Deficiency of the CYLD Impairs Fear Memory of Mice and Disrupts Neuronal Activity and Synaptic Transmission in the Basolateral Amygdala

Fear learning and memory are crucial for animal survival. Abnormal fear memory is a hallmark of many neuropsychiatric disorders. Appropriate neuronal activation and excitability in the basolateral amygdala (BLA) are necessary for the formation of fear memory. The gene cylindromatosis (Cyld), which encodes a lysine-63 deubiquitinase, is expressed in several brain regions including the amygdala. The functions of the cylindromatosis protein (CYLD) in the regulation of the neuronal activity, neural circuits and fear memory, remain largely unknown, however. Here, we report that Cyld knockout impairs amygdala-dependent tone-cued fear memory. The number of c-Fos+ neurons responding to the tone-cued fear test was reduced in the BLA of Cyld–/– mice, suggesting that the absence of CYLD causes aberrant neuronal activation. We found that this aberrant neuronal activation in the BLA of Cyld–/– mice may relate to the decreased excitability of principal neurons. Another possibility of aberrant neuronal activation could be the impaired excitatory synaptic transmission in the BLA of Cyld–/– mice. Specifically, both the frequency of spontaneous excitatory postsynaptic currents and the amplitude of miniature excitatory postsynaptic currents in BLA principal neurons were decreased. In addition, Cyld mutation caused an increase in both the frequency of miniature inhibitory postsynaptic currents in principal neurons and the number of parvalbumin+ interneurons, consistent with excessive local circuit inhibition in the BLA of Cyld–/– mice. Taken together, these results suggest that CYLD deficiency disrupts the neuronal activity and synaptic transmission in the BLA of mice which may contribute to the impaired fear memory observed in Cyld–/– mice.

Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 μM. Second, they responded to 5-HT (50 μM) with a response ratio similar to that for NA, but prazosin (10 μM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 μM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Environmental enrichment mitigates the long-lasting sequelae of perinatal fentanyl exposure

The opioid epidemic is a rapidly evolving societal issue that stems from the abuse of prescription and illicit opioids, including increasing use of synthetic opioids like fentanyl. Fentanyl use among women has increased substantially in the last decade, leading to a 40-fold increase in the number of perinatally-exposed infants. This exposure can result in neuropsychiatric abnormalities that persist into adolescence and, in some cases, adulthood. We previously developed a preclinical model to establish the consequences of perinatal fentanyl exposure and identified a pattern of synaptic pathophysiology that involves lasting impairments in primary somatosensory (S1) circuit function and behavior. Here, we ask if these long-lasting effects can be restored by a non-invasive intervention. We demonstrate that developmental exposure to environmental enrichment ameliorates many of fentanyl's deleterious behavioral effects, including hyperactivity, enhanced sensitivity to anxiogenic environments, and sensory maladaptation. As an extension of our past work, we found that perinatal fentanyl alters the frequency of miniature excitatory postsynaptic currents and impairs long-term potentiation in S1 layer 2/3 neurons. These deficits in synaptic function were restored by environmental enrichment. Environmental enrichment also affected neurons in control mice, reducing long-term potentiation and depression, and increasing frequency of miniature excitatory postsynaptic currents. These results demonstrate that the lasting somatosensory-related effects of fentanyl can be ameliorated with a non-invasive intervention introduced during early development. These findings can inform ongoing efforts to develop actionable steps toward mitigating the consequences of opioid abuse among pregnant women.

Selective filtering of excitatory inputs to nucleus accumbens by dopamine and serotonin

The detailed mechanisms by which dopamine (DA) and serotonin (5-HT) act in the nucleus accumbens (NAc) to influence motivated behaviors in distinct ways remain largely unknown. Here, we examined whether DA and 5-HT selectively modulate excitatory synaptic transmission in NAc medium spiny neurons in an input-specific manner. DA reduced excitatory postsynaptic currents (EPSCs) generated by paraventricular thalamus (PVT) inputs but not by ventral hippocampus (vHip), basolateral amygdala (BLA), or medial prefrontal cortex (mPFC) inputs. In contrast, 5-HT reduced EPSCs generated by inputs from all areas except the mPFC. Release of endogenous DA and 5-HT by methamphetamine (METH) and (±)3,4-methylenedioxymethamphetamine (MDMA), respectively, recapitulated these input-specific synaptic effects. Optogenetic inhibition of PVT inputs enhanced cocaine-conditioned place preference, whereas mPFC input inhibition reduced the enhancement of sociability elicited by MDMA. These findings suggest that the distinct, input-specific filtering of excitatory inputs in the NAc by DA and 5-HT contribute to their discrete behavioral effects.

Recordings from neuron–HEK cell cocultures reveal the determinants of miniature excitatory postsynaptic currents

Spontaneous exocytosis of single synaptic vesicles generates miniature synaptic currents, which provide a window into the dynamic control of synaptic transmission. To resolve the impact of different factors on the dynamics and variability of synaptic transmission, we recorded miniature excitatory postsynaptic currents (mEPSCs) from cocultures of mouse hippocampal neurons with HEK cells expressing the postsynaptic proteins GluA2, neuroligin 1, PSD-95, and stargazin. Synapses between neurons and these heterologous cells have a molecularly defined postsynaptic apparatus, while the compact morphology of HEK cells eliminates the distorting effect of dendritic filtering. HEK cells in coculture produced mEPSCs with a higher frequency, larger amplitude, and more rapid rise and decay than neurons from the same culture. However, mEPSC area indicated that nerve terminals in synapses with both neurons and HEK cells release similar populations of vesicles. Modulation by the glutamate receptor ligand aniracetam revealed receptor contributions to mEPSC shape. Dendritic cable effects account for the slower mEPSC rise in neurons, whereas the slower decay also depends on other factors. Lastly, expression of synaptobrevin transmembrane domain mutants in neurons slowed the rise of HEK cell mEPSCs, thus revealing the impact of synaptic fusion pores. In summary, we show that cocultures of neurons with heterologous cells provide a geometrically simplified and molecularly defined system to investigate the time course of synaptic transmission and to resolve the contribution of vesicles, fusion pores, dendrites, and receptors to this process.

Context-dependent plasticity of adult-born neurons regulated by cortical feedback

In a complex and dynamic environment, the brain flexibly adjusts its circuits to preferentially process behaviorally relevant information. Here, we investigated how the olfactory bulb copes with this demand by examining the plasticity of adult-born granule cells (abGCs). We found that learning of olfactory discrimination elevates odor responses of young abGCs and increases their apical dendritic spines. This plasticity did not occur in abGCs during passive odor experience nor in resident granule cells (rGCs) during learning. Furthermore, we found that feedback projections from the piriform cortex show elevated activity during learning, and activating piriform feedback elicited stronger excitatory postsynaptic currents in abGCs than rGCs. Inactivation of piriform feedback blocked abGC plasticity during learning, and activation of piriform feedback during passive experience induced learning-like plasticity of abGCs. Our work describes a neural circuit mechanism that uses adult neurogenesis to update a sensory circuit to flexibly adapt to new behavioral demands.

Cortical potentiation induced by calcitonin gene-related peptide (CGRP) in the insular cortex of adult mice

Recent studies demonstrate that calcitonin gene-related peptide (CGRP) plays critical roles in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas that are critical for pain perception including the anterior cingulate cortex (ACC) and insular cortex (IC). Recent studies reported that CGRP enhanced excitatory transmission in the ACC. However, little is known about the possible effect of CGRP on excitatory transmission in the IC. In the present study, we investigated the role of CGRP on synaptic transmission in the IC slices of adult male mice. Bath application of CGRP produced dose-dependent potentiation of evoked excitatory postsynaptic currents (eEPSCs). This potentiation was NMDA receptor (NMDAR) independent. After application of CGRP1 receptor antagonist CGRP8-37 or BIBN 4096, CGRP produced potentiation was significantly reduced. Paired-pulse facilitation was significantly decreased by CGRP, suggesting possible presynaptic mechanisms. Consistently, bath application of CGRP significantly increased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. Finally, adenylyl cyclase subtype 1 (AC1) and protein kinase A (PKA) are critical for CGRP-produced potentiation, since both selective AC1 inhibitor NB001 and the PKA inhibitor KT5720 completely blocked the potentiation. Our results provide direct evidence that CGRP contributes to synaptic potentiation in the IC, and the AC1 inhibitor NB001 may be beneficial for the treatment of migraine in the future.

Maturation of NMDA receptor-mediated spontaneous postsynaptic currents in the rat locus coeruleus neurons

IntroductionDuring mammalian brain development, neural activity leads to maturation of glutamatergic innervations to locus coeruleus. In this study, fast excitatory postsynaptic currents mediated by N-methyl-d-aspartate (NMDA) receptors were evaluated to investigate the maturation of excitatory postsynaptic currents in locus coeruleus (LC) neurons.MethodsNMDA receptor-mediated synaptic currents in LC neurons were evaluated using whole-cell voltage-clamp recording during the primary postnatal weeks. This technique was used to calculate the optimum holding potential for NMDA receptor-mediated currents and the best frequency for detecting spontaneous excitatory postsynaptic currents (sEPSC).ResultsThe optimum holding potential for detecting NMDA receptor-mediated currents was + 40 to + 50 mV in LC neurons. The frequency, amplitude, rise time, and decay time constant of synaptic responses depended on the age of the animal and increased during postnatal maturation.ConclusionThese findings suggest that most nascent glutamatergic synapses express functional NMDA receptors in the postnatal coerulear neurons, and that the activities of the neurons in this region demonstrate an age-dependent variation.

Cortical potentiation induced by calcitonin gene-related peptide (CGRP) in the insular cortex of adult mice

Recent studies demonstrate that calcitonin gene-related peptide (CGRP) plays critical roles in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas that are critical for pain perception including the anterior cingulate cortex (ACC) and insular cortex (IC). Recent studies reported that CGRP enhanced excitatory transmission in the ACC. However, little is known about the possible effect of CGRP on excitatory transmission in the IC. In the present study, we investigated the role of CGRP on synaptic transmission in the IC slices of adult male mice. Bath application of CGRP produced dose-dependent potentiation of evoked excitatory postsynaptic currents (eEPSCs). This potentiation was NMDA receptor (NMDAR) independent. After application of CGRP1 receptor antagonist CGRP8-37 or BIBN 4096, CGRP produced potentiation was significantly reduced. Paired-pulse facilitation was significantly decreased by CGRP, suggesting possible presynaptic mechanisms. Consistently, bath application of CGRP significantly increased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. Finally, adenylyl cyclase subtype 1 (AC1) and protein kinase A (PKA) are critical for CGRP-produced potentiation, since both selective AC1 inhibitor NB001 and the PKA inhibitor KT5720 completely blocked the potentiation. Our results provide direct evidence that CGRP contributes to synaptic potentiation in the IC, and the AC1 inhibitor NB001 may be beneficial for the treatment of migraine in the future.