From Cultured Rodent Neurons to Human Brain Tissue: Model Systems for Pharmacological and Translational Neuroscience.

To investigate the enormous complexity of the functional and pathological brain there are a number of possible experimental model systems to choose from. Depending on the research question choosing the appropriate model may not be a trivial task, and given the dynamic and intricate nature of an intact living brain several models might be needed to properly address certain questions. In this review, we aim to provide an overview of neural cell and tissue culture, reflecting on historic methodological milestones and providing a brief overview of the state-of-the-art. We additionally present an example of an effective model system pipeline, composed of dissociated mouse cultures, organotypics, acute mouse brain slices, and acute human brain slices, in that order. The sequential use of these four model systems allows a balance and progression from experimental control to human applicability, and provides a meta-model that can help validate basic research findings in a translational setting. We then conclude with a few remarks regarding the necessity of an integrated approach when performing translational and neuropharmacological studies

Control of Glutamate Transport by Extracellular Potassium: Basis for a Negative Feedback on Synaptic Transmission.

Glutamate and K+, both released during neuronal firing, need to be tightly regulated to ensure accurate synaptic transmission. Extracellular glutamate and K+ ([K+]o) are rapidly taken up by glutamate transporters and K+-transporters or channels, respectively. Glutamate transport includes the exchange of one glutamate, 3 Na+, and one proton, in exchange for one K+. This K+ efflux allows the glutamate binding site to reorient in the outwardly facing position and start a new transport cycle. Here, we demonstrate the sensitivity of the transport process to [K+]o changes. Increasing [K+]o over the physiological range had an immediate and reversible inhibitory action on glutamate transporters. This K+-dependent transporter inhibition was revealed using microspectrofluorimetry in primary astrocytes, and whole-cell patch-clamp in acute brain slices and HEK293 cells expressing glutamate transporters. Previous studies demonstrated that pharmacological inhibition of glutamate transporters decreases neuronal transmission via extrasynaptic glutamate spillover and subsequent activation of metabotropic glutamate receptors (mGluRs). Here, we demonstrate that increasing [K+]o also causes a decrease in neuronal mEPSC frequency, which is prevented by group II mGluR inhibition. These findings highlight a novel, previously unreported physiological negative feedback mechanism in which [K+]o elevations inhibit glutamate transporters, unveiling a new mechanism for activity-dependent modulation of synaptic activity

Extracellular Potassium and Glutamate Interact To Modulate Mitochondria in Astrocytes.

Astrocytes clear glutamate and potassium, both of which are released into the extracellular space during neuronal activity. These processes are intimately linked with energy metabolism. Whereas astrocyte glutamate uptake causes cytosolic and mitochondrial acidification, extracellular potassium induces bicarbonate-dependent cellular alkalinization. This study aimed at quantifying the combined impact of glutamate and extracellular potassium on mitochondrial parameters of primary cultured astrocytes. Glutamate in 3 mM potassium caused a stronger acidification of mitochondria compared to cytosol. 15 mM potassium caused alkalinization that was stronger in the cytosol than in mitochondria. While the combined application of 15 mM potassium and glutamate led to a marked cytosolic alkalinization, pH only marginally increased in mitochondria. Thus, potassium and glutamate effects cannot be arithmetically summed, which also applies to their effects on mitochondrial potential and respiration. The data implies that, because of the nonlinear interaction between the effects of potassium and glutamate, astrocytic energy metabolism will be differentially regulated

Activation of lactate receptor HCAR1 down-modulates neuronal activity in rodent and human brain tissue.

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca <sup>2+</sup> spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy

Imaging extracellular potassium dynamics in brain tissue using a potassium-sensitive nanosensor.

Neuronal activity results in the release of [Formula: see text] into the extracellular space (ECS). Classically, measurements of extracellular [Formula: see text] ([Formula: see text]) are carried out using [Formula: see text]-sensitive microelectrodes, which provide a single point measurement with undefined spatial resolution. An imaging approach would enable the spatiotemporal mapping of [Formula: see text]. Here, we report on the design and characterization of a fluorescence imaging-based [Formula: see text]-sensitive nanosensor for the ECS based on dendrimer nanotechnology. Spectral characterization, sensitivity, and selectivity of the nanosensor were assessed by spectrofluorimetry, as well as in both wide-field and two-photon microscopy settings, demonstrating the nanosensor efficacy over the physiologically relevant ion concentration range. Spatial and temporal kinetics of the nanosensor responses were assessed using a localized iontophoretic [Formula: see text] application on a two-photon imaging setup. Using acute mouse brain slices, we demonstrate that the nanosensor is retained in the ECS for extended periods of time. In addition, we present a ratiometric version of the nanosensor, validate its sensitivity in brain tissue in response to elicited neuronal activity and correlate the responses to the extracellular field potential. Together, this study demonstrates the efficacy of the [Formula: see text]-sensitive nanosensor approach and validates the possibility of creating multimodal nanosensors

Evaluation of Hydroxycarboxylic Acid Receptor 1 (HCAR1) as a Building Block for Genetically Encoded Extracellular Lactate Biosensors.

The status of lactate has evolved from being considered a waste product of cellular metabolism to a useful metabolic substrate and, more recently, to a signaling molecule. The fluctuations of lactate levels within biological tissues, in particular in the interstitial space, are crucial to assess with high spatial and temporal resolution, and this is best achieved using cellular imaging approaches. In this study, we evaluated the suitability of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1, formerly named GPR81), as a basis for the development of a genetically encoded fluorescent lactate biosensor. We used a biosensor strategy that was successfully applied to molecules such as dopamine, serotonin, and norepinephrine, based on their respective G-protein-coupled receptors. In this study, a set of intensiometric sensors was constructed and expressed in living cells. They showed selective expression at the plasma membrane and responded to physiological concentrations of lactate. However, these sensors lost the original ability of HCAR1 to selectively respond to lactate versus other related small carboxylic acid molecules. Therefore, while representing a promising building block for a lactate biosensor, HCAR1 was found to be sensitive to perturbations of its structure, affecting its ability to distinguish between related carboxylic molecules

Within-Mice Comparison of Microdialysis and Fiber Photometry-Recorded Dopamine Biosensor during Amphetamine Response

A fundamental concept in neuroscience is the transmission of information between neurons via neurotransmitters, -modulators, and -peptides. For the past decades, the gold standard for measuring neurochemicals in awake animals has been microdialysis (MD). The emergence of genetically encoded fluorescence-based biosensors, as well as in vivo optical techniques such as fiber photometry (FP), has introduced technologically distinct means of measuring neurotransmission. To directly compare MD and FP, we performed concurrent within-animal recordings of extracellular dopamine (DA) in the dorsal striatum (DS) before and after administration of amphetamine in awake, freely behaving mice expressing the dopamine sensor dLight1.3b. We show that despite temporal differences, MD- and FP-based readouts of DA correlate well within mice. Down-sampling of FP data showed temporal correlation to MD data, with less variance observed using FP. We also present evidence that DA fluctuations periodically reach low levels, and naïve animals have rapid, predrug DA dynamics measured with FP that correlate to the subsequent pharmacodynamics of amphetamine as measured with MD and FP