Author response: Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease
2019-08-22

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling ‘high throughput’ fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry. https://doi.org/10.7554/eLife.48417.001 Introduction The human brain is composed of an intricate network of diverse cell types with complex interactions and connections (DeFelipe et al., 2002; Somogyi et al., 1998). The microstructure within the brain was already appreciated in the early pioneering work by Santiago Ramón y Cajal and has been further investigated in the last decades in post mortem human brain samples (Elston and DeFelipe, 2002; Elston et al., 2001) and in numerous animal models (Branco and Häusser, 2011; Somogyi et al., 1998). Neurons function as a core component of the microstructure of CNS circuits. The computation and integration of signals by these cells and the output is believed to underlie higher brain functions such as cognition and learning. Two broad classes of neurons, glutamatergic neurons (pyramidal neurons, 80%) and GABAergic interneurons (20%), populate the human neocortex. Cortical pyramidal neurons can be further categorized into four classes according to their output connectivity. Intratelencephalic pyramidal neurons are distributed throughout cortical layers 2–6 and build associative connections with other cortical areas (within one hemisphere or across the corpus callosum) or with the striatum. Pyramidal tract neurons reside in layer five and project their axons to the brain stem and spinal cord. Corticothalamic neurons establish axonal output connectivity from layer six to area related thalamic nuclei. The fourth category comprises layer four short-axon intratelencephalic neurons, which locally relay thalamic input (Shepherd and Rowe, 2017). With the exception of layer four intratelencephalic neurons in sensory areas (spiny stellate cells) pyramidal neurons typically show a long apical dendrite. Overall morphology and complexity of apical and basal dendrites can substantially differ between pyramidal neurons and throughout layers. Furthermore, work by colleagues in the field provides direct evidence that there also exist marked species-specific differences of structural features and functional properties of pyramidal neurons (Mohan et al., 2015) as well as regarding basic cortical properties (Eyal et al., 2016), cell types (Boldog et al., 2018; Wang et al., 2015), morphology (Elston et al., 2001; Mohan et al., 2015), functional divergence (Napoli and Obeid, 2016) and plasticity mechanisms (Verhoog et al., 2013). Such differences underscore the challenges of modeling human disease by cellular and animal models and call for the development of new translational approaches based on human nervous tissue. Spare human CNS tissue obtained from neurosurgical procedures has proven valuable throughout the years, for example for electrophysiological studies in acute slice preparations (Bernard et al., 2004; Kalmbach et al., 2018; Kerkhofs et al., 2017). Unlike with rodent CNS tissue, past attempts by different groups to establish organotypic slice cultures of human CNS tissue had yet repeatedly faced pronounced limitations regarding viability and integrity (O'Connor et al., 1997; Verwer et al., 2002). However, recent efforts challenged these limitations and provided promising proof-of-concept data on electrophysiological function (Eugène et al., 2014) and optogenetic targeting (Andersson et al., 2016) in human brain slice cultures. Subsequent studies established successful neuronal labeling, optical manipulation and calcium imaging by virus-driven rapid expression of respective probes in short term cultures of several days (Ting et al., 2018). Recent parallel work by us in contrast focused on optimization of culture conditions and demonstrated that neuronal viability and network activity of human brain slice cultures can be significantly extended to up to three weeks under specified conditions (Schwarz et al., 2017). Proper function of complex CNS networks, however, critically depends on morphological and functional integrity of neurons structurally underlying circuitry. Neurons need to extend their dendritic processes to correct locations in order to receive appropriate synaptic inputs and relay them to adequate target structures. Specific functional properties are required to compute and integrate information in a meaningful way. Whether this level of morphological complexity along with inherent electrophysiological characteristics is maintained in human brain slice cultures over a period of two to three weeks or whether dendritic arbors rarefy and functional features dissipate is not known and will be investigated in this work. In the last decades, important molecular genetic achievements and subsequent combined application of tools such as the Cre-lox system (Song and Palmiter, 2018), optogenetics (Kim et al., 2017; Madisen et al., 2012) and virus-mediated gene targeting/knockout/delivery (Hendrie and Russell, 2005; Howard et al., 2008; Rao and Craig, 1997) enabled an increasingly better understanding of how distinct cell types assemble and form functional circuitry in the rodent brain. Adapting such strategies to human brain slice cultures reflecting an environment as close as possible to the actual human brain will open new roads toward dissection of human CNS circuitry and could potentially provide an increasingly more accurate understanding of how human CNS disease develops. Toward these aims, we now performed detailed cellular analyses of the somatodendritic and synaptic spine compartments and of characteristic electrophysiological properties of single neurons throughout the course of culturing. These features form the basis of intact network function but whether they are maintained in cortical slice cultures remained still elusive. Further experiments established the conditions for efficient viral transduction with a focus on glutamatergic pyramidal neurons within human brain slice cultures. We find robust structural and electrophysiological stability of human pyramidal neurons and amenability of human brain slice cultures to efficient genetic manipulation, as revealed by successful transduction by GFP-encoding adeno-associated viral vectors (AAV-vectors). GFP fluorescence levels were sufficient for both post-hoc confocal microscopy- and two-photon live cell imaging-based assessment of neuronal morphology including spines emanating from the dendritic arbors. We demonstrate GFP-based cellular 3D morphological analysis with increased efficiency and compatible quality in comparison to classic intracellular biocytin fillings. The presented data indicate feasibility of utilizing human brain slice cultures as a model system as close as possible to the actual human brain and apply them toward studies of human CNS circuitry, disease and therapeutic screening, thereby having the potential to close the translational gap to non-human model systems. Results Electrophysiological properties of pyramidal neurons in human brain slice cultures versus acute slices Besides morphological and structural features intact physiology of neurons constitutes one of the hallmarks of healthy neural tissue. In a first set of experiments we determined the stability of the electrophysiological properties of pyramidal neurons performing whole cell patch clamp recordings (n = 45) from cells in acute (n = 17) or cultured (n = 28) slices (2–14 DIV). The cells recorded from cultured slices were further subdivided into early (2-3 DIV, n = 8) and late in culture (7–14 DIV, n = 20) for statistical group analysis (Figure 1E). Recordings of pyramidal neurons showed a typical regular spiking pattern in acute and cultured slices (Figure 1B,C), as described in previous studies investigating human pyramidal neurons in acute slices (Verhoog et al., 2013). The firing frequency in response to current injections was not significantly different between the cells recorded on the day of the surgery (acute slices = 0 DIV, with a peak firing rate of 50.84 ± 9.90 Hz in response to a current injection of 200 pA) and the cells in cultures between 2 and 3 DIV (peak firing rate: 59.4 ± 12.46) and 7–14 DIV (peak firing rate of 55.79 ± 9.34 Hz), (Kruskal-Wallis test, p=0.74, Figure 1C). Similarly, mean values of AP half width, sag potential amplitude and input resistance did not differ between the tested groups (0 DIV, 2–3 DIV and 7–14 DIV, Kruskal-Wallis test, p>0.05, Figure 1E). This was also reflected by no significant linear regression correlation of these parameters versus the DIV (Figure 1D). Figure 1 with 2 supplements see all Download asset Open asset Electrophysiology of adult human pyramidal neurons. (A) Example of biocytin filled pyramidal neuron (14 DIV) after streptavidin-Cy3 counterstaining, scale bar 150 μm. (B) Typical regular spiking pattern of human pyramidal neurons (acute slice, 3 DIV and 13 DIV) in response to +200 pA positive current injection. (C) Neurons recorded in acute slices (within 12 hr after surgery) and in brain slice cultures (2–3 DIV and 7–14 DIV) showed similar action potential firing frequencies and typical spike frequency adaptation upon 200 pA current injection. (D, E) Quantification of (from left to right) the AP half width, resting membrane potential, sag potential amplitude and input resistance revealed no significant correlation between these values and the days in vitro (DIV), (E) except membrane resting potential which slightly changed to more depolarized values within the first 2–3 days in culture and then stayed stable over the remaining time. https://doi.org/10.7554/eLife.48417.002 For mean values of resting membrane potential we found a small significant difference (Kruskal-Wallis test, p<0.05) for the acutely measured cells (0 DIV, −77.94 ± 1.5 mV) in comparison to early cultured (2–3 DIV, −72.38 ± 0.96 mV, Dunn's multiple comparisons test, *p=0.02) and late in culture cells (7-14 DIV, 71.95 ± 1.43, Dunn's multiple comparisons test, *p=0.01). However, there was no significant difference between early (2–3 DIV) and late in culture (7–14 DIV) measured cells (Dunn's multiple comparisons test, p>0.99). This was also reflected by no significant linear regression correlation of the resting potential versus the DIV for cells of all three groups (Figure 1D). In summary, increasing time in culture does not impact the majority of analyzed intrinsic electrophysiological characteristics of pyramidal neurons, except for the membrane resting potential which slightly changed to more depolarized values within the first 2–3 days in culture and then stayed stable over the remaining time. Maintenance of the glutamatergic neuron population in human brain slice cultures over time In a further set of experiments, we determined the degree of survival of the excitatory neuronal population in human brain slice cultures over time. Satb2 is a transcription factor that is expressed exclusively in excitatory neurons in adult mouse and human cortex (Britanova et al., 2008; Huang et al., 2013; Hodge et al., 2019; http://celltypes.brain-map.org/rnaseq). Taking advantage of Map2, a neuron-specific cytoskeletal protein, in combination with Satb2 (Figure 1—figure supplement 1A) we found no significant changes of the absolute numbers of double-positive neurons in slice cultures between 0 DIV (3 slices), 9 DIV (3 slices) and 14 DIV (2 slices) (Dunn's multiple comparisons test, Figure 1—figure supplement 1B). Double positive neurons were counted in small z-stack projections of confocal images of four to six different regions (each area 290 μm x 290 μm in size) in layers 2/3 of each slice: the absolute number of Map2 and Satb2 double positive neurons per 100 μm × 100 μm (10000 μm2) was on average 8.57 ± 0.60 in acute slices (18 analyzed areas), 10.14 ± 3.52 (16 analyzed areas) in slices analyzed at 9 DIV and 9.49 ± 1.77 (9 analyzed areas) in slices obtained at 14 DIV (Figure 1—figure supplement 1B). To investigate whether the ratio of glutamatergic to GABAergic neurons remained stable we calculated the ratio of Satb2 and Map2 double-positive neurons to all Map2 positive neurons. Ratios were 0.72 ± 0.02 in acute slices, 0.59 ± 0.09 at 9 DIV and 0.65 ± 0.11 at 14 DIV and revealed a discretely lower, albeit statistically significant, ratio at 9 DIV compared to 0 DIV, but no difference between 0 DIV and 14 DIV or 9 DIV compared to 14 DIV (Dunn's multiple comparisons test; Figure 1—figure supplement 1B). These data indicate robust survival of excitatory and inhibitory neurons with a sustained ratio of excitatory to inhibitory neurons throughout the culturing time. Viral transduction of human brain slice cultures To investigate whether neurons in human brain slice cultures can be genetically targeted with viral vectors and whether such strategies could render morphological analysis of human neurons more efficient than approaches solely relying on classic biocytin fillings, we used retrograde adeno-associated virus (AAVrg) encoding GFP under the human synapsin promoter to specifically transduce neurons. The slices were injected with the virus at 3–5 DIV using a picospritzer, cells were then recorded and imaged at 8–16 DIV (n = 20 slices). We found robust virus transduction in all injected slices with variable expression in distinct types of neurons throughout all cortical layers (Figure 2). The GFP expression was clearly present in somata, dendrites including spines and axons of transduced neurons (Figure 2A1 and A2). Figure 2 Download asset Open asset Viral transduction in human brain slice cultures and 3D reconstruction of GFP-labeled pyramidal neurons. (A) Representative example of a human brain slice after viral transduction with AAVrg-hSyn-GFP at 9 DIV, scale bar 1000 μm. (A1) Enlarged confocal image from A: layers 2/3 pyramidal neurons with intact apical dendrites (red arrows) and axons (magenta arrows), scale bar 200 μm. (A2) The soma and axon initial segment (AIS) are clearly visible in the virally transduced neurons, scale bar 50 μm. (B) 3D reconstructions of four GFP transduced pyramidal neurons were performed from confocal z-stack tile scans, scale bar 200 μm. The cells were individually traced and pseudo colored (C). (B1) Example of two neurons within close proximity of each other, which could still be clearly separated for further analysis, scale bar 50 μm. (D) Separation of the four distinct GFP-labeled pyramidal cells for further analysis, scale bar 200 μm. https://doi.org/10.7554/eLife.48417.005 GFP expression levels were found to be very robust, suggesting that several types of analyses including quantification of whole cell morphology, spine density, spine head diameter and spine length (see below) could be achieved based on GFP fluorescence. Furthermore, viral transduction of human neurons will enable direct investigation of the impact of disease causing mutations on neuronal function and morphology as well as on neuronal networks and will enable studies of human CNS circuitry involving optogenetic and chemogenetic tools. Dendritic morphology of pyramidal neurons in vitro Morphologically intact neurons are the basic framework of healthy neural brain tissue and degradation of dendritic arbors may even occur before an impairment of electrophysiological properties. Therefore, we set out to determine whether neurons are subject to morphological changes throughout their time in culture and performed 3D reconstructions of biocytin filled neurons at different time points between 0 DIV and 14 DIV. High-resolution confocal z-stack tile scans were acquired (Figure 1A), stitched with ImageJ and neurons were digitally reconstructed using Imaris software (see Materials and methods for details). Pyramidal neurons were filled with biocytin during electrophysiological recordings and 24 fillings were deemed to be of sufficient quality (clear presence of both apical and basal dendritic compartments, Figure 1A, while incompletely filled neurons were excluded from the analysis) to enable high-resolution morphological analysis (Figure 3). Reconstructed neurons were classified by the distance of their soma to the pia as layers 2/3 (distance to pia: 300–1200 μm, n = 16), layer 4 (distance to pia: 1200–1500 μm, n = 3) or layers 5/6 (distance to pia: 1500–2900 μm, n = 5) pyramidal neurons (Figure 3) (Mohan et al., 2015; Ting et al., 2018; Goriounova et al., 2018). All neurons presented a typical pyramidal neuronal shape with distinct apical dendrites (Figure 3, red), extensive basal dendritic trees (Figure 3, blue), axons (Figure 3, magenta) and spines present on the apical and basal dendrites. The total length of the apical and basal dendrites as well as the combined total length of the reconstructed dendritic filaments were quantified for each analyzed neuron and correlated to the distance of the respective soma to the pia and to the DIV (Figure 5A). To investigate whether neurons undergo degenerative processes or exhibit layer-dependent morphological differences, we calculated a linear regression for the values of total basal, total apical and total combined dendritic length of all neurons to the DIV and the distance to the pia. There was no significant correlation between the total basal, apical or combined dendritic length and the number of days in vitro for these analyzed biocytin filled neurons (n = 24, linear regression, p=0.07). While there was also no significant correlation of all three parameters of total dendritic length with overall soma distance to the pia (n = 24), additional subgroup analysis of superficially and incrementally deeper situated layers 2/3 neurons in acute and cultured cortical tissue revealed a gradually increasing total length of apical and basal dendrites (Figure 5A; n = 16), as has been described in a previous study by Mohan et al. (2015) in non-cultured CNS tissue. Figure 3 Download asset Open asset 3D reconstruction of biocytin filled pyramidal neurons. All 24 biocytin labeled reconstructed pyramidal neurons sorted by their soma distance to the pia ranging from 385 to 2400 μm represented in the colors red (apical dendrites), blue (basal dendrites) and magenta (axons). https://doi.org/10.7554/eLife.48417.006 Next, we asked whether the native GFP signal following viral transduction of cultures could be readily used for structural analysis of groups of neurons (Figure 2B). A typical example of reconstruction and separation of GFP-labeled neurons fur further analysis is shown in Figure 2. We picked a total of 23 neurons from four virally transduced slices (8, 9, 10 and 14 DIV) with strong GFP expression in layers 2–6 (Figure 2A). Similar to biocytin filled neurons all 23 GFP positive neurons were classified by the distance of their soma to the pia as layers 2/3 (distance to pia: 300–1200 μm, n = 12), layer 4 (distance to pia: 1200–1500 μm, n = 3) or layers 5/6 (distance to pia: 1500–2900 μm, n = 8) pyramidal neurons (Figure 4; apical dendrites, red; basal dendrites, blue; axons, magenta). In comparison to biocytin fillings GFP-mediated tracing equally successfully detected the gradually increasing dendritic length of layers 2/3 neurons depending on their soma position (Figure 5B). There was no significant correlation between the combined dendritic length and the DIV, neither for GFP-based tracings not for analysis after all biocytin and GFP reconstructed neurons (Figure combined total dendritic length of all biocytin and GFP-labeled neurons of the DIV revealed a of values by GFP (Figure n = 24 n = 23 very dendritic are to GFP positive processes from neurons and whether to GFP positive processes in the of the neuron reconstructed to the of this neuron or whether to them by them as from but overall successful reconstruction of the of dendritic arbors (Figure 2 and Figure Figure 4 Download asset Open asset 3D reconstruction of GFP-labeled pyramidal neurons. All 23 GFP-labeled reconstructed pyramidal neurons sorted by their soma distance to the pia ranging from to μm represented in the colors red (apical dendrites), blue (basal dendrites) and magenta (axons). Figure Download asset Open asset Quantification of the total length of apical and basal dendrites of biocytin filled and GFP-labeled neurons. (A) Neurons that were and filled in layers 2/3 are represented by red neurons in layer four by red and neurons in layers 5/6 by red biocytin filled neuron is represented by a red (B) GFP-labeled layers 2/3 neurons are represented by neurons in layer four by and neurons in layers 5/6 by (C) of the of biocytin filled and GFP-labeled pyramidal neurons in the different layers. (C) dendritic length of biocytin filled and GFP-labeled pyramidal neurons DIV. (C) of pyramidal neurons based on GFP expression slightly the total dendritic length compared to classic biocytin fillings (n = for n = 23 for In a total of biocytin and GFP-labeled neurons been analyzed structural preservation of pyramidal neurons of morphological These data not indicate of neuronal throughout the time in culture but also demonstrate genetic as a approach to 3D morphological analysis of adult human neurons with significantly increased efficiency in comparison to classic single cell biocytin fillings. Electrophysiological properties of interneurons in human brain slice cultures versus acute slices To further investigate our assessment based on the ratio of Map2 and Satb2 double-positive neurons to all Map2 positive neurons, whether also interneurons in human brain slice cultures we first performed for Unlike for other we were to for in acute and cultured human brain slice tissue. assessment at 0 DIV and at 9 DIV demonstrated survival of at this of interneurons (Figure Next, we performed intracellular recordings of neurons that were or as interneurons (Figure In comparison to pyramidal neurons interneurons a and even more diverse of neurons. While morphology and firing are known to substantially differ between of there are intrinsic properties as resting membrane potential, input AP half and sag Figure which can be more and which were found for an assessment over time in For all these parameters we did not find significant differences between neurons measured in acute (n = and in cultured slices at 7–14 DIV (n = (Figure The firing of the recorded interneurons showed distinct properties as described before for and and from spiking (Figure to spiking firing et al., Figure Download asset Open asset and of interneurons in human slice cultures. (A) Example of 2/3 cell with biocytin and (B) after both scale 200 μm. (C) of revealed presence of a of inhibitory interneurons in acute (0 DIV) and late in culture (9 scale bar 20 μm. (D) Example of spiking firing in acute slices and late in culture DIV). (E) of in acute slice and late in culture DIV, comparable AP half Quantification and of basic properties of in to the DIV. comparison between the properties in acute slice and late in culture measured interneurons revealed no significant differences test, morphology of biocytin filled and GFP-labeled pyramidal neurons in acute and cultured slices The spines of pyramidal neurons are the of excitatory and been shown to be a of the morphology of these cells et al., 2002; and To morphological features of spines we acquired high-resolution confocal scans of dendritic compartments of biocytin filled (Figure or GFP positive (Figure pyramidal neurons, performed 3D reconstructions (Figure and determined spine density, spine length and spine head diameter (see Materials and methods for details). Pyramidal neurons in acute (0 DIV) and cultured slices (2 DIV 14 DIV) revealed no significant differences on average regarding spine (acute and filled with ± n = cultured and filled with ± n = cultured and ± n = spine length (acute ± μm, n = cultured ± μm, n = cultured ± μm, n = or spine head diameter (acute ± μm, n = cultured ± 0.02 μm, n = cultured ± μm, n = (Figure supplement we did not find significant differences between of the groups and also not for biocytin versus we labeled and biocytin filled neurons for linear regression We found a correlation between the spine and the DIV (Figure linear regression, = and a positive correlation between the spine head diameter and the DIV (Figure linear regression, = while the average spine length showed no correlation to the DIV (Figure linear regression, In we found a strong positive correlation between the distance of neuronal to the pia and the spine length (Figure linear regression, = but not to the spine head diameter (Figure linear regression, or spine (Figure linear regression, Figure with 1 supplement see all Download asset Open asset of human cortical pyramidal neurons in acute and cultured slices filled with biocytin and labeled with (A) 3D reconstruction of a typical layers 2/3 pyramidal neuron DIV) in human brain slice cultures, scale bar μm. each neuron five dendritic regions were of the apical dendritic red and two of the basal dendritic blue (B) Typical of spines on apical dendrites of layers 2/3 pyramidal neurons recorded and biocytin filled at 0 DIV (acute at DIV and at 14 DIV, scale bar μm. (C) Typical of spines of GFP-labeled neurons at and 14 DIV, scale bar μm. (D) For the were 3D reconstructed and analyzed using and Imaris scale bar μm (see Materials and methods for details). Neurons that were and filled with biocytin are represented by red and transduced GFP positive neurons by (E) Data a correlation of spine with DIV and a positive correlation of spine head diameter with DIV. of the parameters versus the distance of the soma