Taiju Amano

Synaptic correlates of fear extinction in the amygdala

Anxiety disorders such as post-traumatic stress are characterized by an impaired ability to learn that cues previously associated with danger no longer represent a threat. However, the mechanisms underlying fear extinction remain unclear. We found that fear extinction in rats was associated with increased levels of synaptic inhibition in fear output neurons of the central amygdala (CEA). This increased inhibition resulted from a potentiation of fear input synapses to GABAergic intercalated amygdala neurons that project to the CEA. Enhancement of inputs to intercalated cells required prefrontal activity during extinction training and involved an increased transmitter release probability coupled to an altered expression profile of ionotropic glutamate receptors. Overall, our results suggest that intercalated cells constitute a promising target for pharmacological treatment of anxiety disorders.

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinsons disease and a decreased Parkinsons disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinsons disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinsons disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinsons disease.

The fear circuit revisited: contributions of the basal amygdala nuclei to conditioned fear.

The lateral nucleus (LA) is the input station of the amygdala for information about conditioned stimuli (CSs), whereas the medial sector of the central nucleus (CeM) is the output region that contributes most amygdala projections to brainstem fear effectors. However, there are no direct links between LA and CeM. As the main target of LA and with its strong projection to CeM, the basomedial amygdala (BM) constitutes a good candidate to bridge this gap. Consistent with this notion, it was reported that combined posttraining lesions of the basal nuclei [BM plus basolateral nucleus (BL)] abolish conditioned fear responses, whereas selective BL inactivation does not. Thus, we examined the relative contribution of BM and BL to conditioned fear using unit recordings and inactivation with muscimol microinfusions in rats. Approximately 30% of BM and BL neurons acquired robust responses to auditory CSs predicting footshocks. While most BL cells stopped firing at CS offset, BM responses typically outlasted the CS by ≥40 s, paralleling the persistence of conditioned fear after the CS. This observation suggests that BM neurons are not passive relays of rapidly adapting LA inputs about the CS. Surprisingly, independent inactivation of either BM or BL with muscimol did not cause a reduction of conditioned freezing even though an extinction recall deficit was seen the next day. In contrast, combined BL–BM inactivation did. Overall, there results support the notion that the basal nuclei are involved in conditioned fear expression and extinction but that there is functional redundancy between them.

Physiological identification and infralimbic responsiveness of rat intercalated amygdala neurons

Intercalated (ITC) amygdala neurons are thought to play a critical role in the extinction of conditioned fear. However, several factors hinder progress in studying ITC contributions to extinction. First, although extinction is usually studied in rats and mice, most ITC investigations were performed in guinea pigs or cats. Thus it is unclear whether their connectivity is similar across species. Second, we lack criteria to identify ITC cells on the basis of their discharge pattern. As a result, key predictions of ITC extinction models remain untested. Among these, ITC cells were predicted to be strongly excited by infralimbic inputs, explaining why infralimbic inhibition interferes with extinction. To study the connectivity of ITC cells, we labeled them with neurobiotin during patch recordings in slices of the rat amygdala. This revealed that medially located ITC cells project topographically to the central nucleus and to other ITC clusters located more ventrally. To study the infralimbic responsiveness of ITC cells, we performed juxtacellular recording and labeling of amygdala cells with neurobiotin in anesthetized rats. All ITC cells were orthodromically responsive to infralimbic stimuli, and their responses usually consisted of high-frequency (~350 Hz) trains of four to six spikes, a response pattern never seen in neighboring amygdala nuclei. Overall, our results suggest that the connectivity of ITC cells is conserved across species and that ITC cells are strongly responsive to infralimbic stimuli, as predicted by extinction models. The unique response pattern of ITC cells to infralimbic stimuli can now be used to identify them in fear conditioning experiments.

Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia.

α‐amino‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop‐ and flip‐preferring allosteric modulators of AMPA receptors, 4‐[2‐(phenylsulfonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)‐ or kainite (KA)‐induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu‐receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu‐ and KA‐induced currents were potentiated by PEPA and CTZ in a concentration‐dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell‐to‐cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT‐PCR revealed that GluR1‐3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu‐induced release of tumor necrosis factor‐α (TNF‐α) unpredictably. The increase in TNF‐α release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen‐activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF‐α release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.

Expression and function of bradykinin receptors in microglia

Expression of bradykinin (BK) receptors and their cellular function were investigated in microglia. Microglial cells were isolated from mixed cultures of cerebrocortical cells from postnatal day 3 Wistar rats. Reverse transcription-PCR (RT-PCR) showed that rat primary microglia express mRNAs for the type 2 bradykinin (B(2)) receptor subtype but not the type 1 (B(1)) receptor subtype under our experimental condition. However, the expression of B(1) receptor was greatly up-regulated after the treatment of microglia with BK for 24 hours. The expression of B(2) receptor in microglia was further confirmed by immunocytochemistry. Membrane currents were measured using whole-cell recording under voltage-clamp conditions. In 14% of patched cells (12/85 cells), BK (100-200 nM) induced an outward current at the holding potential of -20 mV, with oscillations in 2 cases. The BK-induced outward current was transient and desensitized rapidly. TEA inhibited the BK-induced outward current in a dose-dependent manner. These results suggest that microglia express B(2) receptors and presumably increase the intracellular Ca(2+) concentration via inositol trisphosphate with the subsequent activation of Ca(2+)-dependent K(+) channels. Our data provide the first evidence that microglia express functional BK receptors and support the idea that microglia play an important role in CNS inflammatory responses.

Kinin receptors in cultured rat microglia.

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.

Impact of infralimbic inputs on intercalated amygdala neurons: A biophysical modeling study

Intercalated (ITC) amygdala neurons regulate fear expression by controlling impulse traffic between the input (basolateral amygdala; BLA) and output (central nucleus; Ce) stations of the amygdala for conditioned fear responses. Previously, stimulation of the infralimbic (IL) cortex was found to reduce fear expression and the responsiveness of Ce neurons to BLA inputs. These effects were hypothesized to result from the activation of ITC cells projecting to Ce. However, ITC cells inhibit each other, leading to the question of how IL inputs could overcome the inter-ITC inhibition to regulate the responses of Ce neurons to aversive conditioned stimuli (CSs). To investigate this, we first developed a compartmental model of a single ITC cell that could reproduce their bistable electroresponsive properties, as observed experimentally. Next, we generated an ITC network that implemented the experimentally observed short-term synaptic plasticity of inhibitory inter-ITC connections. Model experiments showed that strongly adaptive CS-related BLA inputs elicited persistent responses in ITC cells despite the presence of inhibitory interconnections. The sustained CS-evoked activity of ITC cells resulted from an unusual slowly deinactivating K(+) current. Finally, over a wide range of stimulation strengths, brief IL activation caused a marked increase in the firing rate of ITC neurons, leading to a persistent decrease in Ce output, despite inter-ITC inhibition. Simulations revealed that this effect depended on the bistable properties and synaptic heterogeneity of ITC neurons. These results support the notion that IL inputs are in a strategic position to control extinction of conditioned fear via the activation of ITC neurons.

Parkin potentiates ATP‐induced currents due to activation of P2X receptors in PC12 cells

Loss‐of‐function mutations of the parkin gene causes an autosomal recessive juvenile‐onset form of Parkinsons disease (AR‐JP). Parkin was shown to function as a RING‐type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin‐potentiated adenosine triphosphate (ATP)‐induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP‐induced inward currents were measured in mock‐, wild‐type or mutant (T415N)‐parkin‐transfected PC12 cells under the conventional whole‐cell patch clamp configuration. The amplitude of ATP‐induced currents was significantly greater in wild‐type parkin‐transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP‐dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin‐dependent protein kinase (CaMKII). ATP‐induced currents were also regulated by phosphatases and cyclin‐dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP‐regulated phosphoprotein (DARPP‐32), though the phosphorylation at Thr‐34 and Thr‐75 were unchanged or rather attenuated. We also tried to investigate the effect of α‐synuclein, a substrate of parkin and also forming Lysine 63‐linked multiubiquitin chains. Expression of α‐synuclein did not affect the amplitude of ATP‐induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity. J. Cell. Physiol. 209: 172–182, 2006.

Distinct preoptic-BST nuclei dissociate paternal and infanticidal behavior in mice.

Paternal behavior is not innate but arises through social experience. After mating and becoming fathers, male mice change their behavior toward pups from infanticide to paternal care. However, the precise brain areas and circuit mechanisms connecting these social behaviors are largely unknown. Here we demonstrated that the c‐Fos expression pattern in the four nuclei of the preoptic‐bed nuclei of stria terminalis (BST) region could robustly discriminate five kinds of previous social behavior of male mice (parenting, infanticide, mating, inter‐male aggression, solitary control). Specifically, neuronal activation in the central part of the medial preoptic area (cMPOA) and rhomboid nucleus of the BST (BSTrh) retroactively detected paternal and infanticidal motivation with more than 95% accuracy. Moreover, cMPOA lesions switched behavior in fathers from paternal to infanticidal, while BSTrh lesions inhibited infanticide in virgin males. The projections from cMPOA to BSTrh were largely GABAergic. Optogenetic or pharmacogenetic activation of cMPOA attenuated infanticide in virgin males. Taken together, this study identifies the preoptic‐BST nuclei underlying social motivations in male mice and reveals unexpected complexity in the circuit connecting these nuclei.