Functional Analysis of NMDAR Subunit Components in Postsynaptic Currents of Identified Cells and Synapses in Brain Slices.

Epilepsy is one of the most represented neurological diseases worldwide. However, in many cases, the precise molecular mechanisms of epileptogenesis and ictiogenesis are unknown. Because of their important role in synaptic function and neuronal excitability, NMDA receptors are implicated in various epileptogenic mechanisms. Most of these are subunit specific and require a precise analysis of the subunit composition of the NMDARs implicated. Here, we describe an express electrophysiological method to analyze the contribution of NMDAR subunits to spontaneous postsynaptic activity in identified cells in brain slices using patch clamp whole cell recordings.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Estimating the Ca2+ Permeability of NMDA Receptors with Whole-Cell Patch-Clamp Electrophysiology.

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca2+-rich currents. Importantly, their high Ca2+ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca2+ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca2+ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (PCa/PNa) and fractional permeability (Pf), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca2+ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.

Characterizing Functional Contributions of Specific GluN2 Subunits to Individual Postsynaptic NMDAR Responses Using Biophysical Parameters.

The NMDAR is a heterotetramer composed of two GluN1 subunits and two GluN2 and/or GluN3 subunits, with the GluN2 subunits exhibiting significant diversity in their structure and function. Recent studies have highlighted the importance of characterizing the specific roles of each GluN2 subunit across central nervous system regions and developmental stages, as well as their unique contributions to NMDAR-mediated signaling and plasticity. Understanding the distinct functions of GluN2 subunits is critical for the development of targeted therapeutic strategies for NMDAR-related disorders. However, measuring the functional contribution of individual GluN2 subtypes in ex vivo slices is challenging. Conventionally, pharmacological or genetic approaches are used, but, in many cases, this is not possible or is restricted to population-level NMDAR responses. Here, we describe a technique for using biophysical properties of miniature synaptic NMDAR responses as a proxy to measure the functional contribution of specific GluN2-NMDAR subunits to individual synapses within a neuron.

Analysis of Functional NMDA Receptors in Astrocytes.

Neuronal N-methyl-D-aspartate (NMDA) receptors are well known for their pivotal role in memory formation. Originally, they were thought to be exclusive to neurons. However, numerous studies revealed their functional expression also on various types of glial cells in the nervous system. Here, the methodology on how to study the physiology of NMDA receptors selectively on astrocytes will be described in detail. Astrocytes are the main class of neuroglia that control transmitter and ion homeostasis, which link cerebral blood flow and neuronal energy demands, but also affect synaptic transmission directly.

Implications of high homocysteine levels in migraine pain: An experimental study of the excitability of peripheral meningeal afferents in rats with hyperhomocysteinemia.

OBJECTIVES: Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats. BACKGROUND: Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia. METHODS: Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations ex vivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue. RESULTS: The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull ex vivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed. CONCLUSIONS: Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Electrophysiology of Ctenophore Smooth Muscle.

Unlike in the Cnidaria, where muscle cells are coupled together into an epithelium, ctenophore muscles are single, elongated, intramesogleal structures resembling vertebrate smooth muscle. Under voltage-clamp, these fibers can be separated into different classes with different sets of membrane ion channels. The ion channel makeup is related to the muscle's anatomical position and specific function. For example, Beroe ovata radial fibers, which are responsible for maintaining the rigidity of the body wall, generate sequences of brief action potentials whereas longitudinal fibers, which are concerned with mouth opening and body flexions, often produce single longer duration action potentials.Beroe muscle contractions depend on the influx of Ca2+. During an action potential the inward current is carried by Ca2+, and the increase in intracellular Ca2+ concentration generated can be monitored in FLUO-3-loaded cells. Confocal microscopy in line scan mode shows that the Ca2+ spreads from the outer membrane into the core of the fiber and is cleared from there relatively slowly. The rise in intracellular Ca2+ is linked to an increase in a Ca2+-activated K+ conductance (KCa), which can also be elicited by iontophoretic Ca2+ injection. Near the cell membrane, Ca2+ clearance monitored using FLUO3, matches the decline in the KCa conductance. For light loads, Ca2+ is cleared rapidly, but this fast system is insufficient when Ca2+ influx is maintained. Action potential frequency may be regulated by the slowly developing KCa conductance.

Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Actions of remimazolam on inhibitory transmission of rat spinal dorsal horn neurons.

Remimazolam is an ultra-short benzodiazepine that acts on the benzodiazepine site of γ-aminobutyric acid (GABA) receptors in the brain and induces sedation. Although GABA receptors are found localized in the spinal dorsal horn, no previous studies have reported the analgesic effects or investigated the cellular mechanisms of remimazolam on the spinal dorsal horn. Behavioral measures, immunohistochemistry, and in vitro whole-cell patch-clamp recordings of dorsal horn neurons were used to assess synaptic transmission. Intrathecal injection of remimazolam induced behavioral analgesia in inflammatory pain-induced mechanical allodynia (six rats/dose; p < 0.05). Immunohistochemical staining revealed that remimazolam suppressed spinal phosphorylated extracellular signal-regulated kinase activation (five rats/group, p < 0.05). In vitro whole-cell patch-clamp analysis demonstrated that remimazolam increased the frequency of GABAergic miniature inhibitory post-synaptic currents, prolonged the decay time (six rats; p < 0.05), and enhanced GABA currents induced by exogenous GABA (seven rats; p < 0.01). However, remimazolam did not affect miniature excitatory post-synaptic currents or amplitude of monosynaptic excitatory post-synaptic currents evoked by Aδ- and C-fiber stimulation (seven rats; p > 0.05). This study suggests that remimazolam induces analgesia by enhancing GABAergic inhibitory transmission in the spinal dorsal horn, suggesting its potential utility as a spinal analgesic for inflammatory pain.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Electrophysiological and morphological properties of prefrontal pyramidal neurons innervated by mediodorsal thalamus.

The high-order cognitive and executive functions are necessary for an individual to survive. The densely bidirectional innervations between the medial prefrontal cortex (mPFC) and the mediodorsal thalamus (MD) play a vital role in regulating high-order functions. Pyramidal neurons in mPFC have been classified into several subclasses according to their morphological and electrophysiological properties, but the properties of the input-specific pyramidal neurons in mPFC remain poorly understood. The present study aimed to profile the morphological and electrophysiological properties of mPFC pyramidal neurons innervated by MD. In the past, the studies for characterizing the morphological and electrophysiological properties of neurons mainly relied on the electrophysiological recording of a large number of neurons and their morphologic reconstructions. But, it is a low efficient method for characterizing the circuit-specific neurons. The present study combined the advantages of traditional morphological and electrophysiological methods with machine learning to address the shortcomings of the past method, to establish a classification model for the morphological and electrophysiological properties of mPFC pyramidal neurons, and to achieve more accurate and efficient identification of the properties from a small size sample of neurons. We labeled MD-innervated pyramidal neurons of mPFC using the trans-synaptic neural circuitry tracing method and obtained their morphological properties using whole-cell patch-clamp recording and morphologic reconstructions. The results showed that the classification model established in the present study could predict the electrophysiological properties of MD-innervated pyramidal neurons based on their morphology. MD-innervated pyramidal neurons exhibit larger basal dendritic length but lower apical dendrite complexity compared to non-MD-innervated neurons in the mPFC. The morphological characteristics of the two subtypes (ET-1 and ET-2) of mPFC pyramidal neurons innervated by MD are different, with the apical dendrites of ET-1 neurons being longer and more complex than those of ET-2 neurons. These results suggest that the electrophysiological properties of MD- innervated pyramidal neurons within mPFC correlate with their morphological properties, indicating that the different roles of these two subclasses in local circuits within PFC, as well as in PFC-cortical/subcortical brain region circuits.

In Vitro Patch-Clamp.

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development.

In Vivo Whole-Cell Recording from the Mouse Brain.

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface.

Loose Coupling between the Voltage Sensor and the Activation Gate in Mammalian HCN Channels Suggests a Gating Mechanism.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>109) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10-9 of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.

Increased glutamatergic neurotransmission between the retinohypothalamic tract and the suprachiasmatic nucleus of old mice.

Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation, and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here, we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole-cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single-neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55 and 73%, respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.

Electrophysiological Properties of Proprioception-Related Neurons in the Intermediate Thoracolumbar Spinal Cord.

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.

Ensemble learning and ground-truth validation of synaptic connectivity inferred from spike trains.

Probing the architecture of neuronal circuits and the principles that underlie their functional organization remains an important challenge of modern neurosciences. This holds true, in particular, for the inference of neuronal connectivity from large-scale extracellular recordings. Despite the popularity of this approach and a number of elaborate methods to reconstruct networks, the degree to which synaptic connections can be reconstructed from spike-train recordings alone remains controversial. Here, we provide a framework to probe and compare connectivity inference algorithms, using a combination of synthetic ground-truth and in vitro data sets, where the connectivity labels were obtained from simultaneous high-density microelectrode array (HD-MEA) and patch-clamp recordings. We find that reconstruction performance critically depends on the regularity of the recorded spontaneous activity, i.e., their dynamical regime, the type of connectivity, and the amount of available spike-train data. We therefore introduce an ensemble artificial neural network (eANN) to improve connectivity inference. We train the eANN on the validated outputs of six established inference algorithms and show how it improves network reconstruction accuracy and robustness. Overall, the eANN demonstrated strong performance across different dynamical regimes, worked well on smaller datasets, and improved the detection of synaptic connectivity, especially inhibitory connections. Results indicated that the eANN also improved the topological characterization of neuronal networks. The presented methodology contributes to advancing the performance of inference algorithms and facilitates our understanding of how neuronal activity relates to synaptic connectivity.