Recordings In Brain Slices Research Articles

A subthalamo-parabrachial glutamatergic pathway is involved in stress-induced self-grooming in mice.

Excessive self-grooming is an important behavioral phenotype of the stress response in rodents. Elucidating the neural circuit that regulates stress-induced self-grooming may suggest potential treatment to prevent maladaptation to stress that is implicated in emotional disorders. Stimulation of the subthalamic nucleus (STN) has been found to induce strong self-grooming. In this study we investigated the role of the STN and a related neural circuit in mouse stress-related self-grooming. Body-restraint and foot-shock stress-induced self-grooming models were established in mice. We showed that both body restraint and foot shock markedly increased the expression of c-Fos in neurons in the STN and lateral parabrachial nucleus (LPB). Consistent with this, the activity of STN neurons and LPB glutamatergic (Glu) neurons, as assessed with fiber photometry recording, was dramatically elevated during self-grooming in the stressed mice. Using whole-cell patch-clamp recordings in parasagittal brain slices, we identified a monosynaptic projection from STN neurons to LPB Glu neurons that regulates stress-induced self-grooming in mice. Enhanced self-grooming induced by optogenetic activation of the STN-LPB Glu pathway was attenuated by treatment with fluoxetine (18 mg·kg-1·d-1, p.o., for 2 weeks) or in the presence of a cage mate. Furthermore, optogenetic inhibition of the STN-LPB pathway attenuated stress-related but not natural self-grooming. Taken together, these results suggest that the STN-LPB pathway regulates the acute stress response and is a potential target for intervention in stress-related emotional disorders.

Glucagon-Like Peptide-1 Receptors in the Gustatory Cortex Influence Food Intake.

The gustatory cortex (GC) region of the insular cortex processes taste information in manners important for taste-guided behaviors, including food intake itself. In addition to oral gustatory stimuli, GC activity is also influenced by physiological states including hunger. The specific cell types and molecular mechanisms that provide the GC with such abilities are unclear. Glucagon-like peptide 1 (GLP-1) is produced by neurons in the brain, where it can act on GLP-1 receptor-expressing (GLP-1R+) neurons found in several brain regions. In these brain regions, GLP-1R agonism suppresses homeostatic food intake and dampens the hedonic value of food. Here, we report in mice of both sexes that cells within the GC express Glp1r mRNA and further, by ex vivo brain slice recordings, that GC GLP-1R+ neurons are depolarized by the selective GLP-1R agonist, exendin-4. Next we found that chemogenetic stimulation of GLP-1R+ neurons, and also pharmacological stimulation of GC-GLP-1Rs themselves, both reduced homeostatic food intake. When mice were chronically maintained on diets with specific fat contents and then later offered foods with new fat contents, we also found that GLP-1R agonism reduced food intake toward foods with differing fat contents, indicating that GC GLP-1R influences may depend on palatability of the food. Together, these results provide evidence for a specific cell population in the GC that may hold roles in both homeostatic and hedonic food intake.SIGNIFICANCE STATEMENT The present study demonstrates that a population of neurons in the GC region of the insular cortex expresses receptors for GLP-1Rs, these neurons are depolarized by agonism of GLP-1Rs, and GC GLP-1Rs can influence food intake on their activation, including in manners depending on food palatability. This work is significant by adding to our understanding of the brain systems that mediate ingestive behavior, which holds implications for metabolic diseases.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.

The role of selective SATB1 deletion in somatostatin expressing interneurons on endogenous network activity and the transition to epilepsy.

Somatostatin (SST) expressing interneurons are the second most abundant group of inhibitory neurons in the neocortex. They mainly target the apical dendrites of excitatory pyramidal cells and are implicated in feedforward and feedback inhibition. In the present study, we employ a conditional knockout mouse, in which the transcription factor Satb1 is selectively deleted in SST-expressing interneurons resulting to the reduction of their number across the somatosensory barrel field. Our goal was to investigate the effect of the reduced number of Satb1 mutant SST-interneurons on (i) the endogenous cortical network activity (spontaneously recurring Up/Down states), and (ii) the transition to epileptiform activity. By conducting LFP recordings in acute brain slices from young male and female mice, we demonstrate that mutant animals exhibit significant changes in network excitability, reflected in increased Up state occurrence, decreased Up state duration and higher levels of extracellular spiking activity. Epileptiform activity was induced through two distinct and widely used in vitro protocols: the low magnesium and the 4-Aminopyridine (4-AP) model. In the former, slices from mutant animals manifested shorter latency for the expression of stable seizure-like events. In contrast, when epilepsy was induced by 4-AP, no significant differences were reported. We conclude that normal SST-interneuron function has a significant role both in the regulation of the endogenous network activity, and in the transition to seizure-like discharges in a context-dependent manner.

Deficiency of NMDA receptor subunit GluN3A as a pathogenic factor for endogenous amyloid deposition and sporadic Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disease, evolving amyloidosis and tau protein hyperphosphorylation, synapse dysfunction, neuron degeneration and cognitive deficits. Investigations based on the Aβ/tau hypothesis have been performed in transgenic animal models expression familial/mutated AD genes. However, treatments based on this hypothesis have not provided effective clinical therapies. Hyperactivity of the glutamatergic NMDA receptor (NMDAR) is a common pathophysiology of AD and has been viewed as a consequence of Aβ pathology. Among NMDAR subunits, GluN3A is a unique inhibitory subunit that suppresses NMDA currents and Ca2+ influx. Recent evidence from rodent and human studies shows that GluN3A is widely expressed and plays important functional roles in neonatal and adult brains. Ca2+ overload is a trigger of excitotoxicity, while GluN3A plays a regulatory role of Ca2+ homeostasis. The role of GluN3A regulation in AD is unknown.MethodGluN3A knockout mice, gene modifications, immunohistochemistry, Western blotting, MEA recordings, Ca2+ imaging, pathological examinations, functional and behavioral tests.ResultChronic neuronal hyperactivity and Ca2+ dysregulation caused by GluN3A deficiency lead to slowly advanced “neurodegenerative excitotoxicity”, dementia and AD pathology in an age‐dependent manner. Ca2+ imaging and MultiElectrode Array recordings in GluN3A KO brain slices revealed moderate but sustained elevation of cytosolic Ca2+ levels, neuronal hyperactivity, and impaired synaptic plasticity compared to age matched controls. Aging GluN3A KO mice showed increased Ca2+‐dependent signals, chronic inflammation, neuronal loss and disease symptoms including early onset of olfactory dysfunction followed by cognitive deficits. β‐Amyloid deposition and tau protein hyperphosphorylation were identified as a late pathology in the cortex and hippocampus. Expression of GluN3A in the GluN3A KO brain prevented AD progression. Early treatment at the pre‐clinical stage using the NMDAR antagonist memantine prevented symptoms in GluN3A KO mice. Western blot analysis of AD patient’s brains showed lower levels of GluN3A in the cortex compared to normal aging subjects.ConclusionOur results identified a novel amyloid‐independent mechanism that the deficiency of GluN3A alone causes a slight lifelong Ca2+ dyshomeostasis, leading to AD pathophysiology and amyloid pathology. The long‐term GluN3A modulation of NMDARs signifies new therapeutic targets and possible preventive interventions for a sporadic type of AD.

Further Studies on the Role of BTBD9 in the Cerebellum, Sleep-like Behaviors and the Restless Legs Syndrome

Genetic analyses have linked BTBD9 to restless legs syndrome (RLS) and sleep regulation. Btbd9 knockout mice show RLS-like motor restlessness. Previously, we found hyperactivity of cerebellar Purkinje cells (PCs) in Btbd9 knockout mice, which may contribute to the motor restlessness observed. However, underlying mechanisms for PC hyperactivity in Btbd9 knockout mice are unknown. Here, we used dissociated PC recording, brain slice recording and western blot to address this question. Our dissociated recording shows that knockout PCs had increased TEA-sensitive, Ca2+-dependent K+ currents. Applying antagonist to large conductance Ca2+-activated K+ (BK) channels further isolated the increased current as BK current. Consistently, we found increased amplitude of afterhyperpolarization and elevated BK protein levels in the knockout mice. Dissociated recording also shows a decrease in TEA-insensitive, Ca2+-dependent K+ currents. The result is consistent with reduced amplitude of tail currents, mainly composed of small conductance Ca2+-activated K+ (SK) currents, in slice recording. Our results suggest that BK and SK channels may be responsible for the hyperactivity of knockout PCs. Recently, BTBD9 protein was shown to associate with SYNGAP1 protein. We found a decreased cerebellar level of SYNGAP1 in Btbd9 knockout mice. However, Syngap1 heterozygous knockout mice showed nocturnal, instead of diurnal, motor restlessness. Our results suggest that SYNGAP1 deficiency may not contribute directly to the RLS-like motor restlessness observed in Btbd9 knockout mice. Finally, we found that PC-specific Btbd9 knockout mice exhibited deficits in motor coordination and balance similar to Btbd9 knockout mice, suggesting that the motor effect of BTBD9 in PCs is cell-autonomous.

Co-activation of GPCRs facilitate GIRK-dependent current.

The activity of dopamine neurons is dependent on both intrinsic properties and afferent projections. One potent form of inhibition is mediated by the activation of two inhibitory G protein-coupled receptors, D2 and GABAB receptors. Each of these receptors activates G protein-coupled inwardly rectifying potassium (GIRK) channels. Recordings in brain slices have shown that co-activation using saturating concentrations of agonists results in occlusion of the GIRK current. The present study examined the interaction between D2 and GABAB receptors using transient applications of sub-saturating concentrations of agonists where the co-application of one agonist resulted in both facilitation and inhibition (desensitization) of the other. The heterologous facilitation was modelled based on the known cooperative interaction between the G protein βγ subunits and GIRK channels. The results indicate that a low tonic level of G βγ results in facilitation of GIRK current and a high level of G βγ results in occlusion. The kinetics of the current induced by transient receptor activation is prolonged in each case. The results suggest that the cooperative interaction between G βγ subunits and GIRK channels determines both the amplitude and kinetics of GPCR-dependent current. KEY POINTS: Inhibitory D2 and GABAB receptors modulate dopamine neuron activity through shared G protein-coupled inwardly rectifying potassium (GIRK) channels. This study reports robust bidirectional interactions between these two converging receptor pathways. Coincident activation of D2 and GABAB receptors leads to facilitation of GIRK channel currents, augmenting both amplitude and prolonging the duration of phasic responses. Activation of either D2 or GABAB receptors also acutely desensitized the GIRK channel current induced by D2 receptor activation that rapidly recovers following termination of desensitizing stimulus. Results demonstrate that the activity of either G protein-coupled receptor system must be considered in the context of other G protein-coupled receptors.

Early impairment of cortical circuit plasticity and connectivity in the 5XFAD Alzheimer’s disease mouse model

Genetic risk factors for neurodegenerative disorders, such as Alzheimer’s disease (AD), are expressed throughout the life span. How these risk factors affect early brain development and function remain largely unclear. Analysis of animal models with high constructive validity for AD, such as the 5xFAD mouse model, may provide insights on potential early neurodevelopmental effects that impinge on adult brain function and age-dependent degeneration. The 5XFAD mouse model over-expresses human amyloid precursor protein (APP) and presenilin 1 (PS1) harboring five familial AD mutations. It is unclear how the expression of these mutant proteins affects early developing brain circuits. We found that the prefrontal cortex (PFC) layer 5 (L5) neurons in 5XFAD mice exhibit transgenic APP overloading at an early post-weaning age. Impaired synaptic plasticity (long-term potentiation, LTP) was seen at 6–8 weeks age in L5 PFC circuit, which was correlated with increased intracellular APP. APP overloading was also seen in L5 pyramidal neurons in the primary visual cortex (V1) during the critical period of plasticity (4–5 weeks age). Whole-cell patch clamp recording in V1 brain slices revealed reduced intrinsic excitability of L5 neurons in 5XFAD mice, along with decreased spontaneous miniature excitatory and inhibitory inputs. Functional circuit mapping using laser scanning photostimulation (LSPS) combined with glutamate uncaging uncovered reduced excitatory synaptic connectivity onto L5 neurons in V1, and a more pronounced reduction in inhibitory connectivity, indicative of altered excitation and inhibition during VC critical period. Lastly, in vivo single-unit recording in V1 confirmed that monocular visual deprivation-induced ocular dominance plasticity during critical period was impaired in 5XFAD mice. Our study reveals plasticity deficits across multiple cortical regions and indicates altered early cortical circuit developmental trajectory as a result of mutant APP/PS1 over-expression.

Spike Afterhyperpolarizations Govern Persistent Firing Dynamics in Rat Neocortical and Hippocampal Pyramidal Cells.

Persistent firing is commonly reported in both cortical and subcortical neurons under a variety of behavioral conditions. Yet the mechanisms responsible for persistent activity are only partially resolved with support for both intrinsic and synaptic circuit-based mechanisms. Little also is known about physiological factors that enable epochs of persistent firing to continue beyond brief pauses and then spontaneously terminate. In the present study, we used intracellular recordings in rat (both sexes) neocortical and hippocampal brain slices to assess the ionic mechanisms underlying persistent firing dynamics. Previously, we showed that blockade of ether-á-go-go-related gene (ERG) potassium channels abolished intrinsic persistent firing in the presence of low concentrations of muscarinic receptor agonists and following optogenetic activation of cholinergic axons. Here we show the slow dynamics of ERG conductance changes allows persistent firing to outlast the triggering stimulus and even to initiate discharges following ∼7 s poststimulus firing pauses. We find that persistent firing dynamics is regulated by the interaction between ERG conductance and spike afterhyperpolarizations (AHPs). Increasing the amplitude of spike AHPs using either SK channel activators or a closed-loop reactive feedback system allows persistent discharges to spontaneously terminate in both neocortical neurons and hippocampal CA1 pyramidal cells. The interplay between ERG and the potassium channels that mediate spike AHPs grades the duration of persistent firing, providing a novel, generalizable mechanism to explain self-terminating persistent firing modes observed behaving animals.SIGNIFICANCE STATEMENT Many classes of neurons generate prolonged spiking responses to transient stimuli. These discharges often outlast the stimulus by seconds to minutes in some in vitro models of persistent firing. While recent work has identified key synaptic and intrinsic components that enable persistent spiking responses, less is known about mechanisms that can terminate and regulate the dynamics of these responses. The present study identified the spike afterhyperpolarizations as a potent mechanism that regulates the duration of persistent firing. We found that amplifying spike afterpotentials converted bistable persistent firing into self-terminating discharges. Varying the spike AHP amplitude grades the duration of persistent discharges, generating in vitro responses that mimic firing modes associated with neurons associated with short-term memory function.

Interferon-γ augments GABA release in the developing neocortex via nitric oxide synthase/soluble guanylate cyclase and constrains network activity.

Interferon-γ (IFN-γ), a cytokine with neuromodulatory properties, has been shown to enhance inhibitory transmission. Because early inhibitory neurotransmission sculpts functional neuronal circuits, its developmental alteration may have grave consequences. Here, we investigated the acute effects of IFN-γ on γ-amino-butyric acid (GABA)ergic currents in layer 5 pyramidal neurons of the somatosensory cortex of rats at the end of the first postnatal week, a period of GABA-dependent cortical maturation. IFN-γ acutely increased the frequency and amplitude of spontaneous/miniature inhibitory postsynaptic currents (s/mIPSC), and this could not be reversed within 30 min. Neither the increase in amplitude nor frequency of IPSCs was due to upregulated interneuron excitability as revealed by current clamp recordings of layer 5 interneurons labeled with VGAT-Venus in transgenic rats. As we previously reported in more mature animals, IPSC amplitude increase upon IFN-γ activity was dependent on postsynaptic protein kinase C (PKC), indicating a similar activating mechanism. Unlike augmented IPSC amplitude, however, we did not consistently observe an increased IPSC frequency in our previous studies on more mature animals. Focusing on increased IPSC frequency, we have now identified a different activating mechanism—one that is independent of postsynaptic PKC but is dependent on inducible nitric oxide synthase (iNOS) and soluble guanylate cyclase (sGC). In addition, IFN-γ shifted short-term synaptic plasticity toward facilitation as revealed by a paired-pulse paradigm. The latter change in presynaptic function was not reproduced by the application of a nitric oxide donor. Functionally, IFN-γ-mediated alterations in GABAergic transmission overall constrained early neocortical activity in a partly nitric oxide–dependent manner as revealed by microelectrode array field recordings in brain slices analyzed with a spike-sorting algorithm. In summary, with IFN-γ-induced, NO-dependent augmentation of spontaneous GABA release, we have here identified a mechanism by which inflammation in the central nervous system (CNS) plausibly modulates neuronal development.

What the hippocampus tells the HPA axis: Hippocampal output attenuates acute stress responses via disynaptic inhibition of CRF+ PVN neurons

The hippocampus exerts inhibitory feedback on the release of glucocorticoids. Because the major hippocampal efferent projections are excitatory, it has been hypothesized that this inhibition is mediated by populations of inhibitory neurons in the hypothalamus or elsewhere. These regions would be excited by hippocampal efferents and project to corticotropin-releasing factor (CRF) cells in the paraventricular nucleus of the hypothalamus (PVN). A direct demonstration of the synaptic responses elicited by hippocampal outputs in PVN cells or upstream GABAergic interneurons has not been provided previously. Here, we used viral vectors to express channelrhodopsin (ChR) and enhanced yellow fluorescent protein (EYFP) in pyramidal cells in the ventral hippocampus (vHip) in mice expressing tdTomato in GABA- or CRF-expressing neurons. We observed dense innervation of the bed nucleus of the stria terminalis (BNST) by labeled vHip axons and sparse labeling within the PVN. Using whole-cell voltage-clamp recording in parasagittal brain slices containing the BNST and PVN, photostimulation of vHip terminals elicited rapid excitatory postsynaptic currents (EPSCs) and longer-latency inhibitory postsynaptic currents (IPSCs) in both CRF+ and GAD + cells. The ratio of synaptic excitation and inhibition was maintained in CRF + cells during 20 Hz stimulus trains. Photostimulation of hippocampal afferents to the BNST and PVN in vivo inhibited the rise in blood glucocorticoid levels produced by acute restraint stress. We thus provide functional evidence suggesting that hippocampal output to the BNST contributes to a net inhibition of the hypothalamic-pituitary axis, providing further mechanistic insights into this process using methods with enhanced spatial and temporal resolution.

Calcineurin Controls Hypothalamic NMDA Receptor Activity and Sympathetic Outflow.

Hypertension is a common and serious adverse effect of calcineurin inhibitors, including cyclosporine and tacrolimus (FK506). Although increased sympathetic nerve discharges are associated with calcineurin inhibitor-induced hypertension, the sources of excess sympathetic outflow and underlying mechanisms remain elusive. Calcineurin (protein phosphatase-2B) is broadly expressed in the brain, including the paraventricular nuclear (PVN) of the hypothalamus, which is critically involved in regulating sympathetic vasomotor tone. We determined whether prolonged treatment with the calcineurin inhibitor causes elevated sympathetic output and persistent hypertension by potentiating synaptic N-methyl-D-aspartate (NMDA) receptor activity in the PVN. Telemetry recordings showed that systemic administration of FK506 (3 mg/kg per day) for 14 days caused a gradual and profound increase in arterial blood pressure in rats, which lasted at least 7 days after discontinuing FK506 treatment. Correspondingly, systemic treatment with FK506 markedly reduced calcineurin activity in the PVN and circumventricular organs, but not rostral ventrolateral medulla, and increased the phosphorylation level and synaptic trafficking of NMDA receptors in the PVN. Immunocytochemistry labeling showed that calcineurin was expressed in presympathetic neurons in the PVN. Whole-cell patch-clamp recordings in brain slices revealed that treatment with FK506 increased baseline firing activity of PVN presympathetic neurons; this increase was blocked by the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Also, treatment with FK506 markedly increased presynaptic and postsynaptic NMDA receptor activity of PVN presympathetic neurons. Furthermore, microinjection of the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist into the PVN of anesthetized rats preferentially attenuated renal sympathetic nerve discharges and blood pressure elevated by FK506 treatment. In addition, systemic administration of memantine, a clinically used NMDA receptor antagonist, effectively attenuated FK506 treatment-induced hypertension in conscious rats. Our findings reveal that normal calcineurin activity in the PVN constitutively restricts sympathetic vasomotor tone via suppressing NMDA receptor activity, which may be targeted for treating calcineurin inhibitor-induced hypertension.

Local Anesthetics via Multicomponent Reactions.

Local anesthetics occupy a prime position in clinical medicine as they temporarily relieve the pain by blocking voltage-gated sodium channels. However, limited structural diversity, problems with the efficiency of syntheses and increasing toxicity, mean that alternative scaffolds with improved chemical syntheses are urgently needed. Here, we demonstrate a multicomponent reaction (MCR)-based approach both towards the synthesis of commercial local anesthetics and towards novel derivatives as potential anesthesia candidates via scaffold hopping. The reactions are efficient and scalable, and several single-crystal structures have been obtained. In addition, our methodology has been applied to the synthesis of the antianginal drug ranolazine, via an Ugi three-component reaction. Representative derivatives from our libraries were evaluated as neuronal activity inhibitors using local field potential recordings (LFPs) in mouse hippocampal brain slices and showed very promising results. This study highlights new opportunities in drug discovery targeting local anesthetics.

Somatodendritic Release of Cholecystokinin Potentiates GABAergic Synapses Onto Ventral Tegmental Area Dopamine Cells

BackgroundNeuropeptides are contained in nearly every neuron in the central nervous system and can be released not only from nerve terminals but also from somatodendritic sites. Cholecystokinin (CCK), among the most abundant neuropeptides in the brain, is expressed in the majority of midbrain dopamine neurons. Despite this high expression, CCK function within the ventral tegmental area (VTA) is not well understood. MethodsWe confirmed CCK expression in VTA dopamine neurons through immunohistochemistry and in situ hybridization and detected optogenetically induced CCK release using an enzyme-linked immunosorbent assay. To investigate whether CCK modulates VTA circuit activity, we used whole-cell patch clamp recordings in mouse brain slices. We infused CCK locally in vivo and tested food intake and locomotion in fasted mice. We also used in vivo fiber photometry to measure Ca2+ transients in dopamine neurons during feeding. ResultsHere we report that VTA dopamine neurons release CCK from somatodendritic regions, where it triggers long-term potentiation of GABAergic (gamma-aminobutyric acidergic) synapses. The somatodendritic release occurs during trains of optogenetic stimuli or prolonged but modest depolarization and is dependent on synaptotagmin-7 and T-type Ca2+ channels. Depolarization-induced long-term potentiation is blocked by a CCK2 receptor antagonist and mimicked by exogenous CCK. Local infusion of CCK in vivo inhibits food consumption and decreases distance traveled in an open field test. Furthermore, intra-VTA–infused CCK reduced dopamine cell Ca2+ signals during food consumption after an overnight fast and was correlated with reduced food intake. ConclusionsOur experiments introduce somatodendritic neuropeptide release as a previously unknown feedback regulator of VTA dopamine cell excitability and dopamine-related behaviors.

Epilepsy-associated increase in gonadotropin-releasing hormone neuron firing in diestrous female mice is independent of chronic seizure burden severity

Reproductive endocrine disorders are common comorbidities of temporal lobe epilepsy (TLE). Our previous studies using the intrahippocampal kainic acid (IHKA) mouse model of TLE demonstrated that many females show prolonged estrous cycles and hypothalamic gonadotropin-releasing hormone (GnRH) neurons exhibit elevated firing during diestrus. However, it is unknown whether the degree of change in GnRH neuron activity is dependent on epilepsy severity. Here, we used 24/7 in vivo electroencephalography (EEG) and in vitro electrophysiological recordings in acute brain slices to assess GnRH neuron firing in relation to chronic seizure burden in diestrous female mice at two months after IHKA injection. We found that percentage of time in seizure activity in the 24 h prior to slice preparation is an accurate proxy of overall seizure burden. Firing rates of GnRH neurons from EEG-recorded IHKA mice were increased in comparison to controls, but no relationships were found between GnRH neuron firing and seizure burden measured in vivo. The independence of GnRH neuron firing rate in relation to seizure burden was unaffected by GnRH neuron soma location or estrous cycle length. Furthermore, GnRH neuron firing rates were not yet different from control values when measured 1 month after injection, when epileptogenesis is already complete in IHKA mice. These findings indicate that the severity of epilepsy and the degree of downstream disruption to GnRH neuron activity are independent, suggesting that susceptibility to reproductive endocrine comorbidities is driven by other risk factors.

Descending Axonal Projections from the Inferior Colliculus Target Nearly All Excitatory and Inhibitory Cell Types of the Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) integrates auditory nerve input with nonauditory sensory signals and is proposed to function in sound source localization and suppression of self-generated sounds. The DCN also integrates activity from descending auditory pathways, including a particularly large feedback projection from the inferior colliculus (IC), the main ascending target of the DCN. Understanding how these descending feedback signals are integrated into the DCN circuit and what role they play in hearing requires knowing the targeted DCN cell types and their postsynaptic responses. In order to explore these questions, neurons in the DCN that received descending synaptic input from the IC were labeled with a trans-synaptic viral approach in male and female mice, which allowed them to be targeted for whole-cell recording in acute brain slices. We tested their synaptic responses to optogenetic activation of the descending IC projection. Every cell type in the granule cell domain received monosynaptic, glutamatergic input from the IC, indicating that this region, considered an integrator of nonauditory sensory inputs, processes auditory input as well and may have complex and underappreciated roles in hearing. Additionally, we found that DCN cell types outside the granule cell regions also receive descending IC signals, including the principal projection neurons, as well as the neurons that inhibit them, leading to a circuit that may sharpen tuning through feedback excitation and lateral inhibition.SIGNIFICANCE STATEMENT Auditory processing starts in the cochlea and ascends through the dorsal cochlear nucleus (DCN) to the inferior colliculus (IC) and beyond. Here, we investigated the feedback projection from IC to DCN, whose synaptic targets and roles in auditory processing are unclear. We found that all cell types in the granule cell regions, which process multisensory feedback, also process this descending auditory feedback. Surprisingly, all except one cell type in the entire DCN receive IC input. The IC-DCN projection may therefore modulate the multisensory pathway as well as sharpen tuning and gate auditory signals that are sent to downstream areas. This excitatory feedback loop from DCN to IC and back to DCN could underlie hyperexcitability in DCN, widely considered an etiology of tinnitus.

Properties of the epileptiform activity in the cingulate cortex of a mouse model of LIS1 dysfunction

Dysfunction of the LIS1 gene causes lissencephaly, a drastic neurological disorder characterized by a deep disruption of the cortical structure. We aim to uncover alterations of the cortical neuronal networks related with the propagation of epileptiform activity in the Lis1/sLis1 mouse, a model lacking the LisH domain in heterozygosis. We did extracellular field-potential and intracellular recordings in brain slices of the anterior cingulate cortex (ACC) or the retrosplenial cortex (RSC) to study epileptiform activity evoked in the presence of bicuculline (10 µM), a blocker of GABAA receptors. The sensitivity to bicuculline of the generation of epileptiform discharges was similar in wild type (WT) and Lis1/sLis1 cortex (EC50 1.99 and 2.24 µM, respectively). In the Lis1/sLis1 cortex, we observed a decreased frequency of the oscillatory post-discharges of the epileptiform events; also, the propagation of epileptiform events along layer 2/3 was slower in the Lis1/sLis1 cortex (WT 47.69 ± 2.16 mm/s, n = 25; Lis1/sLis1 37.34 ± 2.43 mm/s, n = 15; p = 0.004). The intrinsic electrophysiological properties of layer 2/3 pyramidal neurons were similar in WT and Lis1/sLis1 cortex, but the frequency of the spontaneous EPSCs was lower and their peak amplitude higher in Lis1/sLis1 pyramidal neurons. Finally, the propagation of epileptiform activity was differently affected by AMPA receptor blockers: CNQX had a larger effect in both ACC and RSC while GYKI53655 had a larger effect only in the ACC in the WT and Lis1/sLis1 cortex. All these changes indicate that the dysfunction of the LIS1 gene causes abnormalities in the properties of epileptiform discharges and in their propagation along the layer 2/3 in the anterior cingulate cortex and in the restrosplenial cortex.

Antidepressant-like effects of cinnamamide derivative M2 via D2 receptors in the mouse medial prefrontal cortex.

Major depressive disorder is a global mental illness associated with severe mortality and disability. The dopaminergic system is involved in both the etiology and therapeutics of depression. Distinct functions of dopamine D1 and D2 receptor subtypes have attracted considerable research interest, and their roles in the pathogenesis of depression and interaction with antidepressants need to be comprehensively elucidated. Herein, we investigated the antidepressant effects of a candidate antidepressant from a cinnamamide derivative, M2, and examined underlying neural mechanisms. We observed that a single dose of M2 (30 mg/kg, ip) produced rapid antidepressant-like effects in mice subjected to the forced swim and tail suspension tests. Using whole-cell recordings in mouse coronal brain slices, we found that application of M2 (10-150 μM) concentration-dependently increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) of the pyramidal neurons in the medial prefrontal cortex (mPFC). Furthermore, M2-induced enhancement of sEPSC frequency was abolished by sulpiride (10 µM), a dopamine D2 receptor antagonist, but not by the dopamine receptor D1 antagonist, SCH23390 (10 μM). In addition, M2 administration significantly increased expression levels of synaptogenesis-related proteins, including p-mTOR and p-TrkB, in the mPFC at 30 min, and increased postsynaptic protein PSD-95 at 24 h. Our results demonstrated that M2 produces rapid antidepressant actions through a novel mechanism via dopamine D2 receptor-mediated enhancement of mPFC neurotransmission.

Kinetics and Connectivity Properties of Parvalbumin- and Somatostatin-Positive Inhibition in Layer 2/3 Medial Entorhinal Cortex.

Parvalbumin-positive (Pvalb+) and somatostatin-positive (Sst+) cells are the two largest subgroups of inhibitory interneurons. Studies in visual cortex indicate that synaptic connections between Pvalb+ cells are common while connections between Sst+ interneurons have not been observed. The inhibitory connectivity and kinetics of these two interneuron subpopulations, however, have not been characterized in medial entorhinal cortex (mEC). Using fluorescence-guided paired recordings in mouse brain slices from interneurons and excitatory cells in layer 2/3 mEC, we found that, unlike neocortical measures, Sst+ cells inhibit each other, albeit with a lower probability than Pvalb+ cells (18% vs 36% for unidirectional connections). Gap junction connections were also more frequent between Pvalb+ cells than between Sst+ cells. Pvalb+ cells inhibited each other with larger conductances, smaller decay time constants, and shorter delays. Similarly, synaptic connections between Pvalb+ and excitatory cells were more likely and expressed faster decay times and shorter delays than those between Sst+ and excitatory cells. Inhibitory cells exhibited smaller synaptic decay time constants between interneurons than on their excitatory targets. Inhibition between interneurons also depressed faster, and to a greater extent. Finally, inhibition onto layer 2 pyramidal and stellate cells originating from Pvalb+ interneurons were very similar, with no significant differences in connection likelihood, inhibitory amplitude, and decay time. A model of short-term depression fitted to the data indicates that recovery time constants for refilling the available pool are in the range of 50–150 ms and that the fraction of the available pool released on each spike is in the range 0.2–0.5.