Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Ca2+-permeable AMPA receptor-dependent silencing of neurons by KCa3.1 channels during epileptiform activity.

Ca2+-activated KCa3.1 channels are known to contribute to slow afterhyperpolarization in pyramidal neurons of several brain areas, while Ca2+-permeable AMPA receptors (CP-AMPARs) may provide a subthreshold source of Ca2+ elevation in the cytoplasm. The functionality of these two types of channels has also been shown to be altered by epileptic disorders. However, the link between KCa3.1 channels and CP-AMPARs is poorly understood, and their potential interaction in epilepsy remains unclear. Here, we address this issue by overexpressing the KCNN4 gene, which encodes the KCa3.1 channel, using patch clamp, imaging, and channel blockers in an in vitro model of epilepsy in neuronal culture. We show that KCNN4 overexpression causes strong hyperpolarization and substantial silencing of neurons during epileptiform activity events, which also prevents KCNN4-positive neurons from firing action potentials (APs) during experimentally induced status epilepticus. Intracellular blocker application experiments showed that the amplitude of hyperpolarization was strongly dependent on CP-AMPARs, but not on NMDA receptors. Taken together, our data strongly suggest that subthreshold Ca2+ elevation produced by CP-AMPARs can trigger KCa3.1 channels to hyperpolarize neurons and protect them from seizures.

Five Different Artemisia L. Species Ethanol Extracts' Phytochemical Composition and Their Antimicrobial and Nematocide Activity.

Among the plants that exhibit significant or established pharmacological activity, the genus Artemisia L. deserves special attention. This genus comprises over 500 species belonging to the largest Asteraceae family. Our study aimed at providing a comprehensive evaluation of the phytochemical composition of the ethanol extracts of five different Artemisia L. species (collected from the southwest of the Russian Federation) and their antimicrobial and nematocide activity as follows: A. annua cv. Novichok., A. dracunculus cv. Smaragd, A. santonica cv. Citral, A. abrotanum cv. Euxin, and A. scoparia cv. Tavrida. The study of the ethanol extracts of the five different Artemisia L. species using the methods of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-MS/MS) allowed establishing their phytochemical profile. The obtained data on the of five different Artemisia L. species ethanol extracts' phytochemical composition were used to predict the antibacterial and antifungal activity against phytopathogenic microorganisms and nematocidal activity against the free-living soil nematode Caenorhabditis elegans. The major compounds found in the composition of the Artemisia L. ethanol extracts were monoterpenes, sesquiterpenes, flavonoids, flavonoid glycosides, coumarins, and phenolic acids. The antibacterial and antifungal activity of the extracts began to manifest at a concentration of 150 µg/mL. The A. dracunculus cv. Smaragd extract had a selective effect against Gram-positive R. iranicus and B. subtilis bacteria, whereas the A. scoparia cv. Tavrida extract had a selective effect against Gram-negative A. tumefaciens and X. arboricola bacteria and A. solani, R. solani and F. graminearum fungi. The A. annua cv. Novichok, A. dracunculus cv. Smaragd, and A. santonica cv. Citral extracts in the concentration range of 31.3-1000 µg/mL caused the death of nematodes. It was established that A. annua cv. Novichok affects the UNC-63 protein, the molecular target of which is the nicotine receptor of the N-subtype.

Nav1.6 but not KCa3.1 channels contribute to heterogeneity in coding abilities and dynamics of action potentials in the L5 neocortical pyramidal neurons.

Electrophysiological and genetic studies reveal two major subclasses of layer 5 (L5) neocortical pyramidal neurons that differ in electrical parameters and afterhyperpolarization. KCa3.1 channels are identified as contributors to slow afterhyperpolarization (sAHP), and they are expressed by one subclass of L5 neurons. Yet, the impact of class-specific sAHP and KCa3.1 channels on coding abilities of the L5 neurons and dynamics of their action potentials (APs) remains poorly understood. Here, by comparing sAHP+ neurons to those with weak sAHP we investigate differences between the two groups in coding and AP features to address the question of whether those differences are due to contribution of KCa3.1 or other channels. Using patch clamp electrophysiology, channel blockers, and immunohistochemistry we demonstrate that Nav1.6 channels but not KCa3.1 channels affect the threshold of AP, its dynamics and coding abilities of the L5 cells. Immunohistochemical data show that KCa3.1+ and KCa3.1- neurons share the same pattern of Nav1.6 expression in the soma and axonal initial segment, thus they may differ in quantity of the channels expressed. Our study links the Nav1.6 function underlying regulation of voltage threshold to the abilities of L5 neurons to encode high frequencies.

Cytotoxic and Luminescent Properties of Novel Organotin Complexes with Chelating Antioxidant Ligand.

A novel polydentate chelating antioxidant ligand and series of organotin complexes on its base were synthesized and characterized by NMR 1H, 13C, 119Sn, IR spectroscopy, X-ray, and elemental analysis. Their antioxidant activity was evaluated in DPPH and NBT-tests, and as lipoxygenase inhibitory activity. It was shown that ligand alone is a radical scavenger, while introducing tin in the structure of the compound significantly decreases its activity. For the ligand alone the ability to strongly suppress the formation of advanced glycation end products (AGEs) was shown, which may be associated with the established antiradical activity. All synthesized compounds appeared to be moderate lipoxygenase inhibitors. The stability of compounds to hydrolysis under different pH was estimated. The ligand undergoes decomposition after about an hour, while organotin complexes on its base demonstrate vast stability, showing signs of decomposition only after 5 h of experimentation. Cytotoxicity of compounds was studied by standard MTT-test, which showed unorthodox results: the ligand itself demonstrated noticeable cytotoxicity while the introduction of organotin moiety either did not affect the toxicity levels or reduced them instead of increasing. Organotin complexes possess luminescence both as powders and DMSO solutions, its quantum yields reaching 67% in DMSO. The combination of luminescence with unique cytotoxic properties allows us to propose the synthesized compounds as perspective theranostic agents.

Impacts of OrX and cAMP-insensitive Orco to the insect olfactory heteromer activity.

Insect odorant receptors (ORs) have been suggested to function as ligand-gated cation channels, with OrX/Orco heteromers combining ionotropic and metabotropic activity. The latter is mediated by different G proteins and results in Orco self-activation by cyclic nucleotide binding. In this contribution, we co-express the odor-specific subunits DmOr49b and DmOr59b with either wild-type Orco or an Orco-PKC mutant lacking cAMP activation heterologously in mammalian cells. We show that the characteristics of heteromers strongly depend on both the OrX type and the coreceptor variant. Thus, methyl acetate-sensitive Or59b/Orco demonstrated 25-fold faster response kinetics over o-cresol-specific Or49b/Orco, while the latter required a 10-100 times lower ligand concentration to evoke a similar electrical response. Compared to wild-type Orco, Orco-PKC decreased odorant sensitivity in both heteromers, and blocked an outward current rectification intrinsic to the Or49b/Orco pair. Our observations thus provide an insight into insect OrX/Orco functioning, highlighting their natural and artificial tuning features and laying the groundwork for their application in chemogenetics, drug screening, and repellent design.

Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness.

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188-a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn't show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

BACKGROUND: SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. METHODS: Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. RESULTS: Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. CONCLUSIONS: SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. GENERAL SIGNIFICANCE: The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

Phytochemical Study of Ethanol Extract of Gnaphalium uliginosum L. and Evaluation of Its Antimicrobial Activity.

This study evaluates the antibacterial and antifungal effects of ethanol extracts from Gnaphalium uliginosum L. derived from freshly harvested plant biomass, including stems, leaves, flowers, and roots. The extract was analyzed using gas chromatography-mass spectrometry (GC-MS) to determine its antimicrobial activity against phytopathogenic bacteria and fungi. Two methods were used in the experiments: agar well diffusion and double serial dilution. Extraction was carried out using the maceration method with different temperature regimes (25 °C, 45 °C, and 75 °C) and the ultrasonic method at various powers (63-352 W) for different durations (5 and 10 min). It was found that the 70% ethanol extract obtained through the ultrasonic experiment at 189 W power for 10 min and at 252 W power for 5 min had the highest antimicrobial activity compared to the maceration method. The most sensitive components of the extracts were the Gram-positive phytopathogenic bacteria Clavibacter michiganensis and the Gram-negative phytopathogenic bacteria Erwinia carotovora spp., with MIC values of 156 µg/mL. Among the fungi, the most sensitive were Rhizoctonia solani and Alternaria solani (MIC values in the range of 78-156 µg/mL). The evaluation of the antimicrobial activity of extracts using the diffusion method established the presence of a growth suppression zone in the case of C. michiganensis (15-17 mm for flowers, leaves, and total biomass), which corresponds to the average level of antimicrobial activity. These findings suggest that G. uliginosum has potential as a source of biologically active compounds for agricultural use, particularly for developing novel biopesticides.

CADENCE - Neuroinformatics Tool for Supervised Calcium Events Detection.

CADENCE is an open Python 3-written neuroinformatics tool with Qt6 graphic user interface for supervised calcium events detection. In neuronal ensembles recording during calcium imaging experiments, the output of instruments such as Celena X, Zeiss LSM 5 Live confocal microscope and Miniscope is a movie showing flashing cells somata. There are few pipelines to convert video to relative fluorescence ΔF/F, from simplest ImageJ plugins to sophisticated tools like MiniAn (Dong et al. in Elife 11, https://doi.org/10.7554/eLife.70661 , 2022). Minian, an open-source miniscope analysis pipeline. Elife, 11.). While in some areas of study relative fluorescence ΔF/F may be the desired result in itself, researchers of neuronal ensembles are typically interested in a more detailed analysis of calcium events as indirect proxy of neuronal electrical activity. For such analyses, researchers need a tool to infer calcium events from the continuous ΔF/F curve in order to create a raster representation of calcium events for later use in analysis software, such as Elephant (Denker, M., Yegenoglu, A., & Grün, S. (2018). Collaborative HPC-enabled workflows on the HBP Collaboratory using the Elephant framework. Neuroinformatics, 19.). Here we present such an open tool with supervised calcium events detection.

Current Practice in Using Voltage Imaging to Record Fast Neuronal Activity: Successful Examples from Invertebrate to Mammalian Studies.

The optical imaging of neuronal activity with potentiometric probes has been credited with being able to address key questions in neuroscience via the simultaneous recording of many neurons. This technique, which was pioneered 50 years ago, has allowed researchers to study the dynamics of neural activity, from tiny subthreshold synaptic events in the axon and dendrites at the subcellular level to the fluctuation of field potentials and how they spread across large areas of the brain. Initially, synthetic voltage-sensitive dyes (VSDs) were applied directly to brain tissue via staining, but recent advances in transgenic methods now allow the expression of genetically encoded voltage indicators (GEVIs), specifically in selected neuron types. However, voltage imaging is technically difficult and limited by several methodological constraints that determine its applicability in a given type of experiment. The prevalence of this method is far from being comparable to patch clamp voltage recording or similar routine methods in neuroscience research. There are more than twice as many studies on VSDs as there are on GEVIs. As can be seen from the majority of the papers, most of them are either methodological ones or reviews. However, potentiometric imaging is able to address key questions in neuroscience by recording most or many neurons simultaneously, thus providing unique information that cannot be obtained via other methods. Different types of optical voltage indicators have their advantages and limitations, which we focus on in detail. Here, we summarize the experience of the scientific community in the application of voltage imaging and try to evaluate the contribution of this method to neuroscience research.

Antioxidative and Immunomodulating Properties of Aronia melanocarpa Extract Rich in Anthocyanins.

The fruits of Aronia melanocarpa are well known due to their high anthocyanin content that may be effective in preventing certain health disorders arising from oxidative stress. Various polyphenolic compounds such as anthocyanins and flavonoids are responsible for the multiple effects of chokeberry. The aim of this study was to determine in vitro how active the black chokeberry anthocyanins are in scavenging radicals and to evaluate in vivo their immunomodulating capacity. Using the method of column chromatography, we extracted the anthocyanins of black chokeberries, i.e., cyanidin-3-O-galactoside with a purity of over 93.7%. Using HPLC and spectrophotometric analysis, the flavonoid content was determined. Following the analysis of the tests with AAPH and DPPH, the chokeberry cyanidin-3-O-galactoside was found much better than individual anthocyanins in regard to antioxidant capacity. The range of concentrations was revealed, showing the protective effect of anthocyanins on the RPMI-1788 cell culture against cyclophosphamide, as well as against osmotic and peroxide hemolysis. An immunomodulating effect on the functional activity of phagocytes was revealed in vivo as a result of oral administration of chokeberry cyanidin-3-O-galactoside and a mixture composed of cyanidin-3-O-glucoside and cyanidin-3-O-galactoside standards. Consequently, anthocyanins, in particular cyanidin-3-O-galactoside, play an important role, demonstrating immunomodulating effects when chokeberries are consumed.

Comparative Assessment of the Phytochemical Composition and Biological Activity of Extracts of Flowering Plants of Centaurea cyanus L., Centaurea jacea L. and Centaurea scabiosa L.

The data on the phytochemical composition and biological activity for flowering plant extracts of the genus Centaurea (Knapweed)-cornflower (Centaurea cyanus L.), brown knapweed (Centaurea jacea L.), and greater knapweed (Centaurea scabiosa L.), which are typical representatives of the flora in the middle belt of the Russian Federation, were obtained. For the first time, biologically active substances such as pyranone, coumaran (2,3-dihydrobenzofuran), and 5-hydroxymethylfurfural were identified in ethanol and methanol extracts of Centaurea scabiosa L. by gas chromatography-mass spectrometry. Catechol and α-amyrin were the major components of the ethanol extract from Centaurea cyanus L., and flavone was the major component of Centaurea jacea L. flower extract. The greatest antimicrobial activity against phytopathogens was detected in Centaurea scabiosa L. when extracting freshly harvested flower biomass with methyl tert-butyl ether at room temperature: the minimum inhibitory concentrations were 60-120 µg/mL, the minimum fungicidal concentration was 120 µg/mL, and the minimum bactericidal concentration was 250 µg/mL. The low antioxidant activity of the studied plant extracts was established using the maximum values of Centaurea jacea L. Ethanol extract of Centaurea cyanus L. flowers had low antimicrobial and antioxidant activity. The extracts showed no phytotoxicity to garden cress germination but inhibited the growth of juvenile plants, especially roots. The greatest phytotoxic effect was revealed with methyl tert-butyl ether, where the depression of growth indicators was 35% or more.

A BK channel-mediated feedback pathway links single-synapse activity with action potential sharpening in repetitive firing.

Action potential shape is a major determinant of synaptic transmission, and mechanisms of spike tuning are therefore of key functional significance. We demonstrate that synaptic activity itself modulates future spikes in the same neuron via a rapid feedback pathway. Using Ca2+ imaging and targeted uncaging approaches in layer 5 neocortical pyramidal neurons, we show that the single spike-evoked Ca2+ rise occurring in one proximal bouton or first node of Ranvier drives a significant sharpening of subsequent action potentials recorded at the soma. This form of intrinsic modulation, mediated by the activation of large-conductance Ca2+/voltage-dependent K+ channels (BK channels), acts to maintain high-frequency firing and limit runaway spike broadening during repetitive firing, preventing an otherwise significant escalation of synaptic transmission. Our findings identify a novel short-term presynaptic plasticity mechanism that uses the activity history of a bouton or adjacent axonal site to dynamically tune ongoing signaling properties.

Thermogenetic stimulation of single neocortical pyramidal neurons transfected with TRPV1-L channels.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of single neurons using thermosensitive cation channels and IR stimulation. The main advantage of IR stimulation compared to conventional visible light optogenetics is the depth of penetration (up to millimeters). Due to physiological limitations, thermogenetic molecular tools for mammalian brain stimulation remain poorly developed. Here, we tested the possibility of employment of this new technique for stimulation of neocortical neurons. The method is based on activation gating of TRPV1-L channels selectively expressed in specific cells. Pyramidal neurons of layer 2/3 of neocortex were transfected at an embryonic stage using a pCAG expression vector and electroporation in utero. Depolarization and spiking responses of TRPV1L+ pyramidal neurons to IR radiation were recorded electrophysiologically in acute brain slices of adult animals with help of confocal visualization. As TRPV1L-expressing neurons are not sensitive to visible light, there were no limitations of the use of this technique with conventional fluorescence imaging. Our experiments demonstrated that the TRPV1-L+ pyramidal neurons preserve their electrical excitability in acute brain slices, while IR radiation can be successfully used to induce single neuronal depolarization and spiking at near physiological temperatures. Obtained results provide important information for adaptation of thermogenetic technology to mammalian brain studies in vivo.

Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons.

The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials (APs). Experimental results support this idea, however mechanisms of fast onset of APs in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of AP generation and encoding. Here we characterize properties of AP encoding in neocortical neurons grown for 11-25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and AP generation. The onset of APs becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of APs as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of AP generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of AP generation and encoding, and their maturation.

Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

Thermogenetic neurostimulation with single-cell resolution.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen-vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

GHSR DNA hypermethylation is a common epigenetic alteration of high diagnostic value in a broad spectrum of cancers.

Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Here, we report a DNA methylation mark that is characteristic of seven studied malignancies, namely cancers of lung, breast, prostate, pancreas, colorectum, glioblastoma and B cell chronic lymphocytic leukaemia (CLL) (n = 137). This mark was defined by substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) through bisulfite pyrosequencing. The degree of aberrant methylation was capable of accurate discrimination between cancer and control samples. The highest sensitivity and specificity of cancer detection was achieved for cancers of pancreas, lung, breast and CLL yielding the area under the curve (AUC) values of 1.0000, 0.9952, 0.9800 and 0.9400, respectively. Narrowing to a single CpG site within the gene's promoter or four consecutive CpG units of the highest methylation levels within the first exon improved the detection power. GHSR hypermethylation was detected already at the early stage tumors. The accurate performance of this marker was further replicated in an independent set of pancreatic cancer and control samples (n = 78). These findings support the candidature of GHSR methylation as a highly accurate pan-cancer marker.

Nonsynaptic plasticity underlies a compartmentalized increase in synaptic efficacy after classical conditioning.

It is now well documented in both vertebrates and invertebrates that nonsynaptic as well as synaptic plasticity can be a substrate for long-term memory [1-4]. Little is known, however, about how learning-induced nonsynaptic plasticity can lead to compartmentalized presynaptic changes underlying specific memory traces while leaving other circuit functions of the neuron unaffected. Here, using behavioral, electrophysiological, and optical recording methods, we show that the previously described learning-induced depolarization of a modulatory neuron [5] of the Lymnaea feeding system affects its axonal terminals in a spatially segregated manner. In a side branch of the axon of the cerebral giant cells (CGCs), classical conditioning of intact animals reduced proximal-to-distal attenuation of spike-evoked calcium transients, providing a highly effective mechanism for a compartmentalized increase in synaptic efficacy. Somatic depolarization by current injection, which spreads onto the CGC's axonal side branch [5], and the blocking of A-type potassium channels with 4-aminopyridine had an effect similar to learning on the calcium transients. Both of these experimental manipulations also reduced axonal spike attenuation. These findings suggest that the voltage-dependent inactivation of an A-type potassium current links global nonsynaptic changes to compartmentalized synaptic changes.