Neurons In Slices Research Articles

Regulation of Hippocamposeptal Synaptic Transmission by GABABRs Is Altered in 5XFAD Mice in a Sex- and Age-Dependent Manner

Hippocamposeptal (HS) neurons send GABAergic projections from the hippocampus to the medial septum/diagonal band of Broca (MS/DBB) as part of a reciprocal loop that is critical for memory. HS neurons are proposed to be particularly sensitive to the deleterious effects of pathological exposure to amyloid-β (Aβ), as would occur during Alzheimer’s disease (AD). However, it is not known how HS GABA release in the MS/DBB is altered during the progression of AD. To target HS neurons in a mouse model of AD, we crossed SST-Cre mice to 5XFAD mice and performed stereotaxic injections of Cre-dependent AAV containing mCherry/channelrhodopsin-2 (ChR2) into the hippocampus of offspring at 4, 6, 9, and 12 months. We used optogenetics to selectively stimulate HS terminals while performing whole-cell patch-clamp recordings from MS/DBB neurons in slices. There was a transient reduction in HS-inhibitory postsynaptic current (IPSC) amplitude in female 5XFAD mice at 6 months, but no difference in males at any age, and no difference in paired-pulse ratio in either sex at any age. When bath applying the GABABR agonist, baclofen, we found a larger decrease in HS-IPSC amplitude in 5XFAD females at 9 months and 5XFAD males at 12 months. In 12-month-old 5XFAD females, response to baclofen was significantly reduced. These data suggest that there is a transient increase in responsiveness to GABABR activation in 5XFAD mice that occurs earlier in females than in males. These sex-specific changes to HS function are likely to impact the relay of information between the hippocampus and MS/DBB.

An exact mathematical description of computation with transient spatiotemporal dynamics in a complex-valued neural network

Networks throughout physics and biology leverage spatiotemporal dynamics for computation. However, the connection between structure and computation remains unclear. Here, we study a complex-valued neural network (cv-NN) with linear interactions and phase-delays. We report the cv-NN displays sophisticated spatiotemporal dynamics, which we then use, in combination with a nonlinear readout, for computation. The cv-NN can instantiate dynamics-based logic gates, encode short-term memories, and mediate secure message passing through a combination of interactions and phase-delays. The computations in this system can be fully described in an exact, closed-form mathematical expression. Finally, using direct intracellular recordings of neurons in slices from neocortex, we demonstrate that computations in the cv-NN are decodable by living biological neurons as the nonlinear readout. These results demonstrate that complex-valued linear systems can perform sophisticated computations, while also being exactly solvable. Taken together, these results open future avenues for design of highly adaptable, bio-hybrid computing systems that can interface seamlessly with other neural networks.

N-(4-Bromo-2,5-Dimethoxyphenethyl)-6-(4-Phenylbutoxy)Hexan-1-Amine (XOB): A Novel Phenylalkylamine Antagonist of Serotonin 2A Receptors and Voltage-Gated Sodium Channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Toward this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. SIGNIFICANCE STATEMENT: The authors synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels, in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus.

Millions of people struggle with alcohol use disorder (AUD). Abrupt abstinence after a period of chronic alcohol use can precipitate the alcohol withdrawal syndrome (AWS), which includes hyperexcitability and, potentially, seizures. We have shown that T-type Ca2+ channels are novel, sensitive targets of alcohol, an effect that is dependent upon protein kinase C (PKC). The purpose of this study was to (1) understand midline thalamic neuronal hyperexcitability during alcohol withdrawal and its dependence on PKC; (2) characterize T channel functional changes using both current clamp and voltage clamp methods; and (3) determine which PKC isoform may be responsible for alcohol withdrawal (WD) effects. Whole-cell patch clamp recordings were performed in midline thalamic neurons in brain slices prepared from C57bl/6 mice that underwent chronic intermittent alcohol exposure in a standard vapor chamber model. The recordings were compared to those from air-exposed controls. T-channel inactivation curves and burst responses were acquired through voltage-clamp and current-clamp recordings, respectively. Whole-cell voltage clamp recordings of native T-type current exhibited a depolarizing shift in the voltage-dependency of inactivation during alcohol withdrawal compared to air-exposed controls. A PKCε translocation inhibitor peptide mitigated this change. Current clamp recordings demonstrated more spikes per burst during alcohol withdrawal. Consistent with voltage clamp findings, the PKCɛ translocation inhibitor peptide reduced the number of spikes per burst after WD. We found that alcohol WD produces T channel-mediated hyperexcitability in the midline thalamus, produced in part by a shift in the inactivation curve consistent with greater availability of T current. WD effects on T current inactivation were reduced to control levels by blocking PKCε translocation. Our results demonstrate that PKCε translocation plays an important role in the regulation of alcohol withdrawal-induced hyperexcitability in midline thalamic circuitry.

Perinatal High Fat Diet Exposure Reduces KCC2 Expression and Alters GABAergic Signaling in the Dorsal Motor Nucleus of the Vagus

Perinatal high fat diet (pHFD) exposure alters the development of vagal neurocircuits controlling gastrointestinal (GI) functions resulting in gastric motor dysfunction. GABAergic synaptic inhibition of vagal efferent motoneurons within the dorsal motor nucleus of the vagus (DMV) is critical in the brain’s ability to modulate gastric motility. This inhibition is maintained by KCC2, a Cl− transporter, who determines intracellular chloride concentration. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently decreasing GABAergic inhibition and delaying gastric emptying rates. Pregnant Sprague-Dawley rats were fed either a control or high fat (14% vs 60% kcal from fat, respectively) diet from embryonic day 13, and offspring were used starting postnatal day 28. In vitro electrophysiological recordings were made from adult DMV neurons in brainstem slices. To assess the GABA-mediated inhibition, cell-attached recordings were made to preserve the internal Cl− concentration, and firing rate was quantified before and after application of bicuculline (BIC; 30μM), a GABAA antagonist. To determine the Cl− reversal potential, perforated patch clamp recordings were performed and the response to picospritz application of the GABAA agonist muscimol (100μM) was measured. KCC2 expression was quantified using pharmacology in the in vitro slice preparation, as well as immunofluorescence in brainstem cross-sections. Gastric emptying rates were quantified using the in vivo13C octanoic acid breath test technique. KCC2 transfection and induced expression in the DMV of pHFD rats was achieved using an adeno-associated viral vector. BIC increased firing rate in control DMV neurons (143.1% of baseline), but failed to increase DMV firing rate in DMV neurons from pHFD rats (96.27% of baseline; p=0.0103 using unpaired t-test). When control DMV neurons were treated with VU0240551 (VU; 10μM), a KCC2 blocker, BIC application led to a decrease in the firing rate (3.118 Hz to 1.606 Hz; p=0.0375 via paired t-test), demonstrating that antagonizing KCC2 attenuates GABAergic inhibition at this synapse. In control DMV neurons, the Cl− reversal potential was calculated to be -64.6mV. There was a depolarizing shift in Cl− reversal potential observed in pHFD neurons (-43.0mV; p=0.0007 using unpaired t-test). Application of VU shifted the chloride reversal from -64.6mV to -57.3mV in control neurons (p=0.0416 using a paired t-test). VU application had a reduced impact on pHFD neurons, shifting the reversal from -46.7mV to -45.0mV (p=0.1689 using a paired t-test), suggesting KCC2 downregulation. In fact, compared to control rats, there was a significant reduction in KCC2 expression in the pHFD DMV (2.237% area fluorescence in pHFD vs. 5.335%; p=0.0147 using unpaired t-test). When KCC2 expression was virally induced in young adult pHFD rats, GABA became inhibitory (163.7% of baseline firing rate after BIC application vs. 93.25% in pHFD + empty vector (EV); p=0.0019 using unpaired t-test), the Cl− reversal potential was normalized (-63.0mV vs. -49.7mV in pHFD + EV; p=0.0012 using an unpaired t-test), and gastric emptying rates were accelerated (58.7min vs 72.1min in pHFD + EV). Taken together, these results suggest that the loss of KCC2 function is a driver of the gastric pathophysiology seen in the pHFD rats, and that normalizing KCC2 levels can rescue the cellular and gastric phenotype. DK 111667. 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.

Dysfunction of M channels in the insular cortex is involved in pathogenesis of primary hypertension

Heightened sympathetic nerve activity is critically involved in the pathogenesis of primary hypertension. However, the sources driving increased sympathetic vasomotor tone remain unclear. The posterior insular cortex (PIC) is a brain region involved in regulating blood pressure and sympathetic tone. Neurons in the PIC project to the rostral ventrolateral medulla (RVLM), a key brain stem region in regulating sympathetic nerve activity and blood pressure. The non-inactivating voltage-dependent K+ channels, M channels (Kv7/KCNQ channel family) encoded by KCNQ2/3 genes, play a unique role in stabilizing the neuronal membrane potential and regulating neuronal excitability. Here, we determined if the M channel activity of neurons in the PIC is altered in a primary hypertension. Compared with Wistar-Kyoto (WKY) rats, the mRNA and protein expression levels of Kv7.2/Kv7.3 in the PIC tissue were significantly reduced in spontaneously hypertensive rats (SHRs). Microinjection of M channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) into the PIC increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in anesthetized WKY rats, while induced minimal changes of ABP and RSNA in SHRs. M currents recorded from PIC neurons in brain slices labeled by retrograde tracer, Fluospheres, injected into the RVLM were significantly smaller in SHRs than in WKY rats. Furthermore, bath application of specific M channel blocker XE-991 increased the firing rate of PIC-RVLM output neurons in WKY rats while caused a minimal increase in firing rate of PIC-RVLM output neurons in SHRs. In addition, M currents recorded from PIC-RVLM output neurons in SHRs were significantly smaller than those recorded from WKY rats. These data suggest that M channel activity is diminished in PIC-RVLM output neurons in SHRs, and the diminished Kv7 channel activity contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new a mechanistic insight into the pathogenesis of neurogenic hypertension. Supported by grants from National Institutes of Health (HL139523, HL142133, and HL159157 to DPL). 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.

Glucocorticoid receptors regulate hypothalamic CRH neuron activity in primary hypertension

The hypothalamic-pituitary-adrenal axis is important in maintaining homeostasis in response to physical and psychological stressors. The corticotropin-releasing hormone (CRH) is a neuropeptide synthesized by CRH neurons in brain regions including paraventricular nucleus (PVN) of the hypothalamus. The glucocorticoids synthesized and released in adrenal gland act on hypothalamic CRH-expressing neurons inhibit the CRH production and maintain the level of glucocorticoids. The glucocorticoid receptor (GR) is critical for this negative feedback to regulate CRH synthesis and release. Hypertension is associated with high stress response and CRH mRNA levels. The number of CRH-expression neurons are significantly increased in PVN of the hypothalamus in primary hypertension patients. this study determined if the HPA axis negative feedback is altered and contribute to maintaining hypertension. We measured mRNA expression levels of GRs and MR in PVN tissue obtained from adult Wistar-Kyoto normotensive rats (WKYs) and spontaneously hypertensive rats (SHRs) and found that the GR mRNA expression level was significantly lower in SHRs that in WKY rats, while the MR gene expression did not differ between WKY rats and SHRs. Furthermore, we found that acute perfusion of dexamethasone (DEX), a synthetic glucocorticoid, significantly decreased frequency of spontaneous excitatory postsynaptic synaptic currents (EPSCs) in identified PVN-CRH expressing neurons in brain slices prepared from WKY and SHRs. DEX caused a greater reduction of frequency of EPSCs in SHRs was than in WKY rats. Acute perfusion of DEX significantly increased the firing rate of PVN-CRH neurons in WKY rats but did not change the firing rate in SHRs. In addition, incubation of brain slices with DEX for 1 hr significantly inhibited the firing activity of PVN-CRH neurons in WKY rats but did not change the firing activity of PVN CRH neurons in SHRs. These data suggested that the glucocorticoid excites PVN-CRH neurons in acute phase while inhibits these neurons in chronic phase in normotensive rat. In hypertension, downregulated GR may be responsible for impaired the HPA axis feedback and contribute to hypertension. HL139523, HL142133, and HL159157. 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.

Pathogenic gating pore current conducted by autism-related mutations in the NaV1.2 brain sodium channel

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social and communication deficits and repetitive behaviors. The genetic heterogeneity of ASD presents a challenge to the development of an effective treatment targeting the underlying molecular defects. ASD gating charge mutations in the KCNQ/KV7 potassium channel cause gating pore currents (Igp) and impair action potential (AP) firing of dopaminergic neurons in brain slices. Here, we investigated ASD gating charge mutations of the voltage-gated SCN2A/NaV1.2 brain sodium channel, which ranked high among the ion channel genes with mutations in individuals with ASD. Our results show that ASD mutations in the gating charges R2 in Domain-II (R853Q), and R1 (R1626Q) and R2 (R1629H) in Domain-IV of NaV1.2 caused Igp in the resting state of ~0.1% of the amplitude of central pore current. The R1626Q mutant also caused significant changes in the voltage dependence of fast inactivation, and the R1629H mutant conducted proton-selective Igp. These potentially pathogenic Igp were exacerbated by the absence of the extracellular Mg2+ and Ca2+. In silico simulation of the effects of these mutations in a conductance-based single-compartment cortical neuron model suggests that the inward Igp reduces the time to peak for the first AP in a train, increases AP rates during a train of stimuli, and reduces the interstimulus interval between consecutive APs, consistent with increased neural excitability and altered input/output relationships. Understanding this common pathophysiological mechanism among different voltage-gated ion channels at the circuit level will give insights into the underlying mechanisms of ASD.

Analysis of Ionomic Profiles of Spinal Cords in a Rat Model with Bone Cancer Pain.

Ionomics is used to study levels of ionome in different states of organisms and their correlations. Bone cancer pain (BCP) severely reduces quality of life of patients or their lifespan. However, the relationship between BCP and ionome remains unclear. The BCP rat model was constructed through inoculation of Walker 256 cells into the left tibia. Von Frey test, whole-cell patch-clamp recording and inductively coupled plasma mass spectrometry (ICP-MS) technologies were conducted for measuring tactile hypersensitivity, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) of neurons of spinal slices, and ionome of spinal cord samples, respectively. Principal component analysis (PCA) was used to explore ionomic patterns of the spinal cord. The BCP rat model was successfully constructed through implantation of Walker 256 cells into the left tibia. The frequency and amplitude of mEPSCs of neurons in the spinal cord slices from the BCP model rats were notably greater than those in the sham control. In terms of ionomics, the spinal cord levels of two macroelements (Ca and S), four microelements (Fe, Mn, Li and Sr) and the toxic element Ti in the BCP group of rats were significantly increased by inoculation of Walker 256 cancer cells, compared to the sham control. In addition, the correlation patterns between the elements were greatly changed between the sham control and BCP groups. PCA showed that inoculation of Walker 256 cells into the tibia altered the overall ionomic profiles of the spinal cord. There was a significant separation trend between the two groups. Taken together, inoculation of Walker 256 cells into the left tibia contributes to BCP, which could be closely correlated by some elements. The findings provided novel information on the relationship between the ionome and BCP.

Opportunities for risk-taking during play alters cognitive performance and prefrontal inhibitory signalling in rats of both sexes.

Social play behaviour is a rewarding activity that can entail risks, thus allowing young individuals to test the limits of their capacities and to train their cognitive and emotional adaptability to challenges. Here, we tested in rats how opportunities for risk-taking during play affect the development of cognitive and emotional capacities and medial prefrontal cortex (mPFC) function, a brain structure important for risk-based decision making. Male and female rats were housed socially or social play-deprived (SPD) between postnatal day (P)21 and P42. During this period, half of both groups were daily exposed to a high-risk play environment. Around P85, all rats were tested for cognitive performance and emotional behaviour after which inhibitory currents were recorded in layer 5 pyramidal neurons in mPFC slices. We show that playing in a high-risk environment altered cognitive flexibility in both sexes and improved behavioural inhibition in males. High-risk play altered anxiety-like behaviour in the elevated plus maze in males and in the open field in females, respectively. SPD affected cognitive flexibility in both sexes and decreased anxiety-like behaviour in the elevated plus maze in females. We found that synaptic inhibitory currents in the mPFC were increased in male, but not female, rats after high-risk play, while SPD lowered prefrontal cortex (PFC) synaptic inhibition in both sexes. Together, our data show that exposure to risks during play affects the development of cognition, emotional behaviour and inhibition in the mPFC. Furthermore, our study suggests that the opportunity to take risks during play cannot substitute for social play behaviour.

Development and prenatal exposure to androgens alter potassium currents in gonadotropin-releasing hormone neurons from female mice.

Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for reproduction. Disruptions to GnRH secretion patterns may contribute to polycystic ovary syndrome (PCOS). Prenatally androgenized (PNA) female mice recapitulate many neuroendocrine abnormalities observed in PCOS patients. PNA and development induce changes in spontaneous GnRH neuron firing rate, response to synaptic input, and the afterhyperpolarization potential of the action potential. We hypothesized potassium currents are altered by PNA treatment and/or development. Whole-cell patch-clamp recordings were made of transient and residual potassium currents of GnRH neurons in brain slices from 3-week-old and adult control and PNA females. At 3 weeks of age, PNA treatment increased transient current density versus controls. Development and PNA altered voltage-dependent activation and inactivation of the transient current. In controls, transient current activation and inactivation were depolarized at 3 weeks of age versus in adulthood. In GnRH neurons from 3-week-old mice, transient current activation and inactivation were more depolarized in control than PNA mice. Development and PNA treatment interacted to shift the time-dependence of inactivation and recovery from inactivation. Notably, in cells from adult PNA females, recovery was prolonged compared to all other groups. Activation of the residual current occurred at more depolarized membrane potentials in 3-week-old than adult controls. PNA depolarized activation of the residual current in adults. These findings demonstrate the properties of GnRH neuron potassium currents change during typical development, potentially contributing to puberty, and further suggest PNA treatment may both alter some typical developmental changes and induce additional modifications, which together may underlie aspects of the PNA phenotype. There was not any clinical trial involved in this work.

Robotic Fast Dual-Arm Patch Clamp System for Mechanosensitive Excitability Research of Neurons.

A robotic fast dual-arm patch clamp system with controllable mechanical stimulation is proposed in this paper for mechanosensitive excitability research of neurons in brain slice. First, a kinematic model of a dual-arm patch clamp system combined with Monte Carlo method is developed to calculate the workspaces of recording micropipette and stimulation micropipette, and optimize the length of end effector for reducing collision incidences during operation. Then, a quantitative stimulation method to cells using one micropipette is developed based on pressing depth control. Finally, a fast robotic dual-arm patch clamp operation process is proposed based on a three-stage motion control of dual micropipettes to approach target cells and form whole-cell recording with quantitative mechanical stimulation. Experimental results on 50 pyramidal neurons in the primary visual cortex of mouse brain slices demonstrate that this system achieves a threefold throughput with a 37% improvement in the success rate of the contact process and a 42% improvement in the success rate of whole-cell recording in comparison to manual operation. With these advantages, a mechanical stimulation-regulated increase in neuron excitability is observed in primary visual cortex. The experimental results also show that the sodium ion current may be more sensitive to mechanical stimulation than potassium ion current. Our system significantly improves the efficiency of mechanical stimulation induced excitability research of neurons in brain slices. Our methods have the potential to investigate pathological and pathogenic mechanisms of mechanosensitive ion channel dysfunction-induced diseases in the future.

Increases in anterograde axoplasmic transport in neurons of the hyper-glutamatergic, glutamate dehydrogenase 1 (Glud1) transgenic mouse: Effects of glutamate receptors on transport.

The excitatory neurotransmitter glutamate has a role in neuronal migration and process elongation in the central nervous system (CNS). The effects of chronic glutamate hyperactivity on vesicular and protein transport within CNS neurons, that is, processes necessary for neurite growth, have not been examined previously. In this study, we measured the effects of lifelong hyperactivity of glutamate neurotransmission on axoplasmic transport in CNS neurons. We compared wild-type (wt) to transgenic (Tg) mice over-expressing the glutamate dehydrogenase gene Glud1 in CNS neurons and exhibiting increases in glutamate transmitter formation, release, and synaptic activation in brain throughout the lifespan. We found that Glud1 Tg as compared with wt mice exhibited increases in the rate of anterograde axoplasmic transport in neurons of the hippocampus measured in brain slices exvivo, and in olfactory neurons measured in vivo. We also showed that the in vitro pharmacologic activation of glutamate synapses in wt mice led to moderate increases in axoplasmic transport, while exposure to selective inhibitors of ion channel forming glutamate receptors very significantly suppressed anterograde transport, suggesting a link between synaptic glutamate receptor activation and axoplasmic transport. Finally, axoplasmic transport in olfactory neurons of Tg mice in vivo was partially inhibited following 14-day intake of ethanol, a known suppressor of axoplasmic transport and of glutamate neurotransmission. The same was true for transport in hippocampal neurons in slices from Glud1 Tg mice exposed to ethanol for 2 h exvivo. In conclusion, endogenous activity at glutamate synapses regulates and glutamate synaptic hyperactivity increases intraneuronal transport rates in CNS neurons.

Cholinergic Activation of Corticofugal Circuits in the Adult Mouse Prefrontal Cortex.

Acetylcholine (ACh) promotes neocortical output to the thalamus and brainstem by preferentially enhancing the postsynaptic excitability of layer 5 pyramidal tract (PT) neurons relative to neighboring intratelencephalic (IT) neurons. Less is known about how ACh regulates the excitatory synaptic drive of IT and PT neurons. To address this question, spontaneous excitatory postsynaptic potentials (sEPSPs) were recorded in dual recordings of IT and PT neurons in slices of prelimbic cortex from adult female and male mice. ACh (20 µM) enhanced sEPSP amplitudes, frequencies, rise-times, and half-widths preferentially in PT neurons. These effects were blocked by the muscarinic receptor antagonist atropine (1 µM). When challenged with pirenzepine (1 µM), an antagonist selective for M1-type muscarinic receptors, ACh instead reduced sEPSP frequencies, suggesting that ACh may generally suppress synaptic transmission in the cortex via non-M1 receptors. Cholinergic enhancement of sEPSPs in PT neurons was not sensitive to antagonism of GABA receptors with gabazine (10 µM) and CGP52432 (2.5 µM) but was blocked by tetrodotoxin (1 µM), suggesting that ACh enhances action-potential-dependent excitatory synaptic transmission in PT neurons. ACh also preferentially promoted the occurrence of synchronous sEPSPs in dual recordings of PT neurons relative to IT-PT and IT-IT parings. Finally, selective chemogenetic silencing of hM4Di-expressing PT, but not commissural IT, neurons blocked cholinergic enhancement of sEPSP amplitudes and frequencies in PT neurons. These data suggest that, in addition to selectively enhancing the postsynaptic excitability of PT neurons, M1 receptor activation promotes corticofugal output by amplifying recurrent excitation within networks of PT neurons.

Physiological acetic acid concentrations from ethanol metabolism stimulate accumbens shell medium spiny neurons via NMDAR activation in a sex-dependent manner.

Recent studies have implicated the ethanol metabolite, acetic acid, as neuroactive, perhaps even more so than ethanol itself. In this study, we investigated sex-specific metabolism of ethanol (1, 2, and 4 g/kg) to acetic acid in vivo to guide electrophysiology experiments in the accumbens shell (NAcSh), a key node in the mammalian reward circuit. There was a sex-dependent difference in serum acetate production, quantified via ion chromatography only at the lowest dose of ethanol (males > females). Ex vivo electrophysiology recordings of NAcSh medium spiny neurons (MSN) in brain slices demonstrated that physiological concentrations of acetic acid (2 mM and 4 mM) increased NAcSh MSN excitability in both sexes. N-methyl-D-aspartate receptor (NMDAR) antagonists, AP5 and memantine, robustly attenuated the acetic acid-induced increase in excitability. Acetic acid-induced NMDAR-dependent inward currents were greater in females compared to males and were not estrous cycle dependent. These findings suggest a novel NMDAR-dependent mechanism by which the ethanol metabolite, acetic acid, may influence neurophysiological effects in a key reward circuit in the brain from ethanol consumption. Furthermore, these findings also highlight a specific sex-dependent sensitivity in females to acetic acid-NMDAR interactions. This may underlie their more rapid advancement to alcohol use disorder and increased risk of alcohol related neurodegeneration compared to males.

FRI275 Glutamate Receptor Agonists Stimulate Kisspeptin Neurons In The Arcuate Nucleus Of The Hypothalamus

Abstract Disclosure: R. Bearss: None. R. Piet: None. KNDy neurons co-express kisspeptin, neurokinin B (NKB), dynorphin A (Dyn A), and glutamate. These neurons, located in the arcuate nucleus of the hypothalamus, drive pulsatile gonadotropin releasing hormone (GnRH) secretion. This eventually promotes pulsatile gonadotropin hormone release from the anterior pituitary, important for fertility in both sexes. The current hypothesized mechanism for pulse generation involves KNDy neuron interconnectivity and reciprocal regulation through the combined actions of NKB, DynA and glutamate to coordinate KNDy neuron activity. There is evidence that KNDy neurons express NKB, Dyn A and glutamate receptors and that NKB stimulates KNDy neuron electrical activity while Dyn A suppresses it. There is also evidence that ionotropic glutamate receptor antagonists can inhibit KNDy neuron coordinated activity and pulsatile gonadotropin hormone release, indicating that glutamatergic neurotransmission plays a significant role in activity coordination. The purpose of the following experiments was to investigate the response of KNDy neurons to varied glutamate receptor activation. We used calcium imaging to monitor the activity of KNDy neurons in brain slices. We crossed Kiss1-Cre mice—expressing Cre recombinase (Cre) in kisspeptin neurons—with Cre-dependent GCaMP6f mice to generate Kiss1-Cre x GCaMP6f mice that express GCaMP6f in kisspeptin neurons. GCaMP6f fluorescence increases when binding to intracellular calcium and can, thus, be used as an inferential marker of electrical activity. Coronal brain slices (200 µm thickness) obtained from male Kiss1-cre x GCaMP6f mice and containing the arcuate nucleus were placed in a recording chamber under an epifluorescence microscope. KNDy neurons were exposed for 2 minutes to a bath application of either the ionotropic glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 10µM), the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA, 50µM), or the group 1 metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 50µM). AMPA, NMDA, and DHPG increased KNDy neuron fluorescence by 7.8±0.5% (n=113 in 7 slices from 6 mice), 7.5±0.5% (n=154 in 6 slices from 5 mice), and 6.1±0.7% (n=159 in 7 slices from 5 mice) respectively. All cells that were excited by NMDA or DHPG were also excited by AMPA. These experiments provide evidence that activation of ionotropic AMPA and NMDA receptors as well as group 1 mGluRs stimulate KNDy neuron activity. Further investigation into the precise role of individual glutamate receptor subtypes on KNDy neuron coordinated activity is needed. Presentation: Friday, June 16, 2023

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Gonadal Feedback Alters the Relationship between Action Potentials and Hormone Release in Gonadotropin-Releasing Hormone Neurons in Male Mice.

In vertebrates, the pulsatile release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus triggers secretion of anterior pituitary gonadotropins, which activate steroidogenesis, and steroids in turn exert typically homeostatic negative feedback on GnRH release. Although long-term episodic firing patterns of GnRH neurons in brain slices resemble the pulsatile release of GnRH and LH in vivo, neither the relationship between GnRH neuron firing and release nor whether this relationship is influenced by gonadal feedback are known. We combined fast-scan cyclic voltammetry and patch-clamp to perform simultaneous measurements of neuropeptide release with either spontaneous action potential firing or in response to neuromodulator or action-potential-spike templates in brain slice preparations from male mice. GnRH release increased with higher frequency spontaneous firing to a point; release reached a plateau after which further increases in firing rate did not elicit further increased release. Kisspeptin, a potent GnRH neuron activator via a Gq-coupled signaling pathway, triggered GnRH release before increasing firing rate, whether globally perfused or locally applied. Increasing the number of spikes in an applied burst template increased release; orchidectomized mice had higher sensitivity to the increased action potential number than sham-operated mice. Similarly, Ca2+ currents triggered by these burst templates were increased in GnRH neurons of orchidectomized mice. These results suggest removal of gonadal feedback increases the efficacy of the stimulus-secretion coupling mechanisms, a phenomenon that may extend to other steroid-sensitive regions of the brain.SIGNIFICANCE STATEMENT Pulsatile secretion of GnRH plays a critical role in fertility. The temporal relationship between GnRH neuron action potential firing and GnRH release remains unknown as does whether this relationship is influenced by gonadal feedback. By combining techniques of fast-scan cyclic voltammetry and patch-clamp we, for the first time, monitored GnRH concentration changes during spontaneous and neuromodulator-induced GnRH neuron firing. We also made the novel observation that gonadal factors exert negative feedback on excitation-secretion coupling to reduce release in response to the same stimulus. This has implications for the control of normal fertility, central causes of infertility, and more broadly for the effects of sex steroids in the brain.

Kv12-encoded K+ channels drive the day-night switch in the repetitive firing rates of SCN neurons.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Shedding light on human olfaction: Electrophysiological recordings from sensory neurons in acute slices of olfactory epithelium

The COVID-19 pandemic brought attention to our limited understanding of human olfactory physiology. While the cellular composition of the human olfactory epithelium is similar to that of other vertebrates, its functional properties are largely unknown. We prepared acute slices of human olfactory epithelium from nasal biopsies and used the whole-cell patch-clamp technique to record electrical properties of cells. We measured voltage-gated currents in human olfactory sensory neurons and supporting cells, and action potentials in neurons. Additionally, neuronal inward current and action potentials responses to a phosphodiesterase inhibitor suggested a transduction cascade involving cAMP as a second messenger. Furthermore, responses to odorant mixtures demonstrated that the transduction cascade was intact in this preparation. This study provides the first electrophysiological characterization of olfactory sensory neurons in acute slices of the human olfactory epithelium, paving the way for future research to expand our knowledge of human olfactory physiology.