Intrinsic plasticity of Purkinje cell serves homeostatic regulation of fear memory.

Two forms of plasticity, synaptic and intrinsic, are neural substrates for learning and memory. Abnormalities in homeostatic plasticity cause severe neuropsychiatric diseases, such as schizophrenia and autism. This suggests that the balance between synaptic transmission and intrinsic excitability is important for physiological function in the brain. Despite the established role of synaptic plasticity between parallel fiber (PF) and Purkinje cell (PC) in fear memory, its relationship with intrinsic plasticity is not well understood. Here, patch clamp recording revealed depression of intrinsic excitability in PC following auditory fear conditioning (AFC). Depressed excitability balanced long-term potentiation of PF-PC synapse to serve homeostatic regulation of PF-evoked PC firing. We then optogenetically manipulated PC excitability during the early consolidation period resulting in bidirectional regulation of fear memory. Fear conditioning-induced synaptic plasticity was also regulated following optogenetic manipulation. These results propose intrinsic plasticity in PC as a novel mechanism of fear memory and elucidate that decreased intrinsic excitability in PC counterbalances PF-PC synaptic potentiation to maintain fear memory in a normal range.

Optimization of Plant Growth Regulators for In Vitro Mass Propagation of a Disease-Free 'Shine Muscat' Grapevine Cultivar.

This study addresses the propagation challenges faced by 'Shine Muscat', a newly introduced premium grapevine cultivar in South Korea, where multiple viral infections pose considerable economic loss. The primary objective was to establish a robust in vitro propagation method for producing disease-free grapes and to identify effective plant growth regulators to facilitate large-scale mass cultivation. After experimentation, 2.0 µM 6-benzyladenine (BA) exhibited superior shoot formation in the Murashige and Skoog medium compared with kinetin and thidiazuron. Conversely, α-naphthaleneacetic acid (NAA) hindered shoot growth and induced callus formation, while indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) demonstrated favorable root formation, with IBA showing better results overall. Furthermore, inter simple sequence repeat analysis confirmed the genetic stability of in vitro-cultivated seedlings using 2.0 µM BA and 1.0 µM IBA, validating the suitability of the developed propagation method for generating disease-free 'Shine Muscat' grapes. These findings offer promising prospects for commercial grape cultivation, ensuring a consistent supply of healthy grapes in the market.

Preventing Overgrowth of Cucumber and Tomato Seedlings Using Difference between Day and Night Temperature in a Plant Factory with Artificial Lighting.

This study aimed to determine the feasibility of temperature difference as an overgrowth-prevention technique to influence plant height and internode length in a plant factory with artificial lighting. The control plants were grown in a commercial nursery greenhouse using a growth regulator (Binnari), and +DIF (25 °C/15 °C), 0DIF (20 °C/20 °C), and -DIF (15 °C/25 °C) were the treatments with different day/night temperatures and the same average temperature (20 °C). Cucumbers showed the strongest suppression under the -DIF treatment, with a dwarfism rate of 33.3%. Similarly, tomatoes showed 0.8% and 22.2% inhibition in the 0DIF and -DIF treatments, respectively. The FV/FM of cucumber was approximately 0.81 for all treatments. The OJIP changes differed for cucumbers; however, both cucumbers and tomatoes had similar OJIP curve patterns and no abnormalities. The relative growth rate of cucumbers at the growth stage was 1.48 cm·cm·day-1 for days 6-9 in +DIF stage 3, which was the highest growth rate among all treatments, and 0.71 cm·cm·day-1 for days 3-6 in -DIF stage 1, which was the most growth-inhibited treatment. In tomatoes, we found that days 3-6 of -DIF stage 1 had the most growth inhibition at 0.45 cm·cm·day-1. For cucumber, -DIF days 3-6 had the most growth inhibition, with a relative growth rate of 0.71 cm·cm·day-1, but the fidelity was significantly higher than the other treatments, with a 171% increase. The same was true for tomatoes, with days 3-6 of -DIF stage 1 showing the most inhibited growth at 0.45 cm·cm·day-1 but a 200% increase in fidelity. Therefore, applying the -DIF treatment at the beginning of growth would be most effective for both cucumbers and tomatoes to prevent overgrowth through the DIF in a plant factory with artificial lighting because it does not interfere with the seedling physiology and slows down the growth and development stage.

Global Cerebral Ischemia-induced Depression Accompanies Alteration of Neuronal Excitability in the Infralimbic Cortex Layer 2/3 Pyramidal Neurons.

Cerebral ischemia can lead to a range of sequelae, including depression. The pathogenesis of depression involves neuronal change of the medial prefrontal cortex (mPFC). However, how cerebral ischemia-induced changes manifest across subregions and layers of the mPFC is not well understood. In this study, we induced cerebral ischemia in mice via transient bilateral common carotid artery occlusion (tBCCAO) and observed depressive-like behavior. Using whole-cell patch clamp recording, we identified changes in the excitability of pyramidal neurons in the prelimbic cortex (PL) and infralimbic cortex (IL), the subregions of mPFC. Compared to sham control mice, tBCCAO mice showed significantly reduced neuronal excitability in IL layer 2/3 but not layer 5 pyramidal neurons, accompanied by increased rheobase current and decreased input resistance. In contrast, no changes were observed in the excitability of PL layer 2/3 and layer 5 pyramidal neurons. Our results provide a new direction for studying the pathogenesis of depression following ischemic damage by showing that cerebral ischemia induces subregion- and layer-specific changes in the mPFC pyramidal neurons.

Dynamic alteration of intrinsic properties of the cerebellar Purkinje cell during the motor memory consolidation.

Intrinsic plasticity of the cerebellar Purkinje cell (PC) plays a critical role in motor memory consolidation. However, detailed changes in their intrinsic properties during memory consolidation are not well understood. Here, we report alterations in various properties involved in intrinsic excitability, such as the action potential (AP) threshold, AP width, afterhyperpolarization (AHP), and sag voltage, which are associated with the long-term depression of intrinsic excitability following the motor memory consolidation process. We analyzed data recorded from PCs before and 1, 4, and 24 h after cerebellum-dependent motor learning and found that these properties underwent dynamic changes during the consolidation process. We further analyzed data from PC-specific STIM1 knockout (STIM1PKO) mice, which show memory consolidation deficits, and derived intrinsic properties showing distinct change patterns compared with those of wild-type littermates. The levels of memory retention in the STIM1PKO mice were significantly different compared to wild-type mice between 1 and 4 h after training, and AP width, fast- and medium-AHP, and sag voltage showed different change patterns during this period. Our results provide information regarding alterations in intrinsic properties during a particular period that are critical for memory consolidation.

mGluR1 Regulates the Interspike Interval Threshold for Dendritic Ca2+ Transients in the Cerebellar Purkinje Cells.

Ca2++ transients can be observed in the distal dendrites of Purkinje cells (PCs) despite their lack of action potential backpropagation. These Ca2++ events in distal dendrites require specific patterns of PC firing, such as complex spikes (CS) or simple spikes (SS) of burst mode. Unlike CS, which can act directly on voltage-gated calcium channels in the dendrites through climbing fiber inputs, the condition that can produce the Ca2++ events in distal dendrites with burst mode SS is poorly understood. Here, we propose the interspike interval threshold (ISIT) for Ca2++ transients in the distal dendrites of PC. We found that to induce the Ca2++ transients in distal dendrites the frequency of spike firing of PC should reach 250 Hz (3 ms ISI). Metabotropic glutamate receptor 1 (mGluR1) activation significantly relieved the ISIT and established cellular conditions in which spike firing with 50 Hz (19 ms ISI) could induce Ca2++ transients in the distal dendrites. In contrast, blocking T-type Ca2++ channels or depleting the endoplasmic reticulum Ca2++ store resulted in a stricter condition in which spike firing with 333 Hz (2 ms ISI) was required. Our findings demonstrate that the PC has strict ISIT for dendritic Ca2++ transients, and this ISIT can be relieved by mGluR1 activation. This strict restriction of ISIT could contribute to the reduction of the signal-to-noise ratio in terms of collecting information by preventing excessive dendritic Ca2++ transients through the spontaneous activity of PC.

Transcutaneous Auricular Vagus Nerve Stimulation Enhances Cerebrospinal Fluid Circulation and Restores Cognitive Function in the Rodent Model of Vascular Cognitive Impairment.

Vascular cognitive impairment (VCI) is a common sequela of cerebrovascular disorders. Although transcutaneous auricular vagus nerve stimulation (taVNS) has been considered a complementary treatment for various cognitive disorders, preclinical data on the effect of taVNS on VCI and its mechanism remain ambiguous. To measure cerebrospinal fluid (CSF) circulation during taVNS, we used in vivo two-photon microscopy with CSF and vasculature tracers. VCI was induced by transient bilateral common carotid artery occlusion (tBCCAO) surgery in mice. The animals underwent anesthesia, off-site stimulation, or taVNS for 20 min. Cognitive tests, including the novel object recognition and the Y-maze tests, were performed 24 h after the last treatment. The long-term treatment group received 6 days of treatment and was tested on day 7; the short-term treatment group received 2 days of treatment and was tested 3 days after tBCCAO surgery. CSF circulation increased remarkably in the taVNS group, but not in the anesthesia-control or off-site-stimulation-control groups. The cognitive impairment induced by tBCCAO was significantly restored after both long- and short-term taVNS. In terms of effects, both long- and short-term stimulations showed similar recovery effects. Our findings provide evidence that taVNS can facilitate CSF circulation and that repetitive taVNS can ameliorate VCI symptoms.

The Small GTPase CsRAC1 Is Important for Fungal Development and Pepper Anthracnose in Colletotrichum scovillei.

The pepper anthracnose fungus, Colletotrichum scovillei, causes severe losses of pepper fruit production in the tropical and temperate zones. RAC1 is a highly conserved small GTP-binding protein in the Rho GTPase family. This protein has been demonstrated to play a role in fungal development, and pathogenicity in several plant pathogenic fungi. However, the functional roles of RAC1 are not characterized in C. scovillei causing anthracnose on pepper fruits. Here, we generated a deletion mutant (ΔCsrac1) via homologous recombination to investigate the functional roles of CsRAC1. The ΔCsrac1 showed pleiotropic defects in fungal growth and developments, including vegetative growth, conidiogenesis, conidial germination and appressorium formation, compared to wild-type. Although ΔCsrac1 was able to develop appressoria, it failed to differentiate appressorium pegs. However, ΔCsrac1 still caused anthracnose disease with significantly reduced rate on wounded pepper fruits. Further analyses revealed that ΔCsrac1 was defective in tolerance to oxidative stress and suppression of host-defense genes. Taken together, our results suggest that CsRAC1 plays essential roles in fungal development and pathogenicity in C. scovillei-pepper fruit pathosystem.

Persistent Activity of Metabotropic Glutamate Receptor 5 in the Periaqueductal Gray Constrains Emergence of Chronic Neuropathic Pain.

Pain sensation is powerfully modulated by signal processing in the brain, and pain becomes chronic with the dysfunction of the pain modulatory system; however, the underlying mechanisms are unclear. We found that the metabotropic glutamate receptor 5 (mGluR5) in the periaqueductal gray (PAG), the key area of endogenous pain modulation, is persistently active in normal conditions to maintain an appropriate sensory perception. In the neuropathic pain condition, Homer1a, an activity-dependent immediate early gene product, disrupted the persistent mGluR5 activity resulting in chronic pain. Remarkably a single-time blockage of the mGluR5 resulted in chronic neuropathic pain-like symptoms even in the absence of nerve injury. The decline of mGluR5 activity induced the pain modulatory dysfunction with a profound reduction of excitability of PAG neurons. These findings uncover the role of the persistent mGluR5 activity in vivo and provide new insight into how pain becomes chronic with the maladaptive coping of the PAG to pain sensation.

Intrinsic Plasticity of Cerebellar Purkinje Cells Contributes to Motor Memory Consolidation.

Intrinsic plasticity of cerebellar Purkinje cells (PCs) has recently been demonstrated in cerebellar local circuits; however, its physiological impact on cerebellar learning and memory remains elusive. Here, we suggest that intrinsic plasticity of PCs is tightly involved in motor memory consolidation based on findings from PC-specific STIM1 knockout male mice, which show severe memory consolidation deficiency in vestibulo-ocular reflex memory. Gain-up training of the vestibulo-ocular reflex produced a decrease in the synaptic weight of PCs in both the WT and KO groups. However, intrinsic plasticity was impaired only in the knockout mice. Furthermore, the observed defects in the intrinsic plasticity of PCs led to the formation of aberrant neural plasticity in the vestibular nucleus neurons. Our results suggest that synergistic modulation of intrinsic and synaptic plasticity in PCs is required for the changes in downstream plasticity in the vestibular nucleus, and thereby contributing to the long-term storage of motor memory.SIGNIFICANCE STATEMENT Synaptic plasticity is a well-known mechanism for learning and memory. Although plasticity of excitability, intrinsic plasticity, of the cerebellar Purkinje cell has been reported in both directions (potentiation and depression), the physiological role of intrinsic plasticity still remains ambiguous. In this study, we suggest that both synaptic and intrinsic plasticity are required for successful memory consolidation in cerebellar eye movement learning. Despite successful induction and maintenance of synaptic plasticity, we found deficits of memory consolidation when there were defects in intrinsic plasticity. Our results suggest that intrinsic plasticity of cerebellar Purkinje cell has a significant role in motor memory consolidation.

Plasticity leading to cerebellum-dependent learning: two different regions, two different types.

In memory research, studying cerebellum-dependent memory is advantageous due to its relatively simple neural architecture compared with that of other memory circuits. To understand how cerebellum-dependent memory develops and is stored in this circuit, numerous hypotheses have been proposed. These hypotheses are generally able to adequately explain most learning and memory processes; however, several reported results are still poorly understood. Recently, the importance of intrinsic plasticity (i.e., plasticity of intrinsic excitability) has been highlighted in several studies. Because the classical view of cerebellum-dependent eye movement learning was focused on synaptic plasticity, it is valuable to consider the intrinsic plasticity for deeper understanding. In the present review, we re-examine the utility and limitations of previous hypotheses, from classic to recent, and propose an updated hypothesis. Integrating intrinsic plasticity into current models of the vestibulo-ocular reflex (VOR) circuit may facilitate deeper understanding of the VOR adaptation process. In particular, during the period of memory transfer, dynamic changes in excitability in both cerebellar Purkinje cells and vestibular nuclear neurons illuminate the role of intrinsic plasticity in the circuit.

STIM1 Regulates Somatic Ca2+ Signals and Intrinsic Firing Properties of Cerebellar Purkinje Neurons.

Control of Ca2+ flux between the cytosol and intracellular Ca2+ stores is essential for maintaining normal cellular function. It has been well established in both neuronal and non-neuronal cells that stromal interaction molecule 1 (STIM1) initiates and regulates refilling Ca2+ into the ER. Here, we describe a novel, additional role for STIM1, the regulation of free cytosolic Ca2+, and the consequent control of spike firing in neurons. Among central neurons, cerebellar Purkinje neurons express the highest level of STIM1, and they fire continuously in the absence of stimulation, making somatic Ca2+ homeostasis of particular importance. By using Purkinje neuron-specific STIM1 knock-out (STIM1PKO) male mice, we found that the deletion of STIM1 delayed clearance of cytosolic Ca2+ in the soma during ongoing neuronal firing. Deletion of STIM1 also reduced the Purkinje neuronal excitability and impaired intrinsic plasticity without affecting long-term synaptic plasticity. In vestibulo-ocular reflex learning, STIM1PKO male mice showed severe deficits in memory consolidation, whereas they were normal in memory acquisition. Our results suggest that STIM1 is critically involved in the regulation of the neuronal excitability and the intrinsic plasticity of the Purkinje neurons as well as cerebellar memory consolidation.SIGNIFICANCE STATEMENT Stromal interaction molecule 1 (STIM1), which regulates the refilling of ER Ca2+, has been investigated in several systems including the CNS. In addition to a previous study showing that STIM1 regulates dendritic ER Ca2+ refilling and mGluR1-mediated synaptic transmission, we provide compelling evidence describing a novel role of STIM1 in spike firing Purkinje neurons. We found that STIM1 regulates cytosolic Ca2+ clearance of the soma during spike firing, and the interruption of this cytosolic Ca2+ clearing disrupts neuronal excitability and cerebellar memory consolidation. Our results provide new insights into neuronal functions of STIM1 from single neuronal Ca2+ dynamics to behavior level.

Long-Term Depression of Intrinsic Excitability Accompanied by Synaptic Depression in Cerebellar Purkinje Cells.

Long-term depression (LTD) at the parallel fiber (PF)-to-cerebellar Purkinje cell (PC) synapse is implicated in the output of PCs, the sole output of the cerebellar cortex. In addition to synaptic plasticity, intrinsic excitability is also one of the components that determines PC output. Although long-term potentiation of intrinsic excitability (LTP-IE) has been suggested, it has yet to be investigated how PF-PC LTD modifies intrinsic excitability of PCs. Here, we show that pairing of the PF and climbing fiber (CF) for PF-PC LTD induction evokes LTD-IE in cerebellar PCs from male C57BL/6 mice. Interestingly, this intrinsic plasticity showed different kinetics from synaptic plasticity, but both forms of plasticity share Ca2+ signaling and protein kinase C pathway as their underlying mechanism. Although small-conductance Ca2+-activated K+ channels play important roles in LTP-IE, no direct implication has been found. After PF-PC LTD induction, neither the temporal summation of dendritic EPSP nor the power of spike frequency adaptation is changed, indicating that cerebellar LTD executes the information processing in a quantitative way without quality changes of synaptic integration and generation of output signals. Our results suggest that LTD-IE may have a synergistic effect with synaptic depression on the total net output of neurons by amplifying the modification of PF synaptic transmission.SIGNIFICANCE STATEMENT Although the output of Purkinje cells (PCs) is a critical component of cerebellum-dependent learning and memory, the changes of PC excitability when synaptic LTD occurs are unclear. Here, we show that the induction of PF-PC LTD evokes LTD-IE in PCs. Our observation complements previous intrinsic plasticity phenomenon of long-term potentiation of intrinsic excitability (LTP-IE), providing evidence for the idea that intrinsic plasticity has bidirectionality as synaptic plasticity. LTD-IE occurs together with synaptic LTD and both phenomena are dependent on the Ca2+ signaling pathway. Furthermore, our findings raise the prospect that this synaptic and intrinsic plasticity acts synergistically in PCs to modify neuronal activity in the same direction when learning occurs.

mGlu1 receptor mediates homeostatic control of intrinsic excitability through Ih in cerebellar Purkinje cells.

Homeostatic intrinsic plasticity is a cellular mechanism for maintaining a stable neuronal activity level in response to developmental or activity-dependent changes. Type 1 metabotropic glutamate receptor (mGlu1 receptor) has been widely known to monitor neuronal activity, which plays a role as a modulator of intrinsic and synaptic plasticity of neurons. Whether mGlu1 receptor contributes to the compensatory adjustment of Purkinje cells (PCs), the sole output of the cerebellar cortex, in response to chronic changes in excitability remains unclear. Here, we demonstrate that the mGlu1 receptor is involved in homeostatic intrinsic plasticity through the upregulation of the hyperpolarization-activated current (Ih) in cerebellar PCs. This plasticity was prevented by inhibiting the mGlu1 receptor with Bay 36-7620, an mGlu1 receptor inverse agonist, but not with CPCCOEt, a neutral antagonist. Chronic inactivation with tetrodotoxin (TTX) increased the components of Ih in the PCs, and ZD 7288, a hyperpolarization-activated cyclic nucleotide-gated channel selective inhibitor, fully restored reduction of firing rates in the deprived neurons. The homeostatic elevation of Ih was also prevented by BAY 36-7620, but not CPCCOEt. Furthermore, KT 5720, a blocker of protein kinase A (PKA), prevented the effect of TTX reducing the evoked firing rates, indicating the reduction in excitability of PCs due to PKA activation. Our study shows that both the mGlu1 receptor and the PKA pathway are involved in the homeostatic intrinsic plasticity of PCs after chronic blockade of the network activity, which provides a novel understanding on how cerebellar PCs can preserve the homeostatic state under activity-deprived conditions.

Lipid rafts serve as signaling platforms for mGlu1 receptor-mediated calcium signaling in association with caveolin.

BACKGROUND: Group I metabotropic glutamate receptors (mGlu1/5 receptors) have important roles in synaptic activity in the central nervous system. They modulate neuronal excitability by mobilizing intracellular Ca2+ following receptor activation. Also, accumulating evidence has indicated the association of Ca2+ signaling with lipid rafts. Caveolin, an adaptor protein found in a specialized subset of lipid rafts, has been reported to promote the localization of membrane proteins to lipid rafts. RESULTS: In the present study, we investigated the role of lipid rafts on the mGlu1α receptor-mediated Ca2+ signaling in association with caveolin in hippocampal primary neurons and HEK293 cells. We show that the disruption of lipid rafts using methyl-ß-cyclodextrin markedly decreased mGlu1α receptor-mediated Ca2+ transients and lipid rafts localization of the receptor. Furthermore, transfection of mGlu1α receptor with mutated caveolin-binding domain reduced localization of the receptor to lipid rafts. Also, application of a peptide blocker of mGlu1α receptor and caveolin binding reduced the Ca2+ signaling and the lipid rafts localization. CONCLUSIONS: Taken together, these results suggest that the binding of mGlu1α receptor to caveolin is crucial for its lipid rafts localization and mGlu1α receptor-mediated Ca2+ transients.