mPFC Neurons Research Articles

Isolation of psychedelic-responsive neurons underlying anxiolytic behavioral states

Psychedelics hold promise as alternate treatments for neuropsychiatric disorders. However, the neural mechanisms by which they drive adaptive behavioral effects remain unclear. We isolated the specific neurons modulated by a psychedelic to determine their role in driving behavior. Using a light- and calcium-dependent activity integrator, we genetically tagged psychedelic-responsive neurons in the medial prefrontal cortex (mPFC) of mice. Single-nucleus RNA sequencing revealed that the psychedelic drove network-level activation of multiple cell types beyond just those expressing 5-hydroxytryptamine 2A receptors. We labeled psychedelic-responsive mPFC neurons with an excitatory channelrhodopsin to enable their targeted manipulation. We found that reactivation of these cells recapitulated the anxiolytic effects of the psychedelic without driving its hallucinogenic-like effects. These findings reveal essential insight into the cell-type–specific mechanisms underlying psychedelic-induced behavioral states.

Cannabidiol inhibits transient receptor potential canonical 4 and modulates excitability of pyramidal neurons in mPFC

Cannabidiol (CBD), a non-psychoactive compound derived from the cannabis plant, has been extensively studied for its potential therapeutic effects on various central nervous system (CNS) disorders, including epilepsy, chronic pain, Parkinson’s disease, and stress-related neuropsychiatric disorders. However, the pharmacological mechanisms of CBD have not been fully elucidated due to the complexity of their targets. In this study, we reported that the transient receptor potential canonical 4 (TRPC4) channel, a calcium-permeable, non-selective cation channel, could be inhibited by CBD. TRPC4 is highly abundant in the central nervous system and plays a critical role in regulating axonal regeneration, neurotransmitter release, and neuronal network activity. Here, we used whole-cell electrophysiology and intracellular calcium measurements to identify the inhibitory effects of CBD on TRPC4, in which CBD was found to inhibit TRPC4 channel with an IC50 value of 1.52 μM TRPC4 channels function as receptor-operated channels (ROC) and could be activated by epinephrine (EP) via G proteins. We show that CBD can inhibit EP-evoked TRPC4 current in vitro and EP-evoked neuronal excitability in the medial prefrontal cortex (mPFC). These results are consistent with the action of TRPC4-specific inhibitor Pico145, suggesting that TRPC4 works as a functional ionotropic receptor of CBD. This study identified TRPC4 as a novel target for CBD in the CNS and suggested that CBD could reduce the pyramidal neuron excitability by inhibiting TRPC4-containing channels in the mPFC.

Prenatal exposure to MAM impairs mPFC and hippocampal inhibitory function in mice during adolescence and adulthood.

Neurodevelopmental abnormalities are considered to be one of the important causes of schizophrenia. The offspring of methylazoxymethanol acetate (MAM)-exposed mice are recognized for the dysregulation of neurodevelopment and well-characterized with schizophrenia-like phenotypes. However, the inhibition-related properties of the medial prefrontal cortex (mPFC) and hippocampus throughout adolescence and adulthood have not been systematically elucidated. In this study, both 10 mg/kg and 15 mg/kg MAM-exposed mice exhibited schizophrenia-related phenotypes in both adolescence and adulthood, including spontaneous locomotion hyperactivity and deficits in prepulse inhibition (PPI). We observed that there was an obvious parvalbumin (PV) loss in mPFC and hippocampus of MAM-exposed mice, extending from adolescence to adulthood. Moreover, the frequency of sIPSCs in pyramidal neurons at mPFC and hippocampus was significantly dampened in the 10 mg/kg and 15 mg/kg MAM-exposed mice. Furthermore, the firing rate of putative pyramidal neurons in mPFC and hippocampus was increased, while that of putative inhibitory neurons was decreased during both adolescence and adulthood. In conclusion, PV loss in mPFC and hippocampus of MAM-exposed mice may contribute to the impaired inhibitory function leading to the attenuation of inhibition in the brain both in vitro and in vivo.Significance Statement PV neurons could play an important role in maintaining the balance of neural network homeostasis for its ability to provide strong inhibition. PV neurons were reduced in the mPFC and hippocampus in the schizophrenia patients. In this study, we demonstrated that 10 mg/kg and 15 mg/kg MAM exposure causes a loss of PV neurons in the mPFC and hippocampus of mice, which leads to an impaired inhibitory function. Overall, these results provided the significance of PV neurons modulating neural network homeostasis and involving in the pathogenesis of schizophrenia.

Locus coeruleus modulation of prefrontal dynamics and encoding of flexible rule switching.

Behavioral flexibility, the ability to adjust behavioral strategies in response to changing environmental contingencies and internal demands, is fundamental to cognitive functions. Despite a large body of pharmacology and lesion studies, the underlying neurophysiological correlates and mechanisms that support flexible rule switching are under active investigation. To address this question, we trained mice to distinguish complex sensory cues comprising different perceptual dimensions (set shifting). Endoscopic calcium imaging revealed that medial prefrontal cortex (mPFC) neurons exhibited pronounced dynamic changes during rule switching. Notably, prominent encoding capacity in the mPFC was associated with switching across, but not within perceptual dimensions. We then showed the functional importance of the ascending input from the locus coeruleus (LC), as LC inhibition impaired rule switching behavior and impeded mPFC dynamic processes and encoding. Our results highlight the pivotal role of the mPFC in set shifting processes and demonstrate the profound impact of ascending neuromodulation on shaping prefrontal neural dynamics and behavioral flexibility.

Divergent reward cue representations in prefrontal cortex underlie differences in reward motivation between adolescents and adults.

A prevailing view on postnatal brain development is that brain regions gradually acquire adult functions as they mature. The medial prefrontal cortex (mPFC) regulates reward learning, motivation, and behavioral inhibition, and undergoes a protracted postnatal maturation. During adolescence, reward-seeking behavior is heightened compared to adulthood - a developmental difference that may be driven by a hypoactive mPFC, with decreased top-down control of impulsive reward-seeking. However, this hypothesis has been difficult to test directly, due in part to technical challenges of recording neuronal activity in vivo across this developmental period. Here, using a novel 2-photon imaging-compatible platform for recording mPFC activity during an operant reward conditioning task beginning early in life, we show that the adolescent mPFC is hyper-responsive to reward cues. Distinct populations of mPFC neurons encode reward-predictive cues across development, but representations of no-reward cues and unrewarded outcomes are relatively muted in adolescence. Chemogenetic inhibition of GABAergic neurons decreased motivation in adolescence but not in adulthood. Together, our findings indicate that reward-related activity in the adolescent mPFC does not gradually increase across development. On the contrary, adolescent mPFC neurons are hyper-responsive to reward-related stimuli and encode reward-predictive cues and outcomes through qualitatively different mechanisms relative to the adult mPFC, opening avenues to developing distinct, developmentally informed strategies for modulating reward-seeking behavior in adolescence and adulthood.

Sex differences in neural representations of social and nonsocial reward in the medial prefrontal cortex

The reinforcing nature of social interactions is necessary for the maintenance of appropriate social behavior. However, the neural substrates underlying social reward processing and how they might differ based on the sex and internal state of the animal remains unknown. It is also unclear whether these neural substrates are shared with those involved in nonsocial rewarding processing. We developed a fully automated, two choice (social-sucrose) operant assay in which mice choose between social and nonsocial rewards to directly compare the reward-related behaviors associated with two competing stimuli. We performed cellular resolution calcium imaging of medial prefrontal cortex (mPFC) neurons in male and female mice across varying states of water restriction and social isolation. We found that mPFC neurons maintain largely non-overlapping, flexible representations of social and nonsocial reward that vary with internal state in a sex-dependent manner. Additionally, optogenetic manipulation of mPFC activity during the reward period of the assay disrupted reward-seeking behavior across male and female mice. Thus, using a two choice operant assay, we have identified sex-dependent, non-overlapping neural representations of social and nonsocial reward in the mPFC that vary with internal state and that are essential for appropriate reward-seeking behavior.

Activity in the dorsal hippocampus-mPFC circuit modulates stress-coping strategies during inescapable stress

Anatomical connectivity and lesion-deficit studies have shown that the dorsal and ventral hippocampi contribute to cognitive and emotional processes, respectively. However, the role of the dorsal hippocampus (dHP) in emotional or stress-related behaviors remains unclear. Here, we showed that neuronal activity in the dHP affects stress-coping behaviors in mice via excitatory projections to the medial prefrontal cortex (mPFC). The antidepressant ketamine rapidly induced c-Fos expression in both the dorsal and ventral hippocampi. The suppression of GABAergic transmission in the dHP-induced molecular changes similar to those induced by ketamine administration, including eukaryotic elongation factor 2 (eEF2) dephosphorylation, brain-derived neurotrophic factor (BDNF) elevation, and extracellular signal-regulated kinase (ERK) phosphorylation. These synaptic and molecular changes in the dHP induced a reduction in the immobility time of the mice in the tail-suspension and forced swim tests without affecting anxiety-related behavior. Conversely, pharmacological and chemogenetic potentiation of inhibitory neurotransmission in the dHP CA1 region induced passive coping behaviors during the tests. Transneuronal tracing and electrophysiology revealed monosynaptic excitatory connections between dHP CA1 neurons and mPFC neurons. Optogenetic stimulation of dHP CA1 neurons in freely behaving mice produced c-Fos induction and spike firing in the mPFC neurons. Chemogenetic activation of the dHP-recipient mPFC neurons reversed the passive coping behaviors induced by suppression of dHP CA1 neuronal activity. Collectively, these results indicate that neuronal activity in the dHP modulates stress-coping strategies to inescapable stress and contributes to the antidepressant effects of ketamine via the dHP-mPFC circuit.

Correlation between compulsive behaviors and plastic changes in the dendritic spines of the prefrontal cortex and dorsolateral striatum of male rats

Obsessive-compulsive disorder (OCD) is a mental affliction characterized by compulsive behaviors often manifested in intrusive thoughts and repetitive actions. The quinpirole model has been used with rats to replicate compulsive behaviors and study the neurophysiological processes associated with this pathology. Several changes in the dendritic spines of the medial prefrontal cortex (mPFC) and dorsolateral striatum (DLS) have been related to the occurrence of compulsive behaviors. Dendritic spines regulate excitatory synaptic contacts, and their morphology is associated with various brain pathologies. The present study was designed to correlate the occurrence of compulsive behaviors (generated by administering the drug quinpirole) with the morphology of the different types of dendritic spines in the mPFC and DLS. A total of 18 male rats were used. Half were assigned to the experimental group, the other half to the control group. The former received injections of quinpirole, while the latter rats were injected with physiological saline solution, for 10 days in both cases. After the experimental treatment, the quinpirole rats exhibited all the parameters indicative of compulsive behavior and a significant correlation with the density of stubby and wide neckless spines in both the mPFC and DLS. Dendritic spines from both mPFC and DLS neurons showed plastic changes correlatively with the expression of compulsive behavior induced by quinpirole. Further studies are suggested to evaluate the involvement of glutamatergic neurotransmission in the neurobiology of OCD.

Prefrontal and lateral entorhinal neurons co-dependently learn item-outcome rules.

The ability to learn novel items depends on brain functions that store information about items classified by their associated meanings and outcomes1-4, but the underlying neural circuit mechanisms of this process remain poorly understood. Here we show that deep layers of the lateral entorhinal cortex (LEC) contain two groups of 'item-outcome neurons': one developing activity for rewarded items during learning, and another for punished items. As mice learned an olfactory item-outcome association, we found that the neuronal population of LEC layers 5/6 (LECL5/6) formed an internal map of pre-learned and novel items, classified into dichotomic rewarded versus punished groups. Neurons in the medial prefrontal cortex (mPFC), which form a bidirectional loop circuit with LECL5/6, developed an equivalent item-outcome rule map during learning. When LECL5/6 neurons were optogenetically inhibited, tangled mPFC representations of novel items failed to split into rewarded versus punished groups, impairing new learning by mice. Conversely, when mPFC neurons were inhibited, LECL5/6 representations of individual items were held completely separate, disrupting both learning and retrieval of associations. These results suggest that LECL5/6 neurons and mPFC neurons co-dependently encode item memory as a map of associated outcome rules.

Deep brain stimulation targeted at lateral hypothalamus-medial forebrain bundle reverses depressive-like symptoms and related cognitive deficits in rat: Role of serotoninergic system

The aim of the study is to understand the rationale behind the application of deep brain stimulation (DBS) in the treatment of depression. Male Wistar rats, rendered depressive with chronic unpredictable mild stress (CUMS) were implanted with electrode in the lateral hypothalamus-medial forebrain bundle (LH-MFB) and subjected to deep brain stimulation (DBS) for 4 h each day for 14 days. DBS rats, as well as controls, were screened for a range of parameters indicative of depressive state. Symptomatic features noticed in CUMS rats like the memory deficit, anhedonia, reduction in body weight and 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels in mPFC and elevated plasma corticosterone were reversed in rats subjected to DBS. DBS arrested CUMS induced degeneration of 5-HT cells in interfascicular region of dorsal raphe nucleus (DRif) and fibers in LH-MFB and induced dendritic proliferation in mPFC neurons. MFB is known to serve as a major conduit for the DRif-mPFC serotoninergic pathway. While the density of serotonin fibers in the LH-MFB circuit was reduced in CUMS, it was upregulated in DBS-treated rats. Furthermore, microinjection of 5-HT1A receptor antagonist, WAY100635 into mPFC countered the positive effects of DBS like the antidepressant and memory-enhancing action. In this background, we suggest that DBS at LH-MFB may exercise positive effect in depressive rats via upregulation of the serotoninergic system. While these data drawn from the experiments on rat provide meaningful clues, we suggest that further studies aimed at understanding the usefulness of DBS at LH-MFB in humans may be rewarding.

Structural plasticity of pyramidal cell neurons measured after FLASH and conventional dose-rate irradiation.

Evidence shows that ultra-high dose-rate FLASH-radiotherapy (FLASH-RT) protects against normal tissue complications and functional decrements in the irradiated brain. Past work has shown that radiation-induced cognitive impairment, neuroinflammation and reduced structural complexity of granule cell neurons were not observed to the same extent after FLASH-RT (> MGy/s) compared to conventional dose-rate (CONV, 0.1 Gy/s) delivery. To explore the sensitivity of different neuronal populations to cranial irradiation and dose-rate modulation, hippocampal CA1 and medial prefrontal cortex (PFC) pyramidal neurons were analyzed by electron and confocal microscopy. Neuron ultrastructural analyses by electron microscopy after 10 Gy FLASH- or CONV-RT exposures indicated that irradiation had little impact on dendritic complexity and synapse density in the CA1, but did increase length and head diameter of smaller non-perforated synapses. Similarly, irradiation caused no change in PFC prelimbic/infralimbic axospinous synapse density, but reductions in non-perforated synapse diameters. While irradiation resulted in thinner myelin sheaths compared to controls, none of these metrics were dose-rate sensitive. Analysis of fluorescently labeled CA1 neurons revealed no radiation-induced or dose-rate-dependent changes in overall dendritic complexity or spine density, in contrast to our past analysis of granule cell neurons. Super-resolution confocal microscopy following a clinical dosing paradigm (3×10Gy) showed significant reductions in excitatory vesicular glutamate transporter 1 and inhibitory vesicular GABA transporter puncta density within the CA1 that were largely dose-rate independent. Collectively, these data reveal that, compared to granule cell neurons, CA1 and mPFC neurons are more radioresistant irrespective of radiation dose-rate.

Sex differences in change-of-mind neuroeconomic decision-making is modulated by LINC00473 in medial prefrontal cortex.

Changing one's mind is a complex cognitive phenomenon involving a continuous re-appraisal of the trade-off between past costs and future value. Recent work modeling this behavior across species has established associations between aspects of this choice process and their contributions to altered decision-making in psychopathology. Here, we investigated the actions in medial prefrontal cortex (mPFC) neurons of long intergenic non-coding RNA, LINC00473, known to induce stress resilience in a striking sex-dependent manner, but whose role in cognitive function is unknown. We characterized complex decision-making behavior in male and female mice longitudinally in our neuroeconomic foraging paradigm, Restaurant Row, following virus-mediated LINC00473 expression in mPFC neurons. On this task, mice foraged for their primary source of food among varying costs (delays) and subjective value (flavors) while on a limited time-budget during which decisions to accept and wait for rewards were separated into discrete stages of primary commitments and secondary re-evaluations. We discovered important differences in decision-making behavior between female and male mice. LINC00473 expression selectively influenced multiple features of re-evaluative choices, without affecting primary decisions, in female mice only. These behavioral effects included changing how mice (i) cached the value of the passage of time and (ii) weighed their history of economically disadvantageous choices. Both processes were uniquely linked to change-of-mind decisions and underlie the computational bases of distinct aspects of counterfactual thinking. These findings reveal a key bridge between a molecular driver of stress resilience and psychological mechanisms underlying sex-specific decision-making proclivities.

A prefrontal-habenular circuitry regulates social fear behaviour.

The medial prefrontal cortex (mPFC) has been implicated in the pathophysiology of social impairments including social fear. However, the precise subcortical partners that mediate mPFC dysfunction on social fear behaviour have not been identified. Employing a social fear conditioning paradigm, we induced robust social fear in mice and found that the lateral habenula (LHb) neurons and LHb-projecting mPFC neurons are synchronously activated during social fear expression. Moreover, optogenetic inhibition of the mPFC-LHb projection significantly reduced social fear responses. Importantly, consistent with animal studies, we observed an elevated prefrontal-habenular functional connectivity in subclinical individuals with higher social anxiety characterized by heightened social fear. These results unravel a crucial role of the prefrontal-habenular circuitry in social fear regulation and suggest that this pathway could serve as a potential target for the treatment of social fear symptom often observed in many psychiatric disorders.

Unraveling the Pathogenesis of Post-Stroke Depression in a Hemorrhagic Mouse Model through Frontal Lobe Circuitry and JAK-STAT Signaling.

Post-stroke depression is a common complication that imposes significant burdens and challenges on patients. The occurrence of depression is often associated with frontal lobe hemorrhage, however, current understanding of the underlying mechanisms remains limited. Here, the pathogenic mechanisms associated with the circuitry connectivity, electrophysiological alterations, and molecular characteristics are investigated related to the frontal lobe in adult male mice following unilateral injection of blood in the medial prefrontal cortex (mPFC). It is demonstrated that depression is a specific neurological complication in the unilateral hematoma model of the mPFC, and the ventral tegmental area (VTA) shows a higher percentage of connectivity disruption compared to the lateral habenula (LHb) and striatum (STR). Additionally, long-range projections originating from the frontal lobe demonstrate higher damage percentages within the connections between each region and the mPFC. mPFC neurons reveal reduced neuronal excitability and altered synaptic communication. Furthermore, transcriptomic analysis identifies the involvement of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT)signaling pathway, and targeting the JAK-STAT pathway significantly alleviates the severity of depressive symptoms. These findings improve the understanding of post-hemorrhagic depression and may guide the development of efficient treatments.

Prefrontal cortex neurons encode ambient light intensity differentially across regions and layers

While light can affect emotional and cognitive processes of the medial prefrontal cortex (mPFC), no light-encoding was hitherto identified in this region. Here, extracellular recordings in awake mice revealed that over half of studied mPFC neurons showed photosensitivity, that was diminished by inhibition of intrinsically photosensitive retinal ganglion cells (ipRGCs), or of the upstream thalamic perihabenular nucleus (PHb). In 15% of mPFC photosensitive neurons, firing rate changed monotonically along light-intensity steps and gradients. These light-intensity-encoding neurons comprised four types, two enhancing and two suppressing their firing rate with increased light intensity. Similar types were identified in the PHb, where they exhibited shorter latency and increased sensitivity. Light suppressed prelimbic activity but boosted infralimbic activity, mirroring the regions’ contrasting roles in fear-conditioning, drug-seeking, and anxiety. We posit that prefrontal photosensitivity represents a substrate of light-susceptible, mPFC-mediated functions, which could be ultimately studied as a therapeutical target in psychiatric and addiction disorders.

Role of medial prefrontal cortex voltage-dependent potassium 4.3 channels in nicotine-induced enhancement of object recognition memory in male mice

Nicotine has been shown to enhance object recognition memory in the novel object recognition (NOR) test by activating excitatory neurons in the medial prefrontal cortex (mPFC). However, the exact neuronal mechanisms underlying the nicotine-induced activation of mPFC neurons and the resultant memory enhancement remain poorly understood. To address this issue, we performed brain-slice electrophysiology and the NOR test in male C57BL/6J mice. Whole-cell patch-clamp recordings from layer V pyramidal neurons in the mPFC revealed that nicotine augments the summation of evoked excitatory postsynaptic potentials (eEPSPs) and that this effect was suppressed by N-[3,5-Bis(trifluoromethyl)phenyl]-N’-[2,4-dibromo-6-(2H-tetrazol-5-yl)phenyl]urea (NS5806), a voltage-dependent potassium (Kv) 4.3 channel activator. In line with these findings, intra-mPFC infusion of NS5806 suppressed systemically administered nicotine-induced memory enhancement in the NOR test. Additionally, miRNA-mediated knockdown of Kv4.3 channels in mPFC pyramidal neurons enhanced object recognition memory. Furthermore, inhibition of A-type Kv channels by intra-mPFC infusion of 4-aminopyridine was found to enhance object recognition memory, while this effect was abrogated by prior intra-mPFC NS5806 infusion. These results suggest that nicotine augments the summation of eEPSPs via the inhibition of Kv4.3 channels in mPFC layer V pyramidal neurons, resulting in the enhancement of object recognition memory.

Prefrontal cortical network dysfunction from acute neurotoxicant exposure.

Prefrontal cortical (PFC) dysfunction has been linked to disorders exhibiting deficits in cognitive performance, attention, motivation, and impulse control. Neurons of the PFC are susceptible to glutamatergic excitotoxicity, an effect associated with cortical degeneration in frontotemporal disorders (FTDs). PFC susceptibility to environmental toxicant exposure, one possible contributor to sporadic FTD, has not been systematically studied. Here, we tested the ability of a well-known environmental neurotoxicant, methylmercury (MeHg), to induce hyperexcitability in medial prefrontal cortex (mPFC) excitatory pyramidal neurons, using whole cell patch-clamp recording. Acute MeHg exposure (20 μM) produced significant mPFC dysfunction, with a shift in the excitatory to inhibitory (E-I) balance toward increased excitability. Both excitatory postsynaptic current (EPSC) and inhibitory postsynaptic current (IPSC) charges were significantly increased after MeHg exposure. MeHg increased EPSC frequency, but there was no observable effect on IPSC frequency, EPSC amplitude or IPSC amplitude. Neither evoked AMPA receptor- nor NMDA receptor-mediated EPSC amplitudes were affected by MeHg. However, excitatory synapses experienced a significant reduction in paired-pulse depression and probability of release. In addition, MeHg induced temporal synchrony in spontaneous IPSCs, reflecting mPFC inhibitory network dysfunction. MeHg exposure also produced increased intrinsic excitability in mPFC neurons, with an increase in action potential firing rate. The observed effects of MeHg on mPFC reflect key potential mechanisms for neuropsychological symptoms from MeHg poisoning. Therefore, MeHg has a significant effect on mPFC circuits known to contribute to cognitive and emotional function and might contribute to etiology of neurodegenerative diseases, such as FTD.NEW & NOTEWORTHY Prefrontal cortical neurons are highly susceptible to glutamatergic excitotoxicity associated with neuronal degeneration in frontal dementia and to environmental toxicant exposure, one potential contributor to FTD. However, this has not been systematically studied. Our results demonstrate that methylmercury exposure leads to hyperexcitability of prefrontal cortical neurons by shifting excitatory to inhibitory (E-I) balance and raising sensitivity for spiking. Our results provide a mechanism by which environmental neurotoxicants may contribute to pathogenesis of diseases such as FTD.

Sex-Dependent Effects of Angiotensin Type 2 Receptor–Expressing Medial Prefrontal Cortex Interneurons in Fear Extinction Learning

BackgroundThe renin-angiotensin system has been identified as a potential therapeutic target for posttraumatic stress disorder, although its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC), but their function and mechanism in the mPFC remain unexplored. Therefore, we used a combination of imaging, cre/lox, and behavioral methods to investigate mPFC-AT2R–expressing neurons in fear and stess related behavior. MethodsTo characterize mPFC-AT2R–expressing neurons in the mPFC, AT2R-Cre/tdTomato male and female mice were used for immunohistochemistry. mPFC brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker were quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using a Cre-expressing adeno-associated virus. Mice then underwent Pavlovian auditory fear conditioning, elevated plus maze, and open field testing. ResultsImmunohistochemistry results revealed that AT2R was densely expressed throughout the mPFC and primarily expressed in somatostatin interneurons in a sex-dependent manner. Following fear conditioning, mPFC-AT2R Cre-lox deletion impaired extinction and increased exploratory behavior in female but not male mice, while locomotion was unaltered by mPFC-AT2R deletion in both sexes. ConclusionsThese results identify mPFC-AT2R+ neurons as a novel subgroup of somatostatin interneurons and reveal their role in regulating fear learning in a sex-dependent manner, potentially offering insights into novel therapeutic targets for posttraumatic stress disorder.

Prophylactic nicotinamide mononucleotide (NMN) mitigates CSDS-induced depressive-like behaviors in mice via preserving of ATP level in the mPFC

Depression is a prevalent psychiatric disorder with accumulating evidence implicating dysregulation of extracellular adenosine triphosphate (ATP) levels in the medial prefrontal cortex (mPFC). It remains unclear whether facilitating endogenous ATP production and subsequently increasing extracellular ATP level in the mPFC can exert a prophylactic effect against chronic social defeat stress (CSDS)-induced depressive-like behaviors and enhance stress resilience. Here, we found that nicotinamide mononucleotide (NMN) treatment effectively elevated nicotinamide adenine dinucleotide (NAD+) biosynthesis and extracellular ATP levels in the mPFC. Moreover, both the 2-week intraperitoneal (i.p.) injection and 3-week oral gavage of NMN prior to exposure to CSDS effectively prevented the development of depressive-like behavior in mice. These protective effects were accompanied with the preservation of both NAD+ biosynthesis and extracellular ATP level in the mPFC. Furthermore, catalyzing ATP hydrolysis by mPFC injection of the ATPase apyrase negated the prophylactic effects of NMN on CSDS-induced depressive-like behaviors. Prophylactic NMN treatment also prevented the reduction in GABAergic inhibition and the increase in excitability in mPFC neurons projecting to the lateral habenula (LHb). Collectively, these findings demonstrate that the prophylactic effects of NMN on depressive-like behaviors are mediated by preventing extracellular ATP loss in the mPFC, which highlights the potential of NMN supplementation as a novel approach for protecting and preventing stress-induced depression in susceptible individuals.

Sex-Dependent Effects of Angiotensin Type 2 Receptor Expressing Medial Prefrontal Cortex (mPFC) Interneurons in Fear Extinction Learning

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC). Their function and mechanism in the mPFC, however, remain unexplored. We therefore used a combination of neuroanatomical, chemogenetic, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female brains were perfused and used for immunohistochemistry. Brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus. Mice then underwent Pavlovian auditory fear conditioning and anxiety-like and locomotor behavior testing using open field (OF) and elevated plus maze (EPM) tests. Results: IHC revealed that AT2R is expressed throughout the mPFC in males ( 208.6±48.8 cells/mm2) and females ( 139.0±59.8 cells/mm2) and has low co-expression on glutamatergic neurons ( TBR1 co-staining, males: 7.1%±1.8%; females: 13.2%±4.2%). Of the interneuron markers tested, AT2R is primarily expressed on somatostatin interneurons, and has higher colocalization in the mPFC of females than males ( males: 16.5%±1.2%, females: 31.8%±4.2%, p=0.02). Following fear conditioning and extinction, mPFC-AT2R deletion impaired extinction in female ( p=0.03) but not male ( p=0.76) mice. Locomotion in the OF was unaltered by mPFC-AT2R deletion in males or females ( total distance travelled; males: GFP 44.11m±5.92m, Cre 56.00m±10.29m, p=0.35; females: GFP 42.86m±4.85m, Cre 42.39m±4.97m, p=0.95), while AT2R-deleted females had increased exploratory behavior in the EPM ( open arm entries; males: GFP 12.38±1.02, Cre 13.22±1.54, p=0.66; females: GFP 12.50±0.96, Cre 20.00±2.81, p=0.02). Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel somatostatin subgroup that influences fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences, and increases the understanding of circuitry and function of the brain RAS in disordered fear learning. This may lead to improved therapeutic treatments for PTSD. This research and PJM was supported by NIH1R01HL137103-01A1 and the American Heart Association 15CSA24340001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.