Passive Conductance Research Articles

Astrocytic NHERF-1 Increases Seizure Susceptibility by Inhibiting Surface Expression of TREK-1.

Mature hippocampal astrocytes exhibit a linear current-to-voltage (I-V) K+ membrane conductance called passive conductance. It is estimated to enable astrocytes to keep potassium homeostasis in the brain. We previously reported that the TWIK-1/TREK-1 heterodimeric channels are crucial for astrocytic passive conductance. However, the regulatory mechanism of these channels by other binding proteins remains elusive. Here, we identified Na+/H+ exchange regulator-1 (NHERF-1), a protein highly expressed in astrocytes, as a novel interaction partner for these channels. NHERF-1 endogenously bound to TWIK-1/TREK-1 in hippocampal cultured astrocytes. When NHERF-1 is overexpressed or silenced, surface expression and activity of TWIK-1/TREK-1 heterodimeric channels are inhibited or enhanced, respectively. Furthermore, we confirmed that reduced astrocytic passive conductance by NHERF-1 overexpressing in the hippocampus increases kainic acid (KA)-induced seizure sensitivity. Taken together, these results suggest that NHERF-1 is a key regulator of TWIK-1/TREK-1 heterodimeric channels in astrocytes and suppression of TREK-1 surface expression by NHERF-1 increases KA-induced seizure susceptibility via reduction of astrocytic passive conductance.

β-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes.

Mature astrocytes are characterized by a K+ conductance (passive conductance) that changes with a constant slope with voltage, which is involved in K+ homeostasis in the brain. Recently, we reported that the tandem of pore domains in a weak inward rectifying K+ channel (TWIK1 or KCNK1) and TWIK-related K+ channel 1 (TREK1 or KCNK2) form heterodimeric channels that mediate passive conductance in astrocytes. However, little is known about the binding proteins that regulate the function of the TWIK1/TREK1 heterodimeric channels. Here, we found that β-coat protein (COP) regulated the surface expression and activity of the TWIK1/TREK1 heterodimeric channels in astrocytes. β-COP binds directly to TREK1 but not TWIK1 in a heterologous expression system. However, β-COP also interacts with the TWIK1/TREK1 heterodimeric channel in a TREK1 dependent manner and enhances the surface expression of the heterodimeric channel in astrocytes. Consequently, it regulates TWIK1/TREK1 heterodimeric channel-mediated passive conductance in astrocytes in the mouse brain. Taken together, these results suggest that β-COP is a potential regulator of astrocytic passive conductance in the brain.

A Deafness Associated Protein TMEM43 Interacts with KCNK3 (TASK-1) Two-pore Domain K+ (K2P) Channel in the Cochlea.

The TMEM43 has been studied in human diseases such as arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5) and auditory neuropathy spectrum disorder (ANSD). In the heart, the p.(Ser358Leu) mutation has been shown to alter intercalated disc protein function and disturb beating rhythms. In the cochlea, the p.(Arg372Ter) mutation has been shown to disrupt connexin-linked function in glia-like supporting cells (GLSs), which maintain inner ear homeostasis for hearing. The TMEM43-p.(Arg372Ter) mutant knock-in mice displayed a significantly reduced passive conductance current in the cochlear GLSs, raising a possibility that TMEM43 is essential for mediating the passive conductance current in GLSs. In the brain, the two-pore-domain potassium (K2P) channels are generally known as the “leak channels” to mediate background conductance current, raising another possibility that K2P channels might contribute to the passive conductance current in GLSs. However, the possible association between TMEM43 and K2P channels has not been investigated yet. In this study, we examined whether TMEM43 physically interacts with one of the K2P channels in the cochlea, KCNK3 (TASK-1). Utilizing co-immunoprecipitation (IP) assay and Duolink proximity ligation assay (PLA), we revealed that TMEM43 and TASK-1 proteins could directly interact. Genetic modifications further delineated that the intracellular loop domain of TMEM43 is responsible for TASK-1 binding. In the end, gene-silencing of Task-1 resulted in significantly reduced passive conductance current in GLSs. Together, our findings demonstrate that TMEM43 and TASK-1 form a protein-protein interaction in the cochlea and provide the possibility that TASK-1 is a potential contributor to the passive conductance current in GLSs.

TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression.

TWIK-1 is the first identified member of the two-pore domain potassium (K2P) channels that are involved in neuronal excitability and astrocytic passive conductance in the brain. Despite the physiological roles of TWIK-1, there is still a lack of information on the basic expression patterns of TWIK-1 proteins in the brain. Here, using a modified bacterial artificial chromosome (BAC), we generated a transgenic mouse (Tg mouse) line expressing green fluorescent protein (GFP) under the control of the TWIK-1 promoter (TWIK-1 BAC-GFP Tg mice). We confirmed that nearly all GFP-producing cells co-expressed endogenous TWIK-1 in the brain of TWIK-1 BAC-GFP Tg mice. GFP signals were highly expressed in various brain areas, including the dentate gyrus (DG), lateral entorhinal cortex (LEC), and cerebellum (Cb). In addition, we found that GFP signals were highly expressed in immature granule cells in the DG. Finally, our TWIK-1 BAC-GFP Tg mice mimic the upregulation of TWIK-1 mRNA expression in the hippocampus following the injection of kainic acid (KA). Our data clearly showed that TWIK-1 BAC-GFP Tg mice are a useful animal model for studying the mechanisms regulating TWIK-1 gene expression and the physiological roles of TWIK-1 channels in the brain.

On the electrical passivity of astrocyte potassium conductance.

Predominant expression of leak-type K+ channels provides astrocytes a high membrane permeability to K+ ions and a hyperpolarized membrane potential that are crucial for astrocyte function in brain homeostasis. In functionally mature astrocytes, the expression of leak K+ channels creates a unique membrane K+ conductance that lacks voltage-dependent rectification. Accordingly, the conductance is named ohmic or passive K+ conductance. Several inwardly rectifying and two-pore domain K+ channels have been investigated for their contributions to passive conductance. Meanwhile, gap junctional coupling has been postulated to underlie the passive behavior of membrane conductance. It is now clear that the intrinsic properties of K+ channels and gap junctional coupling can each act alone or together to bring about a passive behavior of astrocyte conductance. Additionally, while the passive conductance can generally be viewed as a K+ conductance, the actual representation of this conductance is a combined expression of multiple known and unknown K+ channels, which has been further modified by the intricate morphology of individual astrocytes and syncytial gap junctional coupling. The expression of the inwardly rectifying K+ channels explains the inward-going component of passive conductance disobeying Goldman-Hodgkin-Katz constant field outward rectification. However, the K+ channels encoding the outward-going passive currents remain to be determined in the future. Here, we review our current understanding of ion channels and biophysical mechanisms engaged in the passive astrocyte K+ conductance, propose new studies to resolve this long-standing puzzle in astrocyte physiology, and discuss the functional implication(s) of passive behavior of K+ conductance on astrocyte physiology.

Contribution of Neuronal and Glial Two-Pore-Domain Potassium Channels in Health and Neurological Disorders.

Two-pore-domain potassium (K2P) channels are widespread in the nervous system and play a critical role in maintaining membrane potential in neurons and glia. They have been implicated in many stress-relevant neurological disorders, including pain, sleep disorder, epilepsy, ischemia, and depression. K2P channels give rise to leaky K+ currents, which stabilize cellular membrane potential and regulate cellular excitability. A range of natural and chemical effectors, including temperature, pressure, pH, phospholipids, and intracellular signaling molecules, substantially modulate the activity of K2P channels. In this review, we summarize the contribution of K2P channels to neuronal excitability and to potassium homeostasis in glia. We describe recently discovered functions of K2P channels in glia, such as astrocytic passive conductance and glutamate release, microglial surveillance, and myelin generation by oligodendrocytes. We also discuss the potential role of glial K2P channels in neurological disorders. In the end, we discuss current limitations in K2P channel researches and suggest directions for future studies.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels.

Astrocytes, the most abundant cell type in the brain, are non-excitable cells and play critical roles in brain function. Mature astrocytes typically exhibit a linear current–voltage relationship termed passive conductance, which is believed to enable astrocytes to maintain potassium homeostasis in the brain. We previously demonstrated that TWIK-1/TREK-1 heterodimeric channels mainly contribute to astrocytic passive conductance. However, the molecular identity of astrocytic passive conductance is still controversial and needs to be elucidated. Here, we report that spadin, an inhibitor of TREK-1, can dramatically reduce astrocytic passive conductance in brain slices. A series of gene silencing experiments demonstrated that spadin-sensitive currents are mediated by TWIK-1/TREK-1 heterodimeric channels in cultured astrocytes and hippocampal astrocytes from brain slices. Our study clearly showed that TWIK-1/TREK-1-heterodimeric channels can act as the main molecular machinery of astrocytic passive conductance, and suggested that spadin can be used as a specific inhibitor to control astrocytic passive conductance.

Heterogeneity of astrocytes: Electrophysiological properties of juxtavascular astrocytes before and after brain injury.

Astrocyte heterogeneity is increasingly recognized, but still little is known about juxtavascular astrocytes with their somata directly adjacent to blood vessels, despite their importance after brain injury. As juxtavascular astrocytes originate from common progenitor cells, that is, have a clonal origin, they may intrinsically differ from other, non-juxtavascular astrocytes. To explore this, we examined the electrophysiological properties of these groups of astrocytes and the underlying ion channels. Using brain slices of BAC Aldh1l1-eGFP transgenic mice with astrocytes labeled by GFP expression, we compared juxtavascular and non-juxtavascular astrocytes in the somatosensory cortex by means of whole-cell patch-clamp recordings and immunohistochemical staining. Prior to injury, juxta- and non-juxtavascular astrocytes exhibit comparable electrophysiological properties with characteristic mostly passive conductance and a typical negative resting membrane potential. Immunohistochemical analysis of K+ channels showed that all astrocytes were Kir 4.1+ , but revealed an intriguing difference for Kv 4.3. The expression of Kv 4.3 in sibling astrocytes (non-juxtavascular, juxtavascular and pial) was dependent on their ontogenetic origin with lowest levels in juxtavascular astrocytes located in upper cortical layers. After traumatic brain injury (TBI), we found profound changes in the electrophysiological type of astrocytes with a predominance of non-passive properties and this pattern was significantly enriched in juxtavascular astrocytes. This was accompanied by pronounced down-regulation of Kir 4.1 in proliferating astrocytes, which was significantly more in juxtavascular compared to non-juxtavascular astrocytes. Taken together, TBI induces profound differences in electrophysiological properties between juxtavascular and non-juxtavascular astrocytes that might be related to the preponderance of juxtavascular astrocyte proliferation.

Algorithm for indirect measurement method of parameters of passive complex conductance of a two-pole in a multi-pole electrical circuit

An algorithm is proposed for indirectly measuring the parameters of the passive complex conductivity of a two-terminal circuit, both separate and located in one of the branches of a multi-pole passive electric circuit of the triangle-type. A block diagram of a microprocessor measurement unit is presented. The algorithm of the indirect method for measuring the parameters of the passive complex conductivity of a two-terminal circuit, the rationale of which is given on the basis of the theory of matrices and undirected graphs, is considered. Equations for calculating the modulus and components of the studied passive complex conductivity of a two-terminal device located in a multi-pole electric circuit of the triangle-type are obtained. The revealed features of the proposed algorithm exclude a methodological error due to the influence of spurious parameters. Parasitic parameters include: input complex conductivities and transmission coefficients of voltage converters; complex conductivity of a harmonic signal source; passive complex conductivity of two-terminal circuits located in other branches of the studied passive multipolar electric circuit. The results of an experimental verification of the developed algorithm of the indirect method for measuring the module of the complex conductivity of a two-terminal device are presented. The developed algorithm can be used to measure the parameters of passive complex conductivity of both an individual two-terminal circuit and a two-terminal circuit located in one of the branches of a multi-pole passive electric circuit of the triangle-type, without the need for its separation into separate two-terminal devices.

Astrocytic AEG-1 regulates expression of TREK-1 under acute hypoxia.

TREK-1 (TWIK-related K+ channel), a member of the two-pore domain K+ (K2P) channel family, is highly expressed in astrocytes, where it plays a key role in glutamate release and passive conductance. In addition, TREK-1 is induced to protect neurons under pathological conditions such as hypoxia. However, the upstream regulation of TREK-1 remains poorly understood. In this study, we found that AEG-1 (astrocyte elevated gene-1) regulates the expression of astrocytic TREK-1 under hypoxic conditions. Upregulation of AEG-1 increased expression of TREK-1 in astrocytes, and knockdown of AEG-1 dramatically decreased the mRNA and protein levels of TREK-1, which were restored by expression of shRNA-insensitive AEG-1. In addition, expression of TREK-1 was not regulated in the absence of AEG-1, even when HIF1α was present. Together, these results suggest that AEG-1 acts as a major upstream regulator of TREK-1 channels in astrocytes under hypoxia. SIGNIFICANCE OF THE STUDY: Previous studies have reported that hypoxia increases the expression of astrocytic TREK-1 and that increased TREK-1 expression protects neuronal cells from apoptosis. However, its cellular mechanism is not clear. In this study we first showed that AEG-1 is a major mediator of hypoxic-regulated TREK-1 expression in normal astrocytes independently of HIF-1α.

TMEM43, a novel passive conductance channel of cochlear glia is critical for maintenance of speech discrimination

TMEM43, a novel passive conductance channel of cochlear glia is critical for maintenance of speech discrimination

Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

Recently, μ-opioid receptor (MOR), one of the well-known Gi-protein coupled receptors (Gi-GPCR), was reported to be highly expressed in the hippocampal astrocytes. However, the role of astrocytic MOR has not been investigated. Here we report that activation of astrocytic MOR by [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO), a selective MOR agonist, causes a fast glutamate release using sniffer patch technique. We also found that the DAMGO-induced glutamate release was not observed in the astrocytes from MOR-deficient mice and MOR-short hairpin RNA (shRNA)-expressed astrocytes. In addition, the glutamate release was significantly reduced by gene silencing of the TREK-1-containing two-pore potassium (K2P) channel, which mediates passive conductance in astrocytes. Our findings were consistent with the previous study demonstrating that activation of Gi-GPCR such as cannabinoid receptor CB1 and adenosine receptor A1 causes a glutamate release through TREK-1-containing K2P channel from hippocampal astrocytes. We also demonstrated that MOR and TREK-1 are significantly co-localized in the hippocampal astrocytes. Furthermore, we found that both MOR and TREK-1-containing K2P channels are localized in the same subcellular compartments, soma and processes, of astrocytes. Our study raises a novel possibility that astrocytic MOR may participate in several physiological and pathological actions of opioids, including analgesia and addiction, through astrocytically released glutamate and its signaling pathway.

Emerging Roles of TWIK-1 Heterodimerization in the Brain.

Two-pore domain K+ (K2P) channels play essential roles in regulating resting membrane potential and cellular excitability. Although TWIK-1 (TWIK—tandem of pore domains in a weak inward rectifying K+ channel) was the first identified member of the K2P channel family, it is only in recent years that the physiological roles of TWIK-1 have been studied in depth. A series of reports suggest that TWIK-1 may underlie diverse functions, such as intrinsic excitability of neurons, astrocytic passive conductance, and astrocytic glutamate release, as a homodimer or heterodimer with other K2P isotypes. Here, we summarize expression patterns and newly identified functions of TWIK-1 in the brain.

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture.

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes.

Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ.

We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K+ channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (VM) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K+ channels remains elusive. TREK-1 two-pore domain K+ channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of heterodimer TWIK-1/TREK-1 channel to the membrane and contributing to astrocyte passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K+ channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, VM, or membrane input resistance (Rin) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K+ channels may shed light on this long-standing and important question in astrocyte physiology.

Freshly dissociated mature hippocampal astrocytes exhibit passive membrane conductance and low membrane resistance similarly to syncytial coupled astrocytes.

Mature astrocytes exhibit a linear current-to-voltage K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.

Retinal origin of electrically evoked potentials in response to transcorneal alternating current stimulation in the rat.

Little is known about the physiological mechanisms underlying the reported therapeutic effects of transorbital alternating current stimulation (ACS) in vision restoration, or the origin of the recorded electrically evoked potentials (EEPs) during such stimulation. We examined the issue of EEP origin and electrode configuration for transorbital ACS and characterized the physiological responses to CS in different structures of the visual system. We recorded visually evoked potentials (VEPs) and EEPs from the rat retina, visual thalamus, tectum, and visual cortex. The VEPs were evoked by light flashes and EEPs were evoked by electric stimuli delivered by two electrodes placed either together on the same eye or on the eyeball and in the neck. Electrically evoked potentials and VEPs were recorded before and after bilateral intraorbital injections of tetrodotoxin that blocked retinal ganglion cell activity. Tetrodotoxin abolished VEPs at all levels in the visual pathway, confirming successful blockage of ganglion cell activity. Tetrodotoxin also abolished EEPs and this effect was independent of the stimulating electrode configurations. Transorbital electrically evoked responses in the visual pathway, irrespective of reference electrode placement, are initiated by activation of the retina and not by passive conductance and direct activation of neurons in other visual structures. Thus, placement of stimulating electrodes exclusively around the eyeball may be sufficient to achieve therapeutic effects.

Linolenic acid provides multi-cellular protective effects after photothrombotic cerebral ischemia in rats.

Alpha-linolenic acid (LIN) has been shown to provide neuroprotective effects against cerebral ischemia. LIN is a potent activator of TREK-1 channel and LIN-induced neuroprotection disappears in Trek1-/- mice, suggesting that this channel is directly related to the LIN-induced resistance of brain against ischemia. However, the cellular mechanism underlying LIN induced neuroprotective effects after ischemia remains unclear. In this study, using a rat photochemical brain ischemia model, we investigated the effects of LIN on the protein abundance of astrocytic glutamate transporter and AQP4, microglia activation, cell apoptosis and behavioral recovery following ischemia. Administration of LIN rescued the protein abundance of astrocytic glutamate transporter GLT-1, decreased the protein abundance of AQP4 and brain edema, inhibited microglia activation, attenuated cell apoptosis and improved behavioral function recovery. Meanwhile, TREK-1 was widely distributed in the cortex and hippocampus, primarily localized in astrocytes and neurons. LIN could potentiate the TREK-1 mediated astrocytic passive conductance and hyperpolarize the membrane potential. Our results suggest that LIN provides multiple cellular neuroprotective effects in cerebral ischemia. TREK-1 may serve as a promising multi-mechanism therapeutic target for the treatment of stroke.

Automatic fitness function selection for compartment model optimization

During normal aging, layer 3 pyramidal neurons of the rhesus monkey prefrontal cortex (PFC) exhibit significant morphological changes, as well as higher action potential firing rates in vitro [1]. Computational modeling of individual neurons can provide insight into the ionic mechanisms underlying the increased excitability, which are currently unknown. A unique database of electrophysiological recordings and morphologic reconstructions from the same neurons, gathered through whole-cell patch clamp recording, confocal microscopy and 3D digital tracing, constrains the models. Initial modeling of six young and six aged neurons demonstrated that morphological features alone do not account entirely for the electrophysiological changes with aging [2]. It is now necessary to explore the parameter space of passive cable properties and active membrane channel conductances and kinetics, to uncover parameter combinations that reproduce the firing patterns observed in neurons of each age group. Differential Evolution (DE) is an evolutionary optimization method capable of identifying a population of candidate models throughout parameter space that closely match empirically observed firing patterns. The quality of fit achieved by an optimization is reliant on the ‘fitness functions’ used to measure the accuracy of the model. Previous neuronal compartment modeling studies using parameter optimization have introduced multiple types of fitness measurement [3], but have not described a general method to determine weights for each type. Here we introduce a novel method for automatically establishing weights of minimally correlated fitness functions, and apply it to optimization of models of young and aged PFC neurons. First, a Latin hypercube design (< 1000 points) provides a space-filling sampling of parameter space; the candidate fitness functions are then evaluated at each of these points. Second, clusters of fitness functions that are highly correlated across the hypercube are pruned to leave one representative member. Third, a principal component analysis of the remaining fitness functions across the hypercube identifies a set of fitness functions representing most of the variability in the parameter space, which are selected for use in the optimization. Fourth, weights for each selected fitness function are calculated based on the combination of coefficients for principal components and variance explained by those components. Finally, DE is conducted on the Neuroscience Gateway [4] using this automatically constructed optimization protocol. We demonstrate the method with a compartment model comprising a simplified pyramidal neuron morphology and three ion channels, optimized to data from representative young and aged neurons. Compared to a manual approach involving iterative generation of fitness functions, our novel method produces better fitting models using a tenth of the computation time. Future work will extend the automatic protocol generation to prioritize which parameters to optimize, a critical step as more ion channels are added to the model to improve fitness. This method will be used to generate morphologically detailed models of 20+ young and aged PFC neurons, predicting which ionic mechanisms underlie age-related physiological changes.