Discovery of a small-molecule inhibitor of the TRIP8b-HCN interaction with efficacy in neurons.

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b-HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b-HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b-HCN interaction.

Heterozygous GABAA receptor ß3 subunit N110D knock-in mice have epileptic spasms.

OBJECTIVE: Infantile spasms is an epileptic encephalopathy of childhood, and its pathophysiology is largely unknown. We generated a heterozygous knock-in mouse with the human infantile spasms-associated de novo mutation GABRB3 (c.A328G, p.N110D) to investigate its molecular mechanisms and to establish the Gabrb3+/N110D knock-in mouse as a model of infantile spasms syndrome. METHODS: We used electroencephalography (EEG) and video monitoring to characterize seizure types, and a suite of behavioral tests to identify neurological and behavioral impairment in Gabrb3+/N110D knock-in mice. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from layer V/VI pyramidal neurons in somatosensory cortex, and extracellular multi-unit recordings from the ventral basal nucleus of the thalamus in a horizontal thalamocortical slice were used to assess spontaneous thalamocortical oscillations. RESULTS: The infantile spasms-associated human de novo mutation GABRB3 (c.A328G, p.N110D) caused epileptic spasms early in development and multiple seizure types in adult Gabrb3+/N110D knock-in mice. Signs of neurological impairment, anxiety, hyperactivity, social impairment, and deficits in spatial learning and memory were also observed. Gabrb3+/N110D mice had reduced cortical mIPSCs and increased duration of spontaneous oscillatory firing in the somatosensory thalamocortical circuit. SIGNIFICANCE: The Gabrb3+/N110D knock-in mouse has epileptic spasms, seizures, and other neurological impairments that are consistent with infantile spasms syndrome in patients. Multiple seizure types and abnormal behaviors indicative of neurological impairment both early and late in development suggest that Gabrb3+/N110D mice can be used to study the pathophysiology of infantile spasms. Reduced cortical inhibition and increased duration of thalamocortical oscillatory firing suggest perturbations in thalamocortical circuits.

Prevention of premature death and seizures in a Depdc5 mouse epilepsy model through inhibition of mTORC1.

Mutations in DEP domain containing 5 (DEPDC5) are increasingly appreciated as one of the most common causes of inherited focal epilepsy. Epilepsies due to DEPDC5 mutations are often associated with brain malformations, tend to be drug-resistant, and have been linked to an increased risk of sudden unexplained death in epilepsy (SUDEP). Generation of epilepsy models to define mechanisms of epileptogenesis remains vital for future therapies. Here, we describe a novel mouse model of Depdc5 deficiency with a severe epilepsy phenotype, generated by conditional deletion of Depdc5 in dorsal telencephalic neuroprogenitor cells. In contrast to control and heterozygous mice, Depdc5-Emx1-Cre conditional knockout (CKO) mice demonstrated macrocephaly, spontaneous seizures and premature death. Consistent with increased mTORC1 activation, targeted neurons were enlarged and both neurons and astrocytes demonstrated increased S6 phosphorylation. Electrophysiologic characterization of miniature inhibitory post-synaptic currents in excitatory neurons was consistent with impaired post-synaptic response to GABAergic input, suggesting a potential mechanism for neuronal hyperexcitability. mTORC1 inhibition with rapamycin significantly improved survival of CKO animals and prevented observed seizures, including for up to 40 days following rapamycin withdrawal. These data not only support a primary role for mTORC1 hyperactivation in epilepsy following homozygous loss of Depdc5, but also suggest a developmental window for treatment which may have a durable benefit for some time even after withdrawal.

Dysregulation of GABAA Receptor-Mediated Neurotransmission during the Auditory Cortex Critical Period in the Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the leading monogenic form of intellectual disability and autism, with patients exhibiting numerous auditory-related phenotypes during their developmental period, including communication, language development, and auditory processing deficits. Despite FXS studies describing excitatory-inhibitory (E-I) imbalance in the auditory circuit and an impaired auditory critical period, evaluation of E-I circuitry maturation in the auditory cortex of FXS models remains limited. Here, we examined GABAA receptor (GABAAR)-mediated inhibitory synaptic transmission within the auditory cortex, characterizing normal intracortical circuit development patterns in wild-type (WT) mice and examining their dysregulation in developing Fmr1 knock-out (KO) mice. Electrophysiological recordings revealed gradual developmental shifts in WT L4-L2/3 connectivity, where circuit excitability significantly increased after critical period onset. KO mice exhibited accelerated developmental shifts related to aberrant GABAergic signaling. Specifically, Fmr1 KO L2/3 pyramidal neurons have enhanced developmental sensitivity to pharmacological GABAAR modulators, altered maturation of GABAAR voltage-dependent conductance, with additional presynaptic GABA release alterations. These differences are further accompanied by alterations in developmental long-term potentiation. Together, our results suggest that altered GABAergic signaling within developing Fmr1 KOs impairs the normal patterning of E-I circuit and synaptic plasticity maturation to contribute to the impaired auditory cortex critical period and functional auditory deficits in FXS.

Impaired State-Dependent Potentiation of GABAergic Synaptic Currents Triggers Seizures in a Genetic Generalized Epilepsy Model.

Epileptic activity in genetic generalized epilepsy (GGE) patients preferentially appears during sleep and its mechanism remains unknown. Here, we found that sleep-like slow-wave oscillations (0.5 Hz SWOs) potentiated excitatory and inhibitory synaptic currents in layer V cortical pyramidal neurons from wild-type (wt) mouse brain slices. In contrast, SWOs potentiated excitatory, but not inhibitory, currents in cortical neurons from a heterozygous (het) knock-in (KI) Gabrg2+Q/390X model of Dravet epilepsy syndrome. This created an imbalance between evoked excitatory and inhibitory currents to effectively prompt neuronal action potential firings. Similarly, physiologically similar up-/down-state induction (present during slow-wave sleep) in cortical neurons also potentiated excitatory synaptic currents within brain slices from wt and het KI mice. Moreover, this state-dependent potentiation of excitatory synaptic currents entailed some signaling pathways of homeostatic synaptic plasticity. Consequently, in het KI mice, in vivo SWO induction (using optogenetic methods) triggered generalized epileptic spike-wave discharges (SWDs), being accompanied by sudden immobility, facial myoclonus, and vibrissa twitching. In contrast, in wt littermates, SWO induction did not cause epileptic SWDs and motor behaviors. To our knowledge, this is the first mechanism to explain why epileptic SWDs preferentially happen during non rapid eye-movement sleep and quiet-wakefulness in human GGE patients.

Cortical activation in generalized seizures.

OBJECTIVE: Patients with generalized epilepsy exhibit different epileptiform events including asymptomatic interictal spikes (IS), absence seizures with spike-wave discharges (SWDs), and myoclonic seizures (MS). Our objective was to determine the spatiotemporal patterns of cortical activation in SWDs, IS, and MS in the Gabra1+/A322D juvenile myoclonic epilepsy mouse. METHODS: We fabricated affordable, flexible high-density electroencephalography (HdEEG) arrays and recorded spontaneous SWD, IS, and MS with video/HdEEG. We determined differences among the events in amplitude spectral density (ASD) in the δ/θ/α/ß/γ frequency bands at baseline (3.5-4.0 seconds before the first spike time, t0 ) and the prespike period (0.1-0.5 seconds before t0 ), and we elucidated the spatiotemporal activation during the t0 spike. RESULTS: All three events had an increase in ASD between baseline and prespike in at least one frequency band. During prespike, MS had the largest δ-band ASD, but SWD had the greatest α/ß/γ band ASD. For all three events, the ASD was largest in the anterior regions. The t0 spike voltage was also greatest in the anterior regions for all three events and IS and MS had larger voltages than SWD. From 7.5 to 17.5 msec after t0 , MS had greater voltage than IS and SWD, and maximal voltage was in the posterior parietal region. SIGNIFICANCE: Changes in spectral density from baseline to prespike indicate that none of these generalized events are instantaneous or entirely unpredictable. Prominent engagement of anterior cortical regions during prespike and at t0 suggest that common anterior neural circuits participate in each event. Differences in prespike ASD signify that although the events may engage similar brain regions, they may arise from distinct proictal states with different neuronal activity or connectivity. Prolonged activation of the posterior parietal area in MS suggests that posterior circuits contribute to the myoclonic jerk. Together, these findings identify brain regions and processes that could be specifically targeted for further recording and modulation.

Overexpressing wild-type γ2 subunits rescued the seizure phenotype in Gabrg2+/Q390X Dravet syndrome mice.

OBJECTIVE: The mutant γ-aminobutyric acid type A (GABAA ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABAA receptors, and affects trafficking of partnering α and ß subunits. Heterozygous Gabrg2+/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy. METHODS: We introduced the GABRG2 allele by crossing Gabrg2+/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2HA subunits, and compared GABAA receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording. RESULTS: Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and ß2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold. SIGNIFICANCE: Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.

Early-life seizures alter synaptic calcium-permeable AMPA receptor function and plasticity.

Calcium (Ca2+)-mediated4 signaling pathways are critical to synaptic plasticity. In adults, the NMDA glutamate receptor (NMDAR) represents a major route for activity-dependent synaptic Ca2+ entry. However, during neonatal development, when synaptic plasticity is particularly high, many AMPA glutamate receptors (AMPARs) are also permeable to Ca2+ (CP-AMPAR) due to low GluA2 subunit expression, providing an additional route for activity- and glutamate-dependent Ca2+ influx and subsequent signaling. Therefore, altered hippocampal Ca2+ signaling may represent an age-specific pathogenic mechanism. We thus aimed to assess Ca2+ responses 48h after hypoxia-induced neonatal seizures (HS) in postnatal day (P)10 rats, a post-seizure time point at which we previously reported LTP attenuation. We found that Ca2+ responses were higher in brain slices from post-HS rats than in controls and that this increase was CP-AMPAR-dependent. To determine whether synaptic CP-AMPAR expression was also altered post-HS, we assessed the expression of GluA2 at hippocampal synapses and the expression of long-term depression (LTD), which has been linked to the presence of synaptic GluA2. Here we report a decrease 48h after HS in synaptic GluA2 expression at synapses and LTD in hippocampal CA1. Given the potentially critical role of AMPAR trafficking in disease progression, we aimed to establish whether post-seizure in vivo AMPAR antagonist treatment prevented the enhanced Ca2+ responses, changes in GluA2 synaptic expression, and diminished LTD. We found that NBQX treatment prevents all three of these post-seizure consequences, further supporting a critical role for AMPARs as an age-specific therapeutic target.

The developmental evolution of the seizure phenotype and cortical inhibition in mouse models of juvenile myoclonic epilepsy.

The GABA(A) receptor (GABA(A)R) α1 subunit mutation, A322D, causes autosomal dominant juvenile myoclonic epilepsy (JME). Previous in vitro studies demonstrated that A322D elicits α1(A322D) protein degradation and that the residual mutant protein causes a dominant-negative effect on wild type GABA(A)Rs. Here, we determined the effects of heterozygous A322D knockin (Het(α1)AD) and deletion (Het(α1)KO) on seizures, GABA(A)R expression, and motor cortex (M1) miniature inhibitory postsynaptic currents (mIPSCs) at two developmental time-points, P35 and P120. Both Het(α1)AD and Het(α1)KO mice experience absence seizures at P35 that persist at P120, but have substantially more frequent spontaneous and evoked polyspike wave discharges and myoclonic seizures at P120. Both mutant mice have increased total and synaptic α3 subunit expression at both time-points and decreased α1 subunit expression at P35, but not P120. There are proportional reductions in α3, ß2, and γ2 subunit expression between P35 and P120 in wild type and mutant mice. In M1, mutants have decreased mIPSC peak amplitudes and prolonged decay constants compared with wild type, and the Het(α1)AD mice have reduced mIPSC frequency and smaller amplitudes than Het(α1)KO mice. Wild type and mutants exhibit proportional increases in mIPSC amplitudes between P35 and P120. We conclude that Het(α1)KO and Het(α1)AD mice model the JME subsyndrome, childhood absence epilepsy persisting and evolving into JME. Both mutants alter GABA(A)R composition and motor cortex physiology in a manner expected to increase neuronal synchrony and excitability to produce seizures. However, developmental changes in M1 GABA(A)Rs do not explain the worsened phenotype at P120 in mutant mice.

Altered intrathalamic GABAA neurotransmission in a mouse model of a human genetic absence epilepsy syndrome.

We previously demonstrated that heterozygous deletion of Gabra1, the mouse homolog of the human absence epilepsy gene that encodes the GABAA receptor (GABAAR) α1 subunit, causes absence seizures. We showed that cortex partially compensates for this deletion by increasing the cell surface expression of residual α1 subunit and by increasing α3 subunit expression. Absence seizures also involve two thalamic nuclei: the ventrobasal (VB) nucleus, which expresses only the α1 and α4 subtypes of GABAAR α subunits, and the reticular (nRT) nucleus, which expresses only the α3 subunit subtype. Here, we found that, unlike cortex, VB exhibited significantly reduced total and synaptic α1 subunit expression. In addition, heterozygous α1 subunit deletion substantially reduced miniature inhibitory postsynaptic current (mIPSC) peak amplitudes and frequency in VB. However, there was no change in the expression of the extrasynaptic α4 or δ subunits in VB and, unlike other models of absence epilepsy, no change in tonic GABAAR currents. Although heterozygous α1 subunit knockout increased α3 subunit expression in medial thalamic nuclei, it did not alter α3 subunit expression in nRT. However, it did enlarge the presynaptic vesicular inhibitory amino acid transporter puncta and lengthen the time constant of mIPSC decay in nRT. We conclude that increased tonic GABAA currents are not necessary for absence seizures. In addition, heterozygous loss of α1 subunit disinhibits VB by substantially reducing phasic GABAergic currents and surprisingly, it also increases nRT inhibition by prolonging phasic currents. The increased inhibition in nRT likely represents a partial compensation that helps reduce absence seizures.

Altered cortical GABAA receptor composition, physiology, and endocytosis in a mouse model of a human genetic absence epilepsy syndrome.

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Hetα1KO) of the human epilepsy gene, the GABAAR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Hetα1KO on the expression and physiology of GABAARs in the mouse cortex. We found that Hetα1KO caused modest reductions in the total and surface expression of the ß2 subunit but did not alter ß1 or ß3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetα1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Hetα1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3ßγ receptors. Patch clamp electrophysiology studies showed that Hetα1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetα1KO reduced base-line GABAAR endocytosis, an effect that probably contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetα1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission: 1) a modest reduction of GABAAR number and 2) a partial compensation with GABAAR isoforms that possess physiological properties different from those of the otherwise predominant α1ßγ GABAARs.

An Open-Source Mouse Chronic EEG Array System with High-Density MXene-Based Skull Surface Electrodes.

Electroencephalography (EEG) is an indispensable tool in epilepsy, sleep, and behavioral research. In rodents, EEG recordings are typically performed with metal electrodes that traverse the skull into the epidural space. In addition to requiring major surgery, intracranial EEG is difficult to perform for more than a few electrodes, is time-intensive, and confounds experiments studying traumatic brain injury. Here, we describe an open-source cost-effective refinement of this technique for chronic mouse EEG recording. Our alternative two-channel (EEG2) and sixteen-channel high-density EEG (HdEEG) arrays use electrodes made of the novel, flexible 2D nanomaterial titanium carbide (Ti3C2T x ) MXene. The MXene electrodes are placed on the surface of the intact skull and establish an electrical connection without conductive gel or paste. Fabrication and implantation times of MXene EEG electrodes are significantly shorter than the standard approach, and recorded resting baseline and epileptiform EEG waveforms are similar to those obtained with traditional epidural electrodes. Applying HdEEG to a mild traumatic brain injury (mTBI) model in mice of both sexes revealed that mTBI significantly increased spike-wave discharge (SWD) preictal network connectivity with frequencies of interest in the ß-spectral band (12-30 Hz). These findings indicate that the fabrication of MXene electrode arrays is a cost-effective, efficient technology for multichannel EEG recording in mice that obviates the need for skull-penetrating surgery. Moreover, increased preictal ß-frequency network connectivity may contribute to the development of early post-mTBI SWDs.

Glutamate receptor 1 phosphorylation at serine 831 and 845 modulates seizure susceptibility and hippocampal hyperexcitability after early life seizures.

Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We showed previously that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of serine-831 (S831) and Serine 845 (S845) sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced posttranslational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild-type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation [GluR1 double-phosphomutant (GluR1 DPM) mice]. Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex after a single episode of pentyleneterazol-induced seizures in postnatal day 7 (P7) wild-type mouse pups and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild-type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex and were associated with enhanced seizure susceptibility to later-life kainic-acid-induced seizures. In contrast, later-life seizure susceptibility after hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for posttranslational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue.

Sleep slow-wave oscillations trigger seizures in a genetic epilepsy model of Dravet syndrome.

Sleep is the preferential period when epileptic spike-wave discharges appear in human epileptic patients, including genetic epileptic seizures such as Dravet syndrome with multiple mutations including SCN1A mutation and GABAA receptor γ2 subunit Gabrg2Q390X mutation in patients, which presents more severe epileptic symptoms in female patients than male patients. However, the seizure onset mechanism during sleep still remains unknown. Our previous work has shown that the sleep-like state-dependent homeostatic synaptic potentiation can trigger epileptic spike-wave discharges in one transgenic heterozygous Gabrg2+/Q390X knock-in mouse model.1 Here, using this heterozygous knock-in mouse model, we hypothesized that slow-wave oscillations themselves in vivo could trigger epileptic seizures. We found that epileptic spike-wave discharges in heterozygous Gabrg2+/Q390X knock-in mice exhibited preferential incidence during non-rapid eye movement sleep period, accompanied by motor immobility/facial myoclonus/vibrissal twitching and more frequent spike-wave discharge incidence appeared in female heterozygous knock-in mice than male heterozygous knock-in mice. Optogenetically induced slow-wave oscillations in vivo significantly increased epileptic spike-wave discharge incidence in heterozygous Gabrg2+/Q390X knock-in mice with longer duration of non-rapid eye movement sleep or quiet-wakeful states. Furthermore, suppression of slow-wave oscillation-related homeostatic synaptic potentiation by 4-(diethylamino)-benzaldehyde injection (i.p.) greatly attenuated spike-wave discharge incidence in heterozygous knock-in mice, suggesting that slow-wave oscillations in vivo did trigger seizure activity in heterozygous knock-in mice. Meanwhile, sleep spindle generation in wild-type littermates and heterozygous Gabrg2+/Q390X knock-in mice involved the slow-wave oscillation-related homeostatic synaptic potentiation that also contributed to epileptic spike-wave discharge generation in heterozygous Gabrg2+/Q390X knock-in mice. In addition, EEG spectral power of delta frequency (0.1-4 Hz) during non-rapid eye movement sleep was significantly larger in female heterozygous Gabrg2+/Q390X knock-in mice than that in male heterozygous Gabrg2+/Q390X knock-in mice, which likely contributes to the gender difference in seizure incidence during non-rapid eye movement sleep/quiet-wake states of human patients. Overall, all these results indicate that slow-wave oscillations in vivo trigger the seizure onset in heterozygous Gabrg2+/Q390X knock-in mice, preferentially during non-rapid eye movement sleep period and likely generate the sex difference in seizure incidence between male and female heterozygous Gabrg2+/Q390X knock-in mice.

Hypoxia-induced neonatal seizures diminish silent synapses and long-term potentiation in hippocampal CA1 neurons.

Neonatal seizures can lead to epilepsy and long-term cognitive deficits into adulthood. Using a rodent model of the most common form of human neonatal seizures, hypoxia-induced seizures (HS), we aimed to determine whether these seizures modify long-term potentiation (LTP) and silent NMDAR-only synapses in hippocampal CA1. At 48-72 h after HS, electrophysiology and immunofluorescent confocal microscopy revealed a significant decrease in the incidence of silent synapses, and an increase in AMPARs at the synapses. Coincident with this decrease in silent synapses, there was an attenuation of LTP elicited by either tetanic stimulation of Schaffer collaterals or a pairing protocol, and persistent attenuation of LTP in slices removed in later adulthood after P10 HS. Furthermore, postseizure treatment in vivo with the AMPAR antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX) protected against the HS-induced depletion of silent synapses and preserved LTP. Thus, this study demonstrates a novel mechanism by which early life seizures could impair synaptic plasticity, suggesting a potential target for therapeutic strategies to prevent long-term cognitive deficits.

Artificial sleep-like up/down-states induce synaptic plasticity in cortical neurons from mouse brain slices.

During non-rapid eye movement (NREM) sleep, cortical neuron activity alternates between a depolarized (firing, up-state) and a hyperpolarized state (down-state) coinciding with delta electroencephalogram (EEG) slow-wave oscillation (SWO, 0. 5-4 Hz) in vivo. Recently, we have found that artificial sleep-like up/down-states can potentiate synaptic strength in layer V cortical neurons ex vivo. Using mouse coronal brain slices, whole cell voltage-clamp recordings were made from layer V cortical pyramidal neurons to record spontaneous excitatory synaptic currents (sEPSCs) and inhibitory synaptic currents (sIPSCs). Artificial sleep-like up/down-states (as SWOs, 0.5 Hz, 10 min, current clamp mode) were induced by injecting sinusoidal currents into layer V cortical neurons. Baseline pre-SWO recordings were recorded for 5 min and post-SWO recordings for at least 25-30 min. Compared to pre-SWO sEPSCs or sIPSCs, post-SWO sEPSCs or sIPSCs in layer V cortical neurons exhibited significantly larger amplitudes and a higher frequency for 30 min. This finding suggests that both sEPSCs and sIPSCs could be potentiated in layer V cortical neurons by the low-level activity of SWOs, and sEPSCs and sIPSCs maintained a balance in layer V cortical neurons during pre- and post-SWO periods. Overall, this study presents an ex vivo method to show SWO's ability to induce synaptic plasticity in layer V cortical neurons, which may underlie sleep-related synaptic potentiation for sleep-related memory consolidation in vivo.

Hippocampal cAMP regulates HCN channel function on two time scales with differential effects on animal behavior.

Hyperpolarization-activated cyclic nucleotide­gated (HCN) channels regulate neuronal excitability and represent a possible therapeutic target for major depressive disorder (MDD). These channels are regulated by intracellular cyclic adenosine monophosphate (cAMP). However, the relationship between cAMP signaling and the influence of HCN channels on behavior remains opaque. In this study, we investigated the role of hippocampal cAMP signaling on behavior using chemogenetic technology in mice. Acutely increasing cAMP limited spatial memory and motivated behavior by increasing HCN function. However, chronically elevated cAMP limited surface trafficking of HCN channels by disrupting the interaction between HCN and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), an auxiliary subunit. Chronically increased cAMP in the dorsal hippocampus was also sufficient to rescue cognitive deficits induced by chronic stress in mice. These results reveal a behaviorally relevant form of regulation of HCN channel surface expression that has potential as a therapeutic target for cognitive deficits related to chronic stress.

Modeling intrahippocampal effects of anterior hippocampal hyperactivity relevant to schizophrenia using chemogenetic excitation of long axis-projecting mossy cells in the mouse dentate gyrus.

BACKGROUND: The anterior hippocampus of individuals with early psychosis or schizophrenia is hyperactive, as is the ventral hippocampus in many rodent models for schizophrenia risk. Mossy cells (MCs) of the ventral dentate gyrus (DG) densely project in the hippocampal long axis, targeting both dorsal DG granule cells and inhibitory interneurons. Mossy cells are responsive to stimulation throughout hippocampal subfields, and thus may be suited to detect hyperactivity in areas where it originates such as CA1. Here we tested the hypothesis that hyperactivation of ventral MCs activates dorsal DG granule cells to influence dorsal hippocampal function. METHODS: In CD-1 mice, we targeted dorsal DG-projecting ventral MCs using an adeno-associated virus intersectional strategy. In vivo fiber photometry recording of ventral MCs was performed during exploratory behaviors. We used excitatory chemogenetic constructs to test the effects of ventral MC hyperactivation on long-term spatial memory during an object location memory task. RESULTS: Photometry revealed ventral MCs were activated during exploratory rearing. Ventral MCs made functional monosynaptic inputs to dorsal DG granule cells, and chemogenetic activation of ventral MCs modestly increased activity of dorsal DG granule cells measured by c-Fos. Finally, chemogenetic activation of ventral MCs during the training phase of an object location memory task impaired test performance 24 hours later, without effects on locomotion or object exploration. CONCLUSIONS: These data suggest that ventral MC activation can directly excite dorsal granule cells and interfere with dorsal DG function, supporting future study of their in vivo activity in animal models for schizophrenia featuring ventral hyperactivity.

Altered development of glutamatergic synapses in layer V pyramidal neurons in NR3A knockout mice.

Expression of the NMDA receptor (NMDAR) subunit NR3A reaches its highest level in layer V of the developing rodent cortex during the second postnatal week, a peak period of synaptogenesis. Incorporation of NR3A leads to the formation of non-canonical, Mg2+-insensitive NMDARs, but it is not known whether they participate in synaptic transmission and maturation. Here we show that in the second postnatal week, layer V pyramidal neurons in the somatosensory cortex of wild type (WT) mice exhibited evoked excitatory postsynaptic currents (eEPSCs) with 3- to 6-fold lower Mg2+ sensitivity than NR3A knockout (KO) mice and their reversal potential was approximately 2 mV more negative compared to KO mice consistent with decreased P(Ca) of NMDARs. Surprisingly, ablation of NR3A also led to a 20-fold reduction of the ratio of AMPAR- to NMDAR-mediated eEPSC amplitudes in KO mice. Insertion of AMPARs at the synapses of layer V pyramidal neurons appears to be facilitated by the expression of Mg2+-insensitive NMDARs. The data indicate that NR3A plays a significant role in the development of excitatory synapses in layer V of the developing neocortex.