Distinct ACC Neural Mechanisms Underlie Authentic and Transmitted Anxiety Induced by Maternal Separation in Mice.

It is known that humans and rodents are capable of transmitting stress to their naive partners via social interaction. However, a comprehensive understanding of transmitted stress, which may differ from authentic stress, thus revealing unique neural mechanisms of social interaction resulting from transmitted stress and the associated anxiety, is missing. We used, in the present study, maternal separation (MS) as a stress model to investigate whether MS causes abnormal behavior in adolescence. A key concern in the analysis of stress transmission is whether the littermates of MS mice who only witness MS stress ("Partners") exhibit behavioral abnormalities similar to those of MS mice themselves. Of special interest is the establishment of the neural mechanisms underlying transmitted stress and authentic stress. The results show that Partners, similar to MS mice, exhibit anxiety-like behavior and hyperalgesia after witnessing littermates being subjected to early-life repetitive MS. Electrophysiological analysis revealed that mice subjected to MS demonstrate a reduction in both the excitatory and inhibitory synaptic activities of parvalbumin interneurons (PVINs) in the anterior cingulate cortex (ACC). However, Partners differed from MS mice in showing an increase in the number and excitability of GABAergic PVINs in the ACC and in the ability of chemogenetic PVIN inactivation to eliminate abnormal behavior. Furthermore, the social transfer of anxiety-like behavior required intact olfactory, but not visual, perception. This study suggests a functional involvement of ACC PVINs in mediating the distinct neural basis of transmitted anxiety.SIGNIFICANCE STATEMENT The anterior cingulate cortex (ACC) is a critical brain area in physical and social pain and contributes to the exhibition of abnormal behavior. ACC glutamatergic neurons have been shown to encode transmitted stress, but it remains unclear whether inhibitory ACC neurons also play a role. We evaluate, in this study, ACC neuronal, synaptic and network activities and uncover a critical role of parvalbumin interneurons (PVINs) in the expression of transmitted stress in adolescent mice who had witnessed MS of littermates in infancy. Furthermore, inactivation of ACC PVINs blocks transmitted stress. The results suggest that emotional contagion has a severe effect on brain function, and identify a potential target for the treatment of transmitted anxiety.

Multichannel Extracellular Recording in Freely Moving Mice.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Corrigendum: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum.

[This corrects the article DOI: 10.3389/fnmol.2023.1107355.].

Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum.

Although the deubiquitinase cylindromatosis (CYLD), an abundant protein in the postsynaptic density fraction, plays a crucial role in mediating the synaptic activity of the striatum, the precise molecular mechanism remains largely unclear. Here, using a Cyld-knockout mouse model, we demonstrate that CYLD regulates dorsolateral striatum (DLS) neuronal morphology, firing activity, excitatory synaptic transmission, and plasticity of striatal medium spiny neurons via, likely, interaction with glutamate receptor 1 (GluA1) and glutamate receptor 2 (GluA2), two key subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). CYLD deficiency reduces levels of GluA1 and GluA2 surface protein and increases K63-linked ubiquitination, resulting in functional impairments both in AMPAR-mediated excitatory postsynaptic currents and in AMPAR-dependent long-term depression. The results demonstrate a functional association of CYLD with AMPAR activity, which strengthens our understanding of the role of CYLD in striatal neuronal activity.

Altered Odor-Evoked Electrophysiological Responses in the Anterior Piriform Cortex of Conscious APP/PS1 Mice.

BACKGROUND: Olfactory decline is an indicator of early-stage Alzheimer's disease (AD). Although the anterior piriform cortex (aPC) is an important brain area involved in processing olfactory input, little is known about how its neuronal activity is affected in early-stage AD. OBJECTIVE: To elucidate whether odor-induced electrophysiological responses are altered in the aPC of 3-5-month-old APP/PS1 mice. METHODS: Using head-fixed multi-channel recording techniques in APP/PS1 AD mouse model to uncover potential aberrance of the aPC neuronal firing and local field potential (LFP) in response to vanillin. RESULTS: We show that the firing rate of aPC neurons evoked by vanillin is significantly reduced in conscious APP/PS1 mice. LFP analysis demonstrates reduced low- and high-gamma (γlow,γhigh) oscillations during both the baseline and odor stimulation periods in APP/PS1 mice. Moreover, according to spike-field coherence (SFC) analysis, APP/PS1 mice show decreased coherence between odor-evoked spikes and γlow rhythms, while the coherence with γhigh rhythms and the ΔSFC of the oscillations is unaffected. Furthermore, APP/PS1 mice show reduced phase-locking strength in the baseline period, such that there is no difference between baseline and odor-stimulation conditions. This contrasts markedly with wild type mice, where phase-locking strength decreases on stimulation. CONCLUSION: The abnormalities in both the neuronal and oscillatory activities of the aPC may serve as electrophysiological indicators of underlying olfactory decline in early AD.

Systemic LPS-induced microglial activation results in increased GABAergic tone: A mechanism of protection against neuroinflammation in the medial prefrontal cortex in mice.

Neuroinflammation with excess microglial activation and synaptic dysfunction are early symptoms of most neurological diseases. However, how microglia-associated neuroinflammation regulates synaptic activity remains obscure. We report here that acute neuroinflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) results in cell-type-specific increases in inhibitory postsynaptic currents in the glutamatergic, but not the GABAergic, neurons of medial prefrontal cortex (mPFC), coinciding with excessive microglial activation. LPS causes upregulation in levels of GABAAR subunits, glutamine synthetase and vesicular GABA transporter, and downregulation in brain-derived neurotrophic factor (BDNF) and its receptor, pTrkB. Blockage of microglial activation by minocycline ameliorates LPS-induced abnormal expression of GABA signaling-related proteins and activity of synaptic and network. Moreover, minocycline prevents the mice from LPS-induced aberrant behavior, such as a reduction in total distance and time spent in the centre in the open field test; decreases in entries into the open arm of elevated-plus maze and in consumption of sucrose; increased immobility in the tail suspension test. Furthermore, upregulation of GABA signaling by tiagabine also prevents LPS-induced microglial activation and aberrant behavior. This study illustrates a mode of bidirectional constitutive signaling between the neural and immune compartments of the brain, and suggests that the mPFC is an important area for brain-immune system communication. Moreover, the present study highlights GABAergic signaling as a key therapeutic target for mitigating neuroinflammation-induced abnormal synaptic activity in the mPFC, together with the associated behavioral abnormalities.

Mitochondrial Deficits With Neural and Social Damage in Early-Stage Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. Mitochondrial dysfunction is thought to be an early event in the onset and progression of AD; however, the precise underlying mechanisms remain unclear. In this study, we investigated mitochondrial proteins involved in organelle dynamics, morphology and energy production in the medial prefrontal cortex (mPFC) and hippocampus (HIPP) of young (1∼2 months), adult (4∼5 months) and aged (9∼10, 12∼18 months) APP/PS1 mice. We observed increased levels of mitochondrial fission protein, Drp1, and decreased levels of ATP synthase subunit, ATP5A, leading to abnormal mitochondrial morphology, increased oxidative stress, glial activation, apoptosis, and altered neuronal morphology as early as 4∼5 months of age in APP/PS1 mice. Electrophysiological recordings revealed abnormal miniature excitatory postsynaptic current in the mPFC together with a minor connectivity change between the mPFC and HIPP, correlating with social deficits. These results suggest that abnormal mitochondrial dynamics, which worsen with disease progression, could be a biomarker of early-stage AD. Therapeutic interventions that improve mitochondrial function thus represent a promising approach for slowing the progression or delaying the onset of AD.

Altered corticostriatal synchronization associated with compulsive-like behavior in APP/PS1 mice.

Mild behavioral impairment (MBI), which can include compulsive behavior, is an early sign of Alzheimer's disease (AD), but its underlying neural mechanisms remain unclear. Here, we show that 3-5-month-old APP/PS1 mice display obsessive-compulsive disorder (OCD)-like behavior. The number of parvalbumin-positive (PV) interneurons and level of high gamma (γhigh) oscillation are significantly decreased in the striatum of AD mice. This is accompanied by enhanced ß-γhigh coupling and firing rates of putative striatal projection neurons (SPNs), indicating decorrelation between PV interneurons and SPNs. Local field potentials (LFPs) simultaneously recorded in prefrontal cortex (PFC) and striatum (Str) demonstrate a decrease in γhigh-band coherent activity and spike-field coherence in corticostriatal circuits of APP/PS1 mice. Furthermore, levels of GABAB receptor (GABABR), but not GABAA receptor (GABAAR), and glutamatergic receptors, were markedly reduced, in line with presymptomatic AD-related behavioral changes. These findings suggest that MBI occurs as early as 3-5 months in APP/PS1 mice and that altered corticostriatal synchronization may play a role in mediating the behavioral phenotypes observed.

Pelvic Pain Alters Functional Connectivity Between Anterior Cingulate Cortex and Hippocampus in Both Humans and a Rat Model.

The anterior cingulate cortex (ACC) and hippocampus (HIPP) are two key brain regions associated with pain and pain-related affective processing. However, whether and how pelvic pain alters the neural activity and connectivity of the ACC and HIPP under baseline and during social pain, and the underlying cellular and molecular mechanisms, remain unclear. Using functional magnetic resonance imaging (fMRI) combined with electrophysiology and biochemistry, we show that pelvic pain, particularly, primary dysmenorrhea (PDM), causes an increase in the functional connectivity between ACC and HIPP in resting-state fMRI, and a smaller reduction in connectivity during social exclusion in PDM females with periovulatory phase. Similarly, model rats demonstrate significantly increased ACC-HIPP synchronization in the gamma band, associating with reduced modulation by ACC-theta on HIPP-gamma and increased levels of receptor proteins and excitation. This study brings together human fMRI and animal research and enables improved therapeutic strategies for ameliorating pain and pain-related affective processing.

Enhancing GABAergic signaling ameliorates aberrant gamma oscillations of olfactory bulb in AD mouse models.

BACKGROUND: Before the deposition of amyloid-beta plaques and the onset of learning memory deficits, patients with Alzheimer's disease (AD) experience olfactory dysfunction, typified by a reduced ability to detect, discriminate, and identify odors. Rodent models of AD, such as the Tg2576 and APP/PS1 mice, also display impaired olfaction, accompanied by aberrant in vivo or in vitro gamma rhythms in the olfactory pathway. However, the mechanistic relationships between the electrophysiological, biochemical and behavioral phenomena remain unclear. METHODS: To address the above issues in AD models, we conducted in vivo measurement of local field potential (LFP) with a combination of in vitro electro-olfactogram (EOG), whole-cell patch and field recordings to evaluate oscillatory and synaptic function and pharmacological regulation in the olfactory pathway, particularly in the olfactory bulb (OB). Levels of protein involved in excitation and inhibition of the OB were investigated by western blotting and fluorescence staining, while behavioral studies assessed olfaction and memory function. RESULTS: LFP measurements demonstrated an increase in gamma oscillations in the OB accompanied by altered olfactory behavior in both APP/PS1 and 3xTg mice at 3-5 months old, i.e. an age before the onset of plaque formation. Fewer olfactory sensory neurons (OSNs) and a reduced EOG contributed to a decrease in the excitatory responses of M/T cells, suggesting a decreased ability of M/T cells to trigger interneuron GABA release indicated by altered paired-pulse ratio (PPR), a presynaptic parameter. Postsynaptically, there was a compensatory increase in levels of GABAAR α1 and ß3 subunits and subsequent higher amplitude of inhibitory responses. Strikingly, the GABA uptake inhibitor tiagabine (TGB) ameliorated abnormal gamma oscillations and levels of GABAAR subunits, suggesting a potential therapeutic strategy for early AD symptoms. These findings reveal increased gamma oscillations in the OB as a core indicator prior to onset of AD and uncover mechanisms underlying aberrant gamma activity in the OB. CONCLUSIONS: This study suggests that the concomitant dysfunction of both olfactory behavior and gamma oscillations have important implications for early AD diagnosis: in particular, awareness of aberrant GABAergic signaling mechanisms might both aid diagnosis and suggest therapeutic strategies for olfactory damage in AD.

Optimized Golgi-Cox Staining Validated in the Hippocampus of Spared Nerve Injury Mouse Model.

Golgi-Cox staining has been used extensively in neuroscience. Despite its unique ability to identify neuronal interconnections and neural processes, its lack of consistency and time-consuming nature reduces its appeal to researchers. Here, using a spared nerve injury (SNI) mouse model and control mice, we present a modified Golgi-Cox staining protocol that can stain mouse hippocampal neurons within 8 days. In this improved procedure, the mouse brain was fixed with 4% paraformaldehyde and then stored in a modified Golgi-Cox solution at 37 ± 2°C. The impregnation period was reduced from 5-14 days to 36-48 h. Brain slices prepared in this way could be preserved long-term at -80°C for up to 8 weeks. In addition to minimizing frequently encountered problems and reducing the time required to conduct the method, our modified protocol maintained, and even improved, the quality of traditional Golgi-Cox staining as applied to hippocampal neuronal morphology in SNI mice.

Exposure to prenatal antidepressant alters medial prefrontal-striatal synchronization in mice.

Citalopram (CTM), a selective serotonin reuptake inhibitor (SSRI), has been widely used to treat panic disorders, such as depression which is one of the most disabling, yet common, psychiatric disorders. Although prenatal antidepressant exposure has a major impact on the neurobehavioral development of the offspring, such as anxiety, depression- and autism-like behaviors, the brain alterations of SSRI influence remained largely unknown. We show here, using electrophysiological recordings, that CTM exposure during the last 7 d of gestation can alter theta- and gamma-band oscillation and synchronization in the corticostriatal loop of 2-3 month-old mouse offspring. The dendritic length and number of dendritic branches in prefrontal neurons of these CTM-exposed mice are significantly reduced, consistent with decreased levels of N-methyl-d-aspartate receptors (NMDARs) and calcium/calmodulin-dependent protein kinase II (CaMKII) in the medial prefrontal cortex (mPFC). In addition, the level of c-Fos in both the mPFC and striatum is significantly increased. Together, these results advance our understanding of a neural network that is potentially responsible for abnormalities induced by prenatal antidepressant exposure.

Memantine rescues prenatal citalopram exposure-induced striatal and social abnormalities in mice.

Prenatal exposure to citalopram (CTM), an antidepressant drug, has been associated with altered behavior, including autism-like symptoms in both human and rodent offspring. However, the neurological basis underlying these abnormal behaviors is not well understood. Here, we examined behavioral, morphological, and biochemical alterations in the male and female offspring of C57BL/6 mouse mothers that had been exposed to CTM during the last trimester of gestation. We observed abnormal behavior such as anxiety, altered locomotion and disordered social interactions in 2-5 months old offspring with prenatal CTM exposure. Using Golgi-Cox staining, we found that CTM caused significantly reduced dendritic length and number of dendritic branches in striatal neurons, as well as altered subunit levels of N-methyl-d-aspartate receptors (NMDARs) and calcium/calmodulin-dependent protein kinase II (CaMKII). Memantine, a selective NMDAR antagonist, improved prenatal CTM-induced abnormal protein levels and social interaction deficits. These results highlight potential mechanisms underlying the abnormal behavior observed in children who are prenatally exposed to CTM.

The role of CA3-LS-VTA loop in the formation of conditioned place preference induced by context-associated reward memory for morphine.

Addiction-related behaviors, such as conditioned place preference (CPP), require animals to remember an association between environmental cue and drug treatment, and exposure to environmental cue is one of the key contributing factors to relapse. However, how central neural circuit participates in the formation of CPP induced by stimulus of morphine-paired environment remains unknown. In the present study, we found that reexposure to morphine-paired environment significantly increased the activity of hippocampal CA3 neurons, increased the excitability of GABAergic neurons and expression of glutamic acid decarboxylase 65/67 in the caudal lateral septum (LSc) and decreased the activity of GABAergic neurons and GAD65/67 expression in ventral tegmental area (VTA), leading to activation (disinhibition) of dopaminergic neurons. Inactivation of CA3 neurons attenuated GABAergic neurons activity and decreased the upregulation of GAD65/67 in LSc, prevented the dopaminergic neurons activation,and GAD65/67 downregulation in VTA and ameliorated the CPP behavior following exposure to morphine-paired context. Blockade of NMDA receptor in LSc also prevented the upregulation of GAD65/67 in LSc and formation of CPP induced by stimulus of morphine-paired environment. Suppression of GAD activity in LSc also remarkably attenuated the dopaminergic neurons activation and the GAD65/67 downregulation in VTA and prevented the formation of CPP induced by reexposure to morphine-associated context. Collectively, these results, for the first time, illustrated the involvement of neural circuitry of CA3-LSc-VTA, through integration of the contexts and reward information, participated in the reinstatement of CPP induced by exposure to morphine-associated context, which advanced our understanding on neurobiological basis for the context-associated memory and rewarding behavior.

The mechanisms underlying olfactory deficits in apolipoprotein E-deficient mice: focus on olfactory epithelium and olfactory bulb.

Apolipoprotein E (ApoE) is highly expressed in the central nervous system including the olfactory epithelium (OE) and olfactory bulb (OB). ApoE induction is beneficial for Alzheimer's disease (AD) treatment, whereas ApoE deficiency results in impaired olfaction, but the timing and underlying molecular and cellular mechanisms of these effects remain unclear. Uncovering the mechanisms underlying olfactory dysfunction in ApoE-deficient mice might provide a potential avenue for the early diagnosis of AD. We used an ApoE knockout (ApoE-/-) mouse model and a cookie-finding test to reveal an olfactory deficit in 3- to 5-month-old, but not 1- to 2-month-old, ApoE-/- mice. Electrophysiological experiments indicated a significant decline in the electroolfactogram (EOG) amplitude, which was associated with an increase in rise time in ApoE-/- mice. Knockout mice also exhibited compromised olfactory adaptation, as well as a reduced number of mature olfactory sensory neurons in the OE. Local field potential recording in the OB showed that gamma oscillation power was enhanced, which might be attributed to an increase in GABAergic inhibition mediated by parvalbumin-expressing (PV) interneurons. This study demonstrates the critical involvement of ApoE in olfactory information processing in the OE and OB. ApoE deficiency results in olfaction deficits in mice as young as 3 months old, which has implications for AD pathogenesis.

[A sketch of the overlap in the neural circuitry underlying psychological- and physical-pain].

From the phenomenological point of view, pain can be classified into psychological-pain and physical-pain. Emerging evidence has shown that the psychological- and physical-pain recruit overlapping neural activity in regions associated with the affective component of pain, and share some common pain circuits, e.g., the dorsal anterior cingulate cortex (dACC) and the anterior insula (AI) play important roles in both psychological- and physical-pain. Therefore, understanding the way in which psychological- and physical-pain demonstrate either similarity or discrepancy may provide new insights into the relationship between the two types of experiences and potential targets for treating psychological suffering. This review summarizes research progress that has been obtained through experiments conducted in human and nonhuman animals to discuss the similarity, discrepancy and interaction between psychological- and physical-pain. The important next steps, e.g., uncovering the mechanisms underlying the overlap of psychological- and physical-pain; and whether chronic psychological-pain shapes brain plasticity as physical-pain does, are also discussed.

Activation of TLR4/STAT3 signaling in VTA contributes to the acquisition and maintenance of morphine-induced conditioned place preference.

Morphine, commonly used to relieve the acute or chronic pain, has a high potential for addiction and exerts rewarding effects via a critical role for mesolimbic dopamine system. Studies suggest that addiction-related behavior is highly associated with inflammatory immune response, but the mechanisms are poorly understood. The present study showed that intra-VTA microinjection of TLR4 antagonist LPS-RS prevented the acquisition and maintenance, but not the expression, of morphine-induced CPP in rats. In addition, chronic morphine treatment significantly activated STAT3 on day 6 and 11 in VTA, and bilateral microinjection of STAT3 inhibitor S3I-201 into the VTA suppressed the acquisition and maintenance of morphine-induced CPP in rats. Furthermore, local knockout of STAT3 by injection of the AAV-Cre-GFP into the VTA area of STAT3flox/flox mice also significantly impaired the acquisition of morphine CPP. Importantly, the TLR4 expression is colocalized with p-STAT3-positive cell in VTA, and repeated injection of LPS-RS significantly attenuated the STAT3 activation in VTA induced by chronic morphine treatment. Collectively, these data suggest that TLR4/STAT3 signaling pathway in VTA might play a critical role in the acquisition and maintenance of morphine CPP, and provides new evidence that TLR4/STAT3 signaling pathway might be a potential target for treatment of morphine addiction.

APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.

Amyloid precursor protein (APP) is enriched at the synapse, but its synaptic function is still poorly understood. We previously showed that GABAergic short-term plasticity is impaired in App knock-out (App-/-) animals, but the precise mechanism by which APP regulates GABAergic synaptic transmission has remained elusive. Using electrophysiological, biochemical, moleculobiological, and pharmacological analysis, here we show that APP can physically interact with KCC2, a neuron-specific K+-Cl- cotransporter that is essential for Cl- homeostasis and fast GABAergic inhibition. APP deficiency results in significant reductions in both total and membrane KCC2 levels, leading to a depolarizing shift in the GABA reversal potential (EGABA). Simultaneous measurement of presynaptic action potentials and inhibitory postsynaptic currents (IPSCs) in hippocampal neurons reveals impaired unitary IPSC amplitudes attributable to a reduction in α1 subunit levels of GABAAR. Importantly, restoration of normal KCC2 expression and function in App-/- mice rescues EGABA, GABAAR α1 levels and GABAAR mediated phasic inhibition. We show that APP functions to limit tyrosine-phosphorylation and ubiquitination and thus subsequent degradation of KCC2, providing a mechanism by which APP influences KCC2 abundance. Together, these experiments elucidate a novel molecular pathway in which APP regulates, via protein-protein interaction with KCC2, GABAAR mediated inhibition in the hippocampus.

Orexin A-mediated AKT signaling in the dentate gyrus contributes to the acquisition, expression and reinstatement of morphine-induced conditioned place preference.

Accumulating evidence indicates that the hippocampal dentate gyrus (DG), a critical brain region contributing to learning and memory, is involved in the addiction and relapse to abused drugs. Emerging studies also suggest the role of orexin signaling in the rewarding behavior induced by repeated exposure to opiates. In the present study, we investigated the dynamic adaptation of orexin signaling in the DG and its functional significance in the acquisition, expression, maintenance of and relapse to rewarding behavior induced by morphine. Repeated place conditioning with morphine significantly increased the orexin A content released from the lateral hypothalamic area projecting neurons into the DG. Local infusions of orexin A into the DG sensitized the acquisition of and relapse to the conditioned place preference induced by morphine. The application of the orexin receptor type 1 (OXR1) antagonist SB334867 significantly abolished the acquisition, expression and maintenance of the conditioned place preference induced by repeated exposure to morphine. Furthermore, the significant increase of the phosphorylation of AKT in the DG was associated with preference for the morphine-paired chamber in rats, which was reversed by the local administration of an OXR1 antagonist. Thus, these findings suggested that the dynamic upregulation of orexin A signaling, via the AKT pathway in the DG, may promote the acquisition and maintenance of opioid-induced craving behaviors and may increase sensitivity to the rewarding effect of subsequent opioids.