidonic acid-derived epoxyeicosatrienoic acids (EETs) in attenuating epileptic seizures. However, their effect on neurotransmission has never been investigated in detail. Here, we studied how 11,12- and 14,15 EET affect excitability and excitatory neurotransmission in mouse hippocampus. 11,12 EET (2 μM), but not 14,15 EET (2 μM), induced the opening of a hyperpolarizing K+ conductance in CA1 pyramidal cells. This action could be blocked by BaCl2, the G protein blocker GDPβ-S and the GIRK1/4 blocker tertiapin Q and the channel was thus identified as a GIRK channel. The 11,12 EET-mediated opening of this channel significantly reduced excitability of CA1 pyramidal cells, which could not be blocked by the functional antagonist EEZE (10 μM). Furthermore, both 11,12 EET and 14,15 EET reduced glutamate release on CA1 pyramidal cells with 14,15 EET being the less potent regioisomer. In CA1 pyramidal cells, 11,12 EET reduced the amplitude of excitatory postsynaptic currents (EPSCs) by 20% and the slope of field excitatory postsynaptic potentials (fEPSPs) by 50%, presumably via a presynaptic mechanism. EEZE increased both EPSC amplitude and fEPSP slope by 40%, also via a presynaptic mechanism, but failed to block 11,12 EET-mediated reduction of EPSCs and fEPSPs. This strongly suggests the existence of distinct targets for 11,12 EET and EEZE in neurons. In summary, 11,12 EET substantially reduced excitation in CA1 pyramidal cells by inhibiting the release of glutamate and opening a GIRK channel. These findings might explain the therapeutic potential of EETs in reducing epileptiform activity

In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells