Hiroki Toyoda

Increased Anxiety-Like Behavior and Enhanced Synaptic Efficacy in the Amygdala of GluR5 Knockout Mice

GABAergic transmission in the amygdala modulates the expression of anxiety. Understanding the interplay between GABAergic transmission and excitatory circuits in the amygdala is, therefore, critical for understanding the neurobiological basis of anxiety. Here, we used a multi-disciplinary approach to demonstrate that GluR5-containing kainate receptors regulate local inhibitory circuits, modulate the excitatory transmission from the basolateral amygdala to the central amygdala, and control behavioral anxiety. Genetic deletion of GluR5 or local injection of a GluR5 antagonist into the basolateral amygdala increases anxiety-like behavior. Activation of GluR5 selectively depolarized inhibitory neurons, thereby increasing GABA release and contributing to tonic GABA current in the basolateral amygdala. The enhanced GABAergic transmission leads to reduced excitatory inputs in the central amygdala. Our results suggest that GluR5 is a key regulator of inhibitory circuits in the amygdala and highlight the potential use of GluR5-specific drugs in the treatment of pathological anxiety.

Genetic Evidence for Adenylyl Cyclase 1 as a Target for Preventing Neuronal Excitotoxicity Mediated by N-Methyl-D-aspartate Receptors

The excessive activation of N-methyl-d-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.

Neuromedin U Receptor 2-Deficient Mice Display Differential Responses in Sensory Perception, Stress, and Feeding

ABSTRACT Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMUs central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding.

Requirement of extracellular signal-regulated kinase/mitogen-activated protein kinase for long-term potentiation in adult mouse anterior cingulate cortex

Long-term potentiation (LTP) in the anterior cingulate cortex (ACC) is believed to be critical for higher brain functions including emotion, learning, memory and chronic pain. N-methyl-D-aspartate (NMDA) receptor-dependent LTP is well studied and is thought to be important for learning and memory in mammalian brains. As the downstream target of NMDA receptors, the extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade has been extensively studied for its involvement in synaptic plasticity, learning and memory in hippocampus. By contrast, the role of ERK in cingulate LTP has not been investigated. In this study, we examined whether LTP in ACC requires the activation of ERK. We found that P42/P44 MAPK inhibitors, PD98059 and U0126, suppressed the induction of cingulate LTP that was induced by presynaptic stimulation with postsynaptic depolarization (the pairing protocol). We also showed that cingulate LTP induced by two other different protocols was also blocked by PD98059. Moreover, we found that these two inhibitors had no effect on the maintenance of cingulate LTP. Inhibitors of c-Jun N-terminal kinase (JNK) and p38, other members of MAPK family, SP600125 and SB203850, suppressed the induction of cingulate LTP generated by the pairing protocol. Thus, our study suggests that the MAPK signaling pathway is involved in the induction of cingulate LTP and plays a critical role in physiological conditions.

Long-term depression requires postsynaptic AMPA GluR2 receptor in adult mouse cingulate cortex†

Synaptic long‐term depression (LTD) is thought to be important for various brain functions such as learning, memory, and development. Although anterior cingulated cortex (ACC) has been demonstrated to contribute to learning and memory, no studies has been reported about the synaptic mechanisms for cingulate LTD. Here, we used integrative genetic, pharmacological and electrophysiological approaches to demonstrate that AMPA GluR2, but not GluR3, subunit is critical for cingulate LTD. We found that LTD was absent in adult cingulate slices of GluR2 knockout mice. Furthermore, postsynaptic injections of peptides that inhibit AMPA GluR2‐PDZ interactions blocked the induction of LTD. To determine if the requirement for AMPA receptor‐PDZ interaction is time‐dependent, we injected the same inhibiting peptide into the postsynaptic cells 5 min after the induction of LTD. We found that LTD was not affected by the peptide, providing the first evidence that postsynaptic AMPA GluR2‐mediated depression occurs rapidly (within t = 5 min). Genetic deletion of GluR3 did not affect cingulate LTD. Our results provide the first study of cingulate LTD mechanism using whole‐cell patch‐clamp recording in adult cortical slices and demonstrate that postsynaptic AMPA GluR2 subunit is crucial for synaptic depression in the ACC of adult mice. J. Cell. Physiol. 211: 336–343, 2007.

Differential Columnar Processing in Local Circuits of Barrel and Insular Cortices

The columnar organization is most apparent in the whisker barrel cortex but seems less apparent in the gustatory insular cortex. We addressed here whether there are any differences between the two cortices in columnar information processing by comparing the spatiotemporal patterns of excitation spread in the two cortices using voltage-sensitive dye imaging. In contrast to the well known excitation spread in the horizontal direction in layer II/III induced in the barrel cortex by layer IV stimulation, the excitation caused in the insular cortex by stimulation of layer IV spread bidirectionally in the vertical direction into layers II/III and V/VI, displaying a columnar image pattern. Bicuculline or picrotoxin markedly extended the horizontal excitation spread in layer II/III in the barrel cortex, leading to a generation of excitation in the underlying layer V/VI, whereas those markedly increased the amplitude of optical responses throughout the whole column in the insular cortex, subsequently widening the columnar image pattern. Such synchronous activities as revealed by the horizontal and vertical excitation spreads were consistently induced in the barrel and insular cortices, respectively, even by stimulation of different layers with varying intensities. Thus, a unique functional column existed in the insular cortex, in which intracolumnar communication between the superficial and deep layers was prominent, and GABAA action is involved in the inhibition of the intracolumnar communication in contrast to its involvement in intercolumnar lateral inhibition in the barrel cortex. These results suggest that the columnar information processing may not be universal across the different cortical areas.

Presynaptic regulation of the inhibitory transmission by GluR5-containing kainate receptors in spinal substantia gelatinosa.

GluR5-containing kainate receptors (KARs) are known to be involved in nociceptive transmission. Our previous work has shown that the activation of presynaptic KARs regulates GABAergic and glycinergic synaptic transmission in cultured dorsal horn neurons. However, the role of GluR5-containing KARs in the modulation of inhibitory transmission in the spinal substantia gelatinosa (SG) in slices remains unknown. In the present study, pharmacological, electrophysiological and genetic methods were used to show that presynaptic GluR5 KARs are involved in the modulation of inhibitory transmission in the SG of spinal slices in vitro. The GluR5 selective agonist, ATPA, facilitated the frequency but not amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in SG neurons. ATPA increased sIPSC frequency in all neurons with different firing patterns as delayed, tonic, initial and single spike patterns. The frequency of either GABAergic or glycinergic sIPSCs was significantly increased by ATPA. ATPA could also induce inward currents in all SG neurons recorded. The frequency, but not amplitude, of action potential-independent miniature IPSCs (mIPSCs) was also facilitated by ATPA in a concentration-dependent manner. However, the effect of ATPA on the frequency of either sIPSCs or mIPSCs was abolished in GluR5-/- mice. Deletion of the GluR5 subunit gene had no effect on the frequency or amplitude of mIPSCs in SG neurons. However, GluR5 antagonist LY293558 reversibly inhibited sIPSC and mIPSC frequencies in spinal SG neurons. Taken together, these results suggest that GluR5 KARs, which may be located at presynaptic terminals, contribute to the modulation of inhibitory transmission in the SG. GluR5-containing KARs are thus important for spinal sensory transmission/modulation in the spinal cord.

Evidence for a role of CaMKIV in the development of opioid analgesic tolerance.

cAMP response‐element binding protein (CREB), a transcription factor involved in learning, memory and drug addiction, is phosphorylated by calcium–calmodulin‐dependent protein kinase IV (CaMKIV). Here, we show that CaMKIV‐knockout (KO) mice developed less analgesic tolerance after chronic morphine administration with no alteration in physical dependence or acute morphine‐induced analgesia. The increase in phosphorylated CREB expression observed in wild‐type mice after chronic morphine was absent in CaMKIV‐KO mice, while there was no difference in the expression or phosphorylation of the µ‐opioid receptor between groups. Morphine‐treated CaMKIV‐KO mice showed less G‐protein uncoupling from the µ‐opioid receptor than did wild‐type mice, while uncoupling was similar in control wild‐type and KO mice. In addition, morphine reduced inhibitory transmission to a greater degree in CaMKIV‐KO mice than in controls after chronic morphine exposure. Our results provide novel evidence for the role of CaMKIV in the development of opioid analgesic tolerance but not physical dependence.

cGMP activates a pH-sensitive leak K+ current in the presumed cholinergic neuron of basal forebrain.

In an earlier study, we demonstrated that nitric oxide (NO) causes the long-lasting membrane hyperpolarization in the presumed basal forebrain cholinergic (BFC) neurons by cGMP-PKG-dependent activation of leak K+ currents in slice preparations. In the present study, we investigated the ionic mechanisms underlying the long-lasting membrane hyperpolarization with special interest in the pH sensitivity because 8-Br-cGMP-induced K+ current displayed Goldman-Hodgkin-Katz rectification characteristic of TWIK-related acid-sensitive K+ (TASK) channels. When examined with the ramp command pulse depolarizing from -130 to -40 mV, the presumed BFC neurons displayed a pH-sensitive leak K+ current that was larger in response to pH decrease from 8.3 to 7.3 than in response to pH decrease from 7.3 to 6.3. This K+ current was similar to TASK1 current in its pH sensitivity, whereas it was highly sensitive to Ba(2+), unlike TASK1 current. The 8-Br-cGMP-induced K+ currents in the presumed BFC neurons were almost completely inhibited by lowering external pH to 6.3 as well as by bath application of 100 microM Ba(2+), consistent with the nature of the leak K+ current expressed in the presumed BFC neurons. After 8-Br-cGMP application, the K+ current obtained by pH decrease from 7.3 to 6.3 was larger than that obtained by pH decrease from pH 8.3 to 7.3, contrary to the case seen in the control condition. These observations strongly suggest that 8-Br-cGMP activates a pH- and Ba(2+)-sensitive leak K+ current expressed in the presumed BFC neurons by modulating its pH sensitivity.

Interaction between h-channels and glutamate receptor channels in the mesencephalic trigeminal neurons

To investigate the physiological roles of glutamate receptors (GluRs) in the mesencephalic trigeminal neurons, the current responses to puff application of glutamate (Glu) were recorded under the whole-cell voltage-clamp condition. When the peak amplitudes were plotted against the holding potentials, an N-shaped I–V relationship was obtained. At <−40 mV, the biphasic responses consisting of inward and outward components were observed. Glu-induced outward component was sensitive to ZD7288 and Cs+, indicating an involvement of h-current (Ih) in generating the outward current. Provided that GluR channels shared the microdomain with h-channels, it is possible that a transient increase in [Na]i in the microdomain by activation of GluR channels would cause a transient reduction of Ih due to the negative shift of its reversal potential, leading to a generation of the outward component of the biphasic response where the N-shaped I–V relationship can be seen. Thus, the biphasic response was seen at <−40 mV.