Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

Contributions of NaV1.8 and NaV1.9 to excitability in human induced pluripotent stem-cell derived somatosensory neurons

The inhibition of voltage-gated sodium (NaV) channels in somatosensory neurons presents a promising novel modality for the treatment of pain. However, the precise contribution of these channels to neuronal excitability, the cellular correlate of pain, is unknown; previous studies using genetic knockout models or pharmacologic block of NaV channels have identified general roles for distinct sodium channel isoforms, but have never quantified their exact contributions to these processes. To address this deficit, we have utilized dynamic clamp electrophysiology to precisely tune in varying levels of NaV1.8 and NaV1.9 currents into induced pluripotent stem cell-derived sensory neurons (iPSC-SNs), allowing us to quantify how graded changes in these currents affect different parameters of neuronal excitability and electrogenesis. We quantify and report direct relationships between NaV1.8 current density and action potential half-width, overshoot, and repetitive firing. We additionally quantify the effect varying NaV1.9 current densities have on neuronal membrane potential and rheobase. Furthermore, we examined the simultaneous interplay between NaV1.8 and NaV1.9 on neuronal excitability. Finally, we show that minor biophysical changes in the gating of NaV1.8 can render human iPSC-SNs hyperexcitable, in a first-of-its-kind investigation of a gain-of-function NaV1.8 mutation in a human neuronal background.

New Insights Into the Anticonvulsant Effects of Essential Oil From Melissa officinalis L. (Lemon Balm)

Melissa officinalis L. is used in traditional European and Iranian folk medicines to treat a plethora of neurological diseases including epilepsy. We utilized the in vitro and in vivo models of epilepsy to probe the anticonvulsant potentials of essential oil from M. officinalis (MO) to gain insight into the scientific basis for its applications in traditional medicine for the management of convulsive disorders. MO was evaluated for effects on maximal electroshock (MES) and pentylenetetrazole (PTZ) -induced seizures in mice, on 4–aminopyridine (4-AP)-brain slice model of epilepsy and sustained repetitive firing of current clamped neurons; and its ameliorative effects were examined on seizure severity, anxiety, depression, cognitive dysfunction, oxidative stress and neuronal cell loss in PTZ-kindled rats. MO reversibly blocked spontaneous ictal-like discharges in the 4-AP-brain slice model of epilepsy and secondary spikes from sustained repetitive firing, suggesting anticonvulsant effects and voltage-gated sodium channel blockade. MO protected mice from PTZ– and MES–induced seizures and mortality, and ameliorated seizure severity, fear-avoidance, depressive-like behavior, cognitive deficits, oxidative stress and neuronal cell loss in PTZ–kindled rats. The findings warrant further study for the potential use of MO and/or its constituent(s) as adjunctive therapy for epileptic patients.

Single Prolonged Stress Reduces Intrinsic Excitability and Excitatory Synaptic Drive Onto Pyramidal Neurons in the Infralimbic Prefrontal Cortex of Adult Male Rats

Post-traumatic stress disorder (PTSD) is a chronic, debilitating mental illness marked by abnormal fear responses and deficits in extinction of fear memories. The pathophysiology of PTSD is linked to decreased activation of the ventromedial prefrontal cortex (vmPFC). This study aims to investigate underlying functional changes in synaptic drive and intrinsic excitability of pyramidal neurons in the rodent homolog of the vmPFC, the infralimbic cortex (IL), following exposure to single prolonged stress (SPS), a paradigm that mimics core symptoms of PTSD in rats. Rats were exposed to SPS and allowed 1 week of recovery, following which brain slices containing the PFC were prepared for whole-cell patch clamp recordings from layer V pyramidal neurons in the IL. Our results indicate that SPS reduces spontaneous excitatory synaptic drive to pyramidal neurons. In addition, SPS decreases the intrinsic membrane excitability of IL PFC pyramidal cells, as indicated by an increase in rheobase, decrease in input resistance, hyperpolarization of resting membrane potential, and a reduction in repetitive firing rate. Our results suggest that SPS causes a lasting reduction in PFC activity, supporting a body of evidence linking traumatic stress with prefrontal hypoactivity.

Serotonergic Modulation of Persistent Inward Currents in Serotonergic Neurons of Medulla in ePet-EYFP Mice

Serotonergic (5-HT) neurons in the medulla play multiple functional roles associated with many symptoms and motor activities. The descending serotonergic pathway from medulla is essential for initiating locomotion. However, the ionic properties of 5-HT neurons in the medulla remain unclear. Using whole-cell patch-clamp technique, we studied the biophysical and modulatory properties of persistent inward currents (PICs) in 5-HT neurons of medulla in ePet-EYFP transgenic mice (P3–P6). PICs were recorded by a family of voltage bi-ramps (10-s duration, 40-mV peak step), and the ascending and descending PICs were mirrored to analyze the PIC hysteresis. PICs were found in 77% of 5-HT neurons (198/258) with no significant difference between parapyramidal region (n = 107) and midline raphe nuclei (MRN) (n = 91) in either PIC onset (−47.4 ± 10 mV and −48.7 ± 7 mV; P = 0.44) or PIC amplitude (226.9 ± 138 pA and 259.2 ± 141 pA; P = 0.29). Ninety-six percentage (191/198) of the 5-HT neurons displayed counterclockwise hysteresis and four percentage (7/198) exhibited the clockwise hysteresis. The composite PICs could be differentiated as calcium component (Ca_PIC) by bath application of nimodipine (25 μM), sodium component (Na_PIC) by tetrodotoxin (TTX, 2 μM), and TTX- and dihydropyridine-resistance component (TDR_PIC) by TTX and nimodipine. Ca_PIC, Na_PIC and TDR_PIC all contributed to upregulation of excitability of 5-HT neurons. 5-HT (15 μM) enhanced the PICs, including a 26% increase in amplitude of the compound currents of Ca_PIC and TDR_PIC (P < 0.001, n = 9), 3.6 ± 5 mV hyperpolarization of Na_PIC and TDR_PIC onset (P < 0.05, n = 12), 30% increase in amplitude of TDR_PIC (P < 0.01), and 2.0 ± 3 mV hyperpolarization of TDR_PIC onset (P < 0.05, n = 18). 5-HT also facilitated repetitive firing of 5-HT neurons through modulation of composite PIC, Na_PIC and TDR_PIC, and Ca_PIC and TDR_PIC, respectively. In particular, the high voltage-activated TDR_PIC facilitated the repetitive firing in higher membrane potential, and this facilitation could be amplified by 5-HT. Morphological data analysis indicated that the dendrites of 5-HT neurons possessed dense spherical varicosities intensively crossing 5-HT neurons in medulla. We characterized the PICs in 5-HT neurons and unveiled the mechanism underlying upregulation of excitability of 5-HT neurons through serotonergic modulation of PICs. This study provided insight into channel mechanisms responsible for the serotonergic modulation of serotonergic neurons in brainstem.

Single Prolonged Stress Reduces Intrinsic Excitability and Excitatory Synaptic Drive onto Pyramidal Neurons in the Infralimbic Prefrontal Cortex of Adult Rats

ABSTRACTPost-traumatic stress disorder (PTSD) is a chronic, debilitating mental illness marked by abnormal fear responses and deficits in extinction of fear memories. The pathophysiology of PTSD is linked to decreased activation of the ventromedial prefrontal cortex (vmPFC). This study aims to investigate underlying functional changes in synaptic drive and intrinsic excitability of pyramidal neurons in the rodent homolog of the vmPFC, the infralimbic cortex (IL), following exposure to single prolonged stress (SPS), a paradigm that mimics core symptoms of PTSD in rats. Rats were exposed to SPS and allowed one week of recovery following which brain slices containing the PFC were prepared for whole-cell patch clamp recordings from layer V pyramidal neurons in the IL. Our results indicate that SPS reduces spontaneous excitatory synaptic drive to pyramidal neurons. In addition, SPS decreases the intrinsic membrane excitability of IL PFC pyramidal cells, as indicated by an increase in rheobase, decrease in input resistance, hyperpolarization of resting membrane potential, and a reduction in repetitive firing rate. Our results suggest that SPS causes a lasting reduction in PFC activity, supporting a body of evidence linking traumatic stress with prefrontal hypoactivity.Graphical AbstractSPS causes a decrease in excitatory synaptic drive and intrinsic excitability of IL pyramidal neurons.

Influence of Additive Firing on the Surface Characteristics, Streptococcus mutans Viability and Optical Properties of Zirconia

The purpose of this study was to observe whether the repetitive firing of dental zirconia caused changes in surface characteristics, S. mutans viability, and optical properties of zirconia. Dental zirconia blocks were sintered and randomly distributed into seven experimental groups: F0–F6. Except for F0, which only went through sintering, the additive firing was performed in order for F1–F6. Surface roughness, contact angle, S. mutans viability by fluorescence, and translucency parameter were measured. They were all highest after sintering (F0) and decreased after additive firings (F1–F6). The additive firing of zirconia after sintering decreased surface roughness, contact angle, S. mutans viability, and translucency. The number of firings after the first firing was not found to be critical in surface characteristics, S. mutans viability, and optical property. Changes in surface characteristics might have led to a decrease in S. mutans viability, while the change of translucency was not clinically significant. This implies that additive firing may prevent secondary caries under zirconia restorations, not compromising esthetic appearance.

Imbalanced Subthreshold Currents Following Sepsis and Chemotherapy: A Shared Mechanism Offering a New Therapeutic Target?

Both sepsis and treatment of cancer with chemotherapy are known to cause neurologic dysfunction. The primary defects seen in both groups of patients are neuropathy and encephalopathy; the underlying mechanisms are poorly understood. Analysis of preclinical models of these disparate conditions reveal similar defects in ion channel function contributing to peripheral neuropathy. The defects in ion channel function extend to the central nervous system where lower motoneurons are affected. In motoneurons the defect involves ion channels responsible for subthreshold currents that convert steady depolarization into repetitive firing. The inability to correctly translate depolarization into steady, repetitive firing has profound effects on motor function, and could be an important contributor to weakness and fatigue experienced by both groups of patients. The possibility that disruption of function, either instead of, or in addition to neurodegeneration, may underlie weakness and fatigue leads to a novel approach to therapy. Activation of serotonin (5HT) receptors in a rat model of sepsis restores the normal balance of subthreshold currents and normal motoneuron firing. If an imbalance of subthreshold currents also occurs in other central nervous system neurons, it could contribute to encephalopathy. We hypothesize that pharmacologically restoring the proper balance of subthreshold currents might provide effective therapy for both neuropathy and encephalopathy in patients recovering from sepsis or treatment with chemotherapy.

Ectopic HCN4 expression drives mTOR-dependent epilepsy in mice

The causative link between focal cortical malformations (FCMs) and epilepsy is well accepted, especially among patients with focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC). However, the mechanisms underlying seizures remain unclear. Using a mouse model of TSC- and FCDII-associated FCM, we showed that FCM neurons were responsible for seizure activity via their unexpected abnormal expression of the hyperpolarization-activated cyclic nucleotide–gated potassium channel isoform 4 (HCN4), which is normally not present in cortical pyramidal neurons after birth. Increasing intracellular cAMP concentrations, which preferentially affects HCN4 gating relative to the other isoforms, drove repetitive firing of FCM neurons but not control pyramidal neurons. Ectopic HCN4 expression was dependent on the mechanistic target of rapamycin (mTOR), preceded the onset of seizures, and was also found in diseased neurons in tissue resected from patients with TSC and FCDII. Last, blocking HCN4 channel activity in FCM neurons prevented epilepsy in the mouse model. These findings suggest that HCN4 play a main role in seizure and identify a cAMP-dependent seizure mechanism in TSC and FCDII. Furthermore, the unique expression of HCN4 exclusively in FCM neurons suggests that gene therapy targeting HCN4 might be effective in reducing seizures in FCDII or TSC.

Inhibition of glutamatergic transmission and neuronal excitability by oxycodone in the rat hippocampal CA3 neurons

Oxycodone, a semisynthetic opioid analgesic with actions similar to morphine, is extensively prescribed for treatment of moderate to severe acute pain. Given that glutamate plays a crucial role in mediating pain transmission, the propose of this study was to investigate the effect of oxycodone on glutamatergic synaptic transmission in rat hippocampal CA3 area, which is associated with the modulation of nociceptive perception. Whole-cell patch-clamp recordings revealed that oxycodone effectively reduced presynaptic glutamate release, as detected by decreased frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs), without eliciting significant changes in the amplitudes of sEPSCs and mEPSCs and glutamate-evoked inward currents. The inhibitory effect of oxycodone on the frequency of sEPSCs was blocked by the nonselective opioid receptor antagonist naloxone. In addition, oxycodone suppressed burst firing induced by 4-aminopyridine and tonic repetitive firing evoked by the applied depolarizing current. These results suggest that oxycodone inhibits spontaneous presynaptic glutamate release possibly by activating opioid receptors and consequently suppressing the neuronal excitability of hippocampal CA3 neurons.