Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology.

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.

Identification of Target Genes of Antiarrhythmic Traditional Chinese Medicine Wenxin Keli.

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.

Sleep Regulation by Neurotensinergic Neurons in a Thalamo-Amygdala Circuit.

A crucial step in understanding the sleep-control mechanism is to identify sleep neurons. Through systematic anatomical screening followed by functional testing, we identified two sleep-promoting neuronal populations along a thalamo-amygdala pathway, both expressing neurotensin (NTS). Rabies-mediated monosynaptic retrograde tracing identified the central nucleus of amygdala (CeA) as a major source of GABAergic inputs to multiple wake-promoting populations; gene profiling revealed NTS as a prominent marker for these CeA neurons. Optogenetic activation and inactivation of NTS-expressing CeA neurons promoted and suppressed non-REM (NREM) sleep, respectively, and optrode recording showed they are sleep active. Further tracing showed that CeA GABAergic NTS neurons are innervated by glutamatergic NTS neurons in a posterior thalamic region, which also promote NREM sleep. CRISPR/Cas9-mediated NTS knockdown in either the thalamic or CeA neurons greatly reduced their sleep-promoting effect. These results reveal a novel thalamo-amygdala circuit for sleep generation in which NTS signaling is essential for both the upstream glutamatergic and downstream GABAergic neurons.

Modulation of thalamocortical oscillations by TRIP8b, an auxiliary subunit for HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I h, in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b-/-). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K+ current, I A, in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.

Social Control of Hypothalamus-Mediated Male Aggression.

How environmental and physiological signals interact to influence neural circuits underlying developmentally programmed social interactions such as male territorial aggression is poorly understood. We have tested the influence of sensory cues, social context, and sex hormones on progesterone receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) that are critical for male territorial aggression. We find that these neurons can drive aggressive displays in solitary males independent of pheromonal input, gonadal hormones, opponents, or social context. By contrast, these neurons cannot elicit aggression in socially housed males that intrude in another male's territory unless their pheromone-sensing is disabled. This modulation of aggression cannot be accounted for by linear integration of environmental and physiological signals. Together, our studies suggest that fundamentally non-linear computations enable social context to exert a dominant influence on developmentally hard-wired hypothalamus-mediated male territorial aggression.

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 febrile seizures worsen adult phenotypes in Scn1a mutants.

Mutations in the voltage-gated sodium channel (VGSC) gene SCN1A, encoding the Nav1.1 channel, are responsible for a number of epilepsy disorders including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). Patients with SCN1A mutations often experience prolonged early-life febrile seizures (FSs), raising the possibility that these events may influence epileptogenesis and lead to more severe adult phenotypes. To test this hypothesis, we subjected 21-23-day-old mice expressing the human SCN1A GEFS+ mutation R1648H to prolonged hyperthermia, and then examined seizure and behavioral phenotypes during adulthood. We found that early-life FSs resulted in lower latencies to induced seizures, increased severity of spontaneous seizures, hyperactivity, and impairments in social behavior and recognition memory during adulthood. Biophysical analysis of brain slice preparations revealed an increase in epileptiform activity in CA3 pyramidal neurons along with increased action potential firing, providing a mechanistic basis for the observed worsening of adult phenotypes. These findings demonstrate the long-term negative impact of early-life FSs on disease outcomes. This has important implications for the clinical management of this patient population and highlights the need for therapeutic interventions that could ameliorate disease progression.

Selective antagonism of TRPA1 produces limited efficacy in models of inflammatory- and neuropathic-induced mechanical hypersensitivity in rats.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund's adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.

Developmental Switch in Spike Timing-Dependent Plasticity and Cannabinoid-Dependent Reorganization of the Thalamocortical Projection in the Barrel Cortex.

UNLABELLED: The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Δ(9)-tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs. SIGNIFICANCE STATEMENT: It has been shown that neural activity is required for initial synapse formation of thalamocortical axons with cortical cells, but precisely what sort of activities in presynaptic and postsynaptic cells are required is not yet clear. In addition, how activity is further translated into structural changes is unclear. We show here that the period during which spike timing-dependent long-term potentiation and depression (t-LTP, t-LTD) can be induced closely matches the time course of synapse formation and retraction, respectively, at the thalamocortical synapse. Moreover, administration of cannabinoid agonists, which mimic t-LTD, caused TCA retraction, suggesting that cannabinoids translate physiological changes into morphological consequences.

Thalamocortical Connections Drive Intracortical Activation of Functional Columns in the Mislaminated Reeler Somatosensory Cortex.

Neuronal wiring is key to proper neural information processing. Tactile information from the rodent's whiskers reaches the cortex via distinct anatomical pathways. The lemniscal pathway relays whisking and touch information from the ventral posteromedial thalamic nucleus to layer IV of the primary somatosensory "barrel" cortex. The disorganized neocortex of the reeler mouse is a model system that should severely compromise the ingrowth of thalamocortical axons (TCAs) into the cortex. Moreover, it could disrupt intracortical wiring. We found that neuronal intermingling within the reeler barrel cortex substantially exceeded previous descriptions, leading to the loss of layers. However, viral tracing revealed that TCAs still specifically targeted transgenically labeled spiny layer IV neurons. Slice electrophysiology and optogenetics proved that these connections represent functional synapses. In addition, we assessed intracortical activation via immediate-early-gene expression resulting from a behavioral exploration task. The cellular composition of activated neuronal ensembles suggests extensive similarities in intracolumnar information processing in the wild-type and reeler brains. We conclude that extensive ectopic positioning of neuronal partners can be compensated for by cell-autonomous mechanisms that allow for the establishment of proper connectivity. Thus, genetic neuronal fate seems to be of greater importance for correct cortical wiring than radial neuronal position.

Docosahexaenoic acid inhibits mechanical allodynia and thermal hyperalgesia in diabetic rats by decreasing the excitability of DRG neurons.

Diabetes mellitus is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. One of the most common complications of early-onset diabetes mellitus is peripheral diabetic neuropathy, which is manifested either by loss of nociception or by allodynia and hyperalgesia. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown the potential of anti-inflammation and modulating neuron excitability. The present study investigated the effects of docosahexaenoic acid (DHA) on the excitability of dorsal root ganglion (DRG) neurons in streptozotocin (STZ)-induced diabetes rats. The effects of DHA on the allodynia and hyperalgesia of diabetic rats were also evaluated. Dietary DHA supplementation effectively attenuated both allodynia and hyperalgesia induced by STZ injection. DHA supplementation decreased the excitability of DRG neurons by decreasing the sodium currents and increasing potassium currents, which may contribute to the effect of alleviating allodynia and hyperalgesia in diabetic rats. The results suggested that DHA might be useful as an adjuvant therapy for the prevention and treatment of painful diabetic neuropathy.

Activation of peripheral KCNQ channels relieves gout pain.

Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate-induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout.

Functional and structural deficits of the dentate gyrus network coincide with emerging spontaneous seizures in an Scn1a mutant Dravet Syndrome model during development.

Dravet syndrome (DS) is characterized by severe infant-onset myoclonic epilepsy along with delayed psychomotor development and heightened premature mortality. A primary monogenic cause is mutation of the SCN1A gene, which encodes the voltage-gated sodium channel subunit Nav1.1. The nature and timing of changes caused by SCN1A mutation in the hippocampal dentate gyrus (DG) network, a core area for gating major excitatory input to hippocampus and a classic epileptogenic zone, are not well known. In particularly, it is still not clear whether the developmental deficit of this epileptogenic neural network temporally matches with the progress of seizure development. Here, we investigated the emerging functional and structural deficits of the DG network in a novel mouse model (Scn1a(E1099X/+)) that mimics the genetic deficit of human DS. Scn1a(E1099X/+) (Het) mice, similarly to human DS patients, exhibited early spontaneous seizures and were more susceptible to hyperthermia-induced seizures starting at postnatal week (PW) 3, with seizures peaking at PW4. During the same period, the Het DG exhibited a greater reduction of Nav1.1-expressing GABAergic neurons compared to other hippocampal areas. Het DG GABAergic neurons showed altered action potential kinetics, reduced excitability, and generated fewer spontaneous inhibitory inputs into DG granule cells. The effect of reduced inhibitory input to DG granule cells was exacerbated by heightened spontaneous excitatory transmission and elevated excitatory release probability in these cells. In addition to electrophysiological deficit, we observed emerging morphological abnormalities of DG granule cells. Het granule cells exhibited progressively reduced dendritic arborization and excessive spines, which coincided with imbalanced network activity and the developmental onset of spontaneous seizures. Taken together, our results establish the existence of significant structural and functional developmental deficits of the DG network and the temporal correlation between emergence of these deficits and the onset of seizures in Het animals. Most importantly, our results uncover the developmental deficits of neural connectivity in Het mice. Such structural abnormalities likely further exacerbate network instability and compromise higher-order cognitive processing later in development, and thus highlight the multifaceted impacts of Scn1a deficiency on neural development.

Neuronal activity of the prefrontal cortex is reduced in rats selectively bred for deficient sensorimotor gating.

Rats selectively bred for deficient prepulse inhibition (PPI), an operant measure of sensorimotor gating in which a weak prepulse stimulus attenuates the response to a subsequent startling stimulus, may be used to study certain pathophysiological mechanisms and therapeutic strategies for neuropsychiatric disorders with abnormalities in information processing, such as schizophrenia and Tourette's syndrome (TS). Little is known about neuronal activity in the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAC), which are involved in the modulation of PPI. Here, we examined neuronal activity in these structures, and also in the entopeduncular nucleus (EPN), since lesions of this region alleviate the PPI deficit. Male rats with breeding-induced high and low expression of PPI (n=7, each) were anesthetized with urethane (1.4 mg/kg). Single-unit activity and local field potentials were recorded in the mPFC, the NAC and in the EPN. In the mPFC discharge rate, measures of irregularity and burst activity were significantly reduced in PPI low compared to PPI high rats (P<0.05), while analysis in the NAC showed approximately inverse behavior. In the EPN no difference between groups was found. Additionally, the oscillatory theta band activity (4-8 Hz) was enhanced and the beta band (13-30 Hz) and gamma band (30-100 Hz) activity was reduced in the NAC in PPI low rats. Reduced neuronal activity in the mPFC and enhanced activity in the NAC of PPI low rats, together with altered oscillatory behavior are clearly associated with reduced PPI. PPI low rats may thus be used to study the pathophysiology and therapeutic strategies for neuropsychiatric disorders accompanied by deficient sensorimotor gating.

Genetic background modulates impaired excitability of inhibitory neurons in a mouse model of Dravet syndrome.

Dominant loss-of-function mutations in voltage-gated sodium channel NaV1.1 cause Dravet Syndrome, an intractable childhood-onset epilepsy. NaV1.1(+/-) Dravet Syndrome mice in C57BL/6 genetic background exhibit severe seizures, cognitive and social impairments, and premature death. Here we show that Dravet Syndrome mice in pure 129/SvJ genetic background have many fewer seizures and much less premature death than in pure C57BL/6 background. These mice also have a higher threshold for thermally induced seizures, fewer myoclonic seizures, and no cognitive impairment, similar to patients with Genetic Epilepsy with Febrile Seizures Plus. Consistent with this mild phenotype, mutation of NaV1.1 channels has much less physiological effect on neuronal excitability in 129/SvJ mice. In hippocampal slices, the excitability of CA1 Stratum Oriens interneurons is selectively impaired, while the excitability of CA1 pyramidal cells is unaffected. NaV1.1 haploinsufficiency results in increased rheobase and threshold for action potential firing and impaired ability to sustain high-frequency firing. Moreover, deletion of NaV1.1 markedly reduces the amplification and integration of synaptic events, further contributing to reduced excitability of interneurons. Excitability is less impaired in inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background. Because specific deletion of NaV1.1 in forebrain GABAergic interneuons is sufficient to cause the symptoms of Dravet Syndrome in mice, our results support the conclusion that the milder phenotype in 129/SvJ mice is caused by lesser impairment of sodium channel function and electrical excitability in their forebrain interneurons. This mild impairment of excitability of interneurons leads to a milder disease phenotype in 129/SvJ mice, similar to Genetic Epilepsy with Febrile Seizures Plus in humans.

Hyper-SUMOylation of the Kv7 potassium channel diminishes the M-current leading to seizures and sudden death.

Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP.

Impaired path integration and grid cell spatial periodicity in mice lacking GluA1-containing AMPA receptors.

The hippocampus and the parahippocampal region have been proposed to contribute to path integration. Mice lacking GluA1-containing AMPA receptors (GluA1(-/-) mice) were previously shown to exhibit impaired hippocampal place cell selectivity. Here we investigated whether path integration performance and the activity of grid cells of the medial entorhinal cortex (MEC) are affected in these mice. We first tested GluA1(-/-) mice on a standard food-carrying homing task and found that they were impaired in processing idiothetic cues. To corroborate these findings, we developed an L-maze task that is less complex and is performed entirely in darkness, thereby reducing numerous confounding variables when testing path integration. Also in this task, the performance of GluA1(-/-) mice was impaired. Next, we performed in vivo recordings in the MEC of GluA1(-/-) mice. MEC neurons exhibited altered grid cell spatial periodicity and reduced spatial selectivity, whereas head direction tuning and speed modulation were not affected. The firing associations between pairs of neurons in GluA1(-/-) mice were stable, both in time and space, indicating that attractor states were still present despite the lack of grid periodicity. Together, these results support the hypothesis that spatial representations in the hippocampal-entorhinal network contribute to path integration.

Translaminar inhibitory cells recruited by layer 6 corticothalamic neurons suppress visual cortex.

In layer 6 (L6), a principal output layer of the mammalian cerebral cortex, a population of excitatory neurons defined by the NTSR1-Cre mouse line inhibit cortical responses to visual stimuli. Here we show that of the two major types of excitatory neurons existing in L6, the NTSR1-Cre line selectively targets those whose axons innervate both cortex and thalamus and not those whose axons remain within the cortex. These corticothalamic neurons mediate widespread inhibition across all cortical layers by recruiting fast-spiking inhibitory neurons whose cell body resides in deep cortical layers yet whose axons arborize throughout all layers. This study reveals a circuit by which L6 modulates cortical activity and identifies an inhibitory neuron able to regulate the strength of cortical responses throughout cortical depth.

Long-range connectivity defines behavioral specificity of amygdala neurons.

Memories are acquired and encoded within large-scale neuronal networks spanning different brain areas. The anatomical and functional specificity of such long-range interactions and their role in learning is poorly understood. The amygdala and the medial prefrontal cortex (mPFC) are interconnected brain structures involved in the extinction of conditioned fear. Here, we show that a defined subpopulation of basal amygdala (BA) projection neurons targeting the prelimbic (PL) subdivision of mPFC is active during states of high fear, whereas BA neurons targeting the infralimbic (IL) subdivision are recruited, and exhibit cell-type-specific plasticity, during fear extinction. Pathway-specific optogenetic manipulations demonstrate that the activity balance between pathways is causally involved in fear extinction. Together, our findings demonstrate that, although intermingled locally, long-range connectivity defines distinct subpopulations of amygdala projection neurons and indicate that the formation of long-term extinction memories depends on the balance of activity between two defined amygdala-prefrontal pathways.

Memory recall and modifications by activating neurons with elevated CREB.

Memory is supported by a specific ensemble of neurons distributed in the brain that form a unique memory trace. We previously showed that neurons in the lateral amygdala expressing elevated levels of cAMP response-element binding protein are preferentially recruited into fear memory traces and are necessary for the expression of those memories. However, it is unknown whether artificially activating just these selected neurons in the absence of behavioral cues is sufficient to recall that fear memory. Using an ectopic rat vanilloid receptor TRPV1 and capsaicin system, we found that activating this specific ensemble of neurons was sufficient to recall established fear memory. Furthermore, this neuronal activation induced a reconsolidation-like reorganization process, or strengthening of the fear memory. Thus, our findings establish a direct link between the activation of specific ensemble of neurons in the lateral amygdala and the recall of fear memory and its subsequent modifications.