# Influence of asymmetric microchannels in the structure and function of engineered neuronal circuits

J C Mateus, P Melo, M Aroso, Benoit Charlot, P Aguiar

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J C Mateus, P Melo, M Aroso, Benoit Charlot, P Aguiar. Influence of asymmetric microchannels in the structure and function of engineered neuronal circuits. Biofabrication, 2025, 17 (2), pp.025022. 10.1088/1758-5090/adb2e5 hal-04997277

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# Influence of asymmetric microchannels in the structure and function of engineered neuronal circuits

To cite this article: J C Mateus et al 2025 *Biofabrication* 17 025022

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PAPER

Influence of asymmetric microchannels in the structure and
function of engineered neuronal circuits

J C Mateus¹,*, P Melo¹,² ID, M Aroso³ ID, B Charlot⁴ ID and P Aguiar¹,²,*, ID

¹ i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

² FEUP—Faculdade de Engenharia da Universidade de Porto, Porto, Portugal

³ INEB—Instituto Nacional de Engenharia Biomédica, Porto, Portugal

⁴ Institut d'Electronique et des Systèmes, CNRS, University Montpellier, Montpellier, France

* Authors to whom any correspondence should be addressed.

E-mail: jose.mateus@i3s.up.pt and pauloaguiar@i3s.up.pt

**Keywords:** axonal outgrowth, signal propagation, microfluidics, microelectrode array (MEA), brain-on-a-chip

Supplementary material for this article is available online

Abstract

Understanding the intricate structure–function relationships of neuronal circuits is crucial for unraveling how the brain achieves efficient information transfer. In specific brain regions, like the hippocampus, neurons are organized in layers and form unidirectional connectivity, which is thought to help ensure controlled signal flow and information processing. In recent years, researchers have tried emulating these structural principles by providing cultured neurons with asymmetric environmental cues, namely microfluidics’ microchannels, which promote directed axonal growth. Even though a few reports have claimed to achieve unidirectional connectivity of *in vitro* neuronal circuits, given the lack of functional characterization, it remains unknown if this structural connectivity correlates with functional connectivity. We have replicated and tested the performance of asymmetric microchannel designs previously reported in the literature to be successful in promoting directed axonal growth, as well as other custom variations. A new variation of ‘Arrowhead’, termed ‘Rams’, was the best-performing motif with a ~76% probability per microchannel of allowing strictly unidirectional connections at 14 d *in vitro*. Importantly, we assessed the functional implications of these different asymmetric microchannel designs. For this purpose, we combined custom microfluidics with microelectrode array technology to record the electrophysiological activity of two segregated populations of hippocampal neurons (‘Source’ and ‘Target’). This functional characterization revealed that up to ~94% of the spiking activity recorded along microchannels with the ‘Rams’ motif propagates towards the ‘Target’ population. Moreover, our results indicate that these engineered circuits also tended to exhibit network-level synchronizations with defined directionality. Overall, this functional characterization of the structure–function relationships promoted by asymmetric microchannels has the potential to provide insights into how neuronal circuits use specific network architectures for effective computations. Moreover, the here-developed devices and approaches may be used in a wide range of applications, such as disease modeling or preclinical drug screening.

**1. Introduction**

A key goal of neuroscience is to understand how
neurons' physical connections (structural connectiv-
ity) and how they communicate with one another
(functional connectivity) work together to produce

the computations underlying cognition and beha-
vior. Theoretical studies suggest that brain networks
tend to maximize the number and variety of func-
tional motifs while using a relatively limited set of
structural motifs (Bassett and Sporns 2017). While
it is virtually impossible to ascertain the prevalence
of a specific motif within the entire connectome, some traits, like ‘feed-forward’ connectivity, are common across diverse brain networks. Examples include the hippocampus’s trisynaptic circuit or the neocortex pathways activated by sensory input (e.g. visual stimuli). However, the difficulty of studying these pathways *in vivo* has hampered our ability to fully understand the dynamic interplay of structure and function.

To address this challenge, researchers have engineered *in vitro* models that take advantage of the axons’ dimensions and cue-sensing capability to impose directional connectivity between neuronal populations. Even though a wide range of patterning techniques has been employed to control axon guidance *in vitro* (reviewed in Raj *et al* 2021, Mateus *et al* 2024), the simplest and most effective at the population level is microfluidics (Habibey *et al* 2022). Importantly, microfluidics can be combined with complementary techniques, such as microelectrode arrays (MEAs), to dissect neuronal function in long-term experiments (Liu *et al* 2024). Pioneered by Taylor *et al* microfluidic technology allows for the compartmentalization of different neuronal populations, interconnected via high-aspect ratio microchannels that only allow axons to grow (Taylor *et al* 2005). Taylor-based microfluidics have been used extensively to obtain insights regarding axon physiology and pathology (Neto *et al* 2016, Mateus *et al* 2024); but also to segregate neuronal populations into bidirectionally connected compartments, modeling brain circuits, such as the cortico-striatal (Virlogeux *et al* 2018), cortico-hippocampal (Brofiga *et al* 2022), or the hippocampus (Vakilna *et al* 2021).

While most of the literature makes use of straight microchannels, a few groups have advanced the state-of-the-art by incorporating asymmetric motifs in the microchannel design. Taking advantage of axonal outgrowth edge guidance (Renault *et al* 2016), these features bias outgrowth in the researcher’s desired direction. Thus, microchannels with asymmetric motifs can be used to establish feed-forward networks in a controlled environment (Habibey *et al* 2022, Mateus *et al* 2024). Among these motifs, ‘Arrowhead’ (Gladkov *et al* 2017, Holloway *et al* 2019) and ‘Tesla valve’-inspired (Na *et al* 2016, Winter-Hjelm *et al* 2023) designs have been reported as capable of enforcing strictly unidirectional connections. However, no detailed comparison of the performance of these designs in shaping the structure and function of *in vitro* neuronal networks has yet been made. Moreover, no study has attempted to describe the directional flow of axonal information, which has proven crucial for the characterization of the structural connectivity between hippocampal subregions (Vakilna *et al* 2021).

In this study, we set out to characterize the influence of different asymmetric motifs in the structure and function of *in vitro* neuronal circuits. We test previously reported, as well as new designs, on their blocking capabilities in the axonal outgrowth of rat hippocampal neurons from embryonic origin. We demonstrate the implications of using these designs on the profiles of activity recorded along the microchannels and in the ratio of forward/backward propagating spikes. Finally, we assess the directional bias in population-to-population network burst (NB) propagation when using these asymmetric microchannels.

## 2. Results

### 2.1. Asymmetric microchannel designs

We set out to design motifs that could bias axonal outgrowth via edge-guidance pathfinding along microchannels. To this end, we based our choice on the most prominent designs in the current literature and tested new variants as well.

We replicated the ‘Arrowhead’ design, which has been reported as capable of 100% unidirectional outgrowth bias at 14 d *in vitro* (DIV) after 5 motifs (Holloway *et al* 2019), by creating a design that comprised 6 repeated motifs of these arrow-shaped structures (here termed ‘Arrows’). We also used this design as the basis to create a variant that, instead of stalling axonal outgrowth at the arrowheads’ corners, leads axons to a diverting loop that traps and re-routes axonal outgrowth. We named this design ‘Rams’, inspired by the shape of rams’ horns. We were also motivated by the Tesla valve concept, originally proposed by Nikola Tesla to allow fluidic control without any moving mechanical parts (Tesla 1920), to design 2 variants that could re-route axonal outgrowth and create a unidirectional bias (‘Tesla’ and ‘Tesla v2’). These 2 variants differ in the number of motifs (8 in ‘Tesla’; 6 in ‘Tesla v2’) and the aggressiveness of the re-routing loop, being ‘Tesla v2’ the variant with the sharpest angle and the one most similar to the original design. A recent study has since proposed a design that is very similar to ‘Tesla’, as well as another Tesla valve-inspired design (Winter-Hjelm *et al* 2023). Each design was implemented in microfluidic devices that comprised 2 compartments, interconnected by 16 microchannels for compatibility with standard 252-recording MEAs. Scanning electron microscopy (SEM) images of the fabricated designs’ main variants can be seen in figure 1(A).

The 4 motifs (‘Rams’; ‘Arrows’; ‘Tesla’; ‘Tesla v2’) were employed in 1030 µm-long microchannels (measured in a straight line) that connected separate neuronal populations (termed ‘Source’ and ‘Target’, as in (Gladkov *et al* 2017)) (figure 1(B)).
<figure><img src="image_6.png" /></figure>

Each microchannel comprised a straight segment (550 µm-long) to facilitate axonal outgrowth in the desired direction and an asymmetric segment (480 µm-long) to block and/or re-route axonal outgrowth in the non-desired direction. All designs kept the same microchannel height (5 µm) throughout their length. At one end of the microchannel, the compartment wall ('Target' compartment) had indentations to prevent axon growth perpendicular to the microchannels' exit (figure 1(C)), as commonly

occurs in Taylor-based microfluidics (Peyrin et al 2011). The rationale for each design is schematized in figure 1(C).

## 2.2. Performance of different designs in creating axonal outgrowth bias

To assess the efficacy of each design at promoting unidirectional axonal growth, we started by culturing hippocampal neurons isolated from E18 Wistar rats in glass coverslips and virally transducing the 'Source'
and ‘Target’ populations with adeno-associated vir-
uses (AAVs) encoding for mRuby3 and EGFP, respect-
ively. Both viral constructs yielded a high transduc-
tion efficacy, which allowed ubiquitous expression of
the different fluorescent proteins in the two neuronal
populations (figure 1(B)). However, despite main-
taining a hydrostatic pressure gradient to hinder viral
leakage between the two populations, we observed
some cross-contamination of viruses that hindered
formal analysis of axon blocking. Thus, to quantitat-
ively compare the axonal outgrowth blocking prob-
ability of each microchannel design, we opted to
use monocultures (‘Target’-only) expressing EGFP
(figures 2(A) and (B)).

We quantified the percentage of microchan-
nels capable of completely blocking axonal out-
growth before reaching the straight segment of the
microchannel. At 14 DIV, no motif was capable of
completely blocking axonal outgrowth consistently.
Per order of effectiveness, the mean ± SD percent-
age of success in complete blocking was 76 ± 20%
for ‘Rams’ (n = 6); 40 ± 20% for ‘Arrows’ (n = 6);
28 ± 25% for ‘Tesla v2’ (n = 5); and 19 ± 19% for
‘Tesla’ (n = 6) (figure 2(B)).

It is important to note that while asymmetric
microchannels might not completely prevent axonal
outgrowth towards the ‘Source’ population; by divert-
ing many axons, they decrease the overall number
of axons that do so. At this stage, given the small
size of axons and their complex growth dynamics
(e.g. ramifications), it is challenging to grasp the
exact number (or fraction) of diverted axons. Still,
to get a better approximation of the filtering capa-
city of each motif, we analyzed the fluorescence
pixel intensity profiles along the better-performing
designs’ microchannels (‘Rams’, ‘Arrows’ and ‘Tesla
v2’). This analysis demonstrated that the pixel intens-
ity decays the most along the microchannels with the
‘Rams’ motif (figure 2(C)), which provides an indir-
ect measure of better performance at blocking axonal
outgrowth with this motif.

## 2.3. Asymmetry in axonal activity along the microchannel

After structural characterization of the motifs' per-
formance in biasing axonal outgrowth direction, we
set out to characterize their influence on the func-
tion of neuronal circuits. To this end, we com-
bined MEAs with microFluidics (μEF devices) to
record neuronal activity in neuronal cultures at
12–21 DIV. The alignment compatibility with 252-
recording electrode MEAs allowed for the probing
of the 16 microchannels, encompassing 5 electrodes
each, per experiment (figure 3(A)). Importantly,
since the number of microchannels matches the
number of electrode columns, every microchannel

is probed electrophysiologically. Thus, theoretic-
ally, every event propagating between populations
('Source' and 'Target') can be recorded. This config-
uration enabled the separation of activity from the
'Source' and 'Target' populations, as well as from the
axons interconnecting them via the microchannels
(figure 3(B)).

Taking advantage of this capability to record
propagating axonal signals (as in (Mateus et al
2021)), we characterized the motifs' influence in the
microchannels' firing rate profile (figure 3) and the
direction of the events' (i.e. action potentials (APs))
propagation (figure 4). For these and the remainder
of the Results, we pooled the data from 'Tesla' and
'Tesla v2' experiments since we did not observe any
significant differences between these designs.

We hypothesized that, given their blocking cap-
abilities, asymmetric microchannels should also
exhibit an asymmetry in firing rate. Whereas con-
trols (i.e. completely straight microchannels) should
not exhibit differences (figure 3(C)). To this end, we
compared the firing rate of the electrodes within the
straight and asymmetric segments of the microchan-
nels. For the ‘Rams’ design, 94 ± 4% (n = 5) of
the microchannels presented higher mean firing rate
(MFR) in the asymmetric segment than in the straight
segment (figure 3(D)). This design also presented
the highest average fold-change in MFR (2.6 ± 1.2)
between segments (figure 3(E)). This increase in MFR
occurs most probably due to an accumulation of sig-
nal sources (i.e. axons) in the vicinity of the electrodes
within the asymmetric segment of the microchannel,
which demonstrates a blockage bias when using these
motifs. This is also supported by the activity pro-
files along the 5 electrodes within the microchannels,
which demonstrated a trend of increasing activity in
the ‘Source’-to-‘Target’ direction (figure 3(F)).

Direct measures of axonal conduction should
provide a clear indication of the preferred direc-
tionality of axonal outgrowth and information flow;
thus, we analyzed the directionality of AP propaga-
tion along the microchannels. We labeled as ‘forward’
propagating events APs propagating in the ‘Source’-
to-‘Target’ direction, whereas events propagating
from the ‘Target’-to-‘Source’ were labeled as ‘back-
ward’ (figures 4(A)–(C)).

We assessed the direction of axonal conduc-
tion during network development. These longitud-
inal experiments (12–21 DIV) revealed that asym-
metric microchannels consistently exhibited a much
higher prevalence of forward propagating events (at
least 4 times more), regardless of the DIV or motif
(figure 4(D)). Still, the ‘Rams’ motif exhibited the
highest fraction of forward propagating events, par-
ticularly at 12 DIV (94 ± 9% (n = 4)). At 21 DIV,
this fraction was 91 ± 6% (n = 4) which supports the
rationale that the forward spiking bias is maintained
<figure><img src="image_7.png" /><figcaption>**Figure 2.** (A) Widefield microscopy mosaic images (20× objective) of representative neuronal monocultures (seeded on the 'Target' compartment) at 14 d *in vitro* (DIV) for each design. All cultures are expressing EGFP (grayscale). Asterisks mark microchannels that failed to completely block axonal outgrowth before the straight segment of the microchannel (dashed line). (B) Percentage of microchannels that completely block axonal outgrowth per design. Filled dots represent the cultures shown on (A). (C) Normalized average pixel intensity values along the microchannels. The average intensity profiles (and colorized shade for the SD) of 5–6 independent experiments (16 microchannels per experiment) are plotted together with a fitted exponential curve. Per design, a representative microchannel is colorized with the Turbo lookup table (LUT).</figcaption></figure>

throughout network development. Given the consistency in these ratios during development, we pooled the data from all DIVs to compare with controls (i.e. straight microchannels). This analysis returned the ‘Rams’ and ‘Arrows’ motifs as performing significantly differently than controls, but not ‘Tesla’ (figure 4(E)).

It is important to note that, throughout this section’s results, we observed the same performance trend (‘Rams’ > ‘Arrows’ > ‘Tesla’) as in the structural studies.

## 2.4. Asymmetry in NB propagation

Following the characterization of spiking activity propagation along the microchannels, we analyzed whether this activity led to a recruitment of network-level activation. NB are hallmarks of neuronal synchronization *in vitro* and are often used as a proxy for assessing inter-population communication (DeMarse *et al* 2016, Barral *et al* 2019, Winter-Hjelm *et al* 2023, Pigareva *et al* 2024). Here, we used a strict causality window (20, 240) ms to identify instances of population-to-population spontaneous NB propagation. If the onset of both populations’ NB fell into this causality window, we considered this as a putative NB propagation (figure 5(A)). Once

again, we distinguished these propagations as ‘forward’ and ‘backward’ depending on the direction of propagation ‘Source’-to-‘Target’ and ‘Target’-to-‘Source’, respectively. Importantly, we generally did not observe significant differences between the overall number of NBs detected for each population category (figure 5(B)), which could bias the results. We also did not observe significant differences in the time delays between NB onsets (110–150 ms on average), regardless of the direction of propagation (forward vs backward) or the microchannel design (figure S1).

We could observe that Source’s NBs were capable of driving Target’s responses at 12 DIV on populations interconnected via asymmetric microchannels (figure 5(C)). Thus, we next estimated the fraction of NBs generated in each population that propagated to its counterpart. Even though most NBs did not propagate (or elicit a NB), independently of the analyzed DIV, we observed that the ‘Rams’ and ‘Arrows’ motifs biased NB propagation to preferentially occur in the forward direction (figure 5(D)). On average, the ratio between forward and backward-propagating NBs was of 2.9 and 3.6 for these motifs, respectively. These results demonstrate, once again, that these designs effectively create a directional bias in the neuronal circuits’ function. The relatively low number of microchannels may explain why most cultures
<figure><img src="image_8.png" /><figcaption>**Figure 3.** (A) Phase-contrast microscopy image (20× objective, mosaic) of a hippocampal co-culture at 21 d *in vitro* (DIV). The asymmetric microchannels comprise the 'Rams' motif. The combination of microElectrode arrays and microFluidics (μEFs) allows the compartmentalization and probing of two neuronal populations ('Target' and 'Source'), as well as the assessment of all event propagation between them. (B) Representative raster plot of 10 min of spontaneous activity for each of the three sections of a μEF: 'Source' population; microchannels; 'Target' population. The summed firing rate (color-coded) is overlayed in each plot. On the microchannels' plot, each line represents a microchannel (i.e. one electrode per microchannel) and only events detected as propagating throughout the whole microchannel are depicted (color-coded depending on the direction as 'forward' or 'backward'). Culture at 12 DIV on a μEF device with the 'Rams' motif. (C) Schematics of the predicted effect of microchannels on axonal outgrowth and representative activity maps (each pixel represents an electrode's mean firing rate (MFR)) for μEFs with (i) symmetric microchannels (control) and (ii) asymmetric microchannels. (D) Horizontal bar graph showing the percentage of microchannels with a higher MFR in the asymmetric segment, when compared to the straight segment of the microchannel (i.e. with backward blockage bias), per design (n = 3–6 independent experiments, Kruskal–Wallis test with Dunn's multiple comparisons, **p < 0.01, mean ± SD). (E) Horizontal bar graph showing the fold-change in MFR of the two last vs two first electrodes (in the 'Source'-to-'Target' direction) within the microchannels for each design (n = 3–6 independent experiments, Kruskal–Wallis test with Dunn's multiple comparisons, **p < 0.01, mean ± SD). (F) Average profile of activity (difference to the mean) along the 5 electrodes within the microchannels, per design (n = 3–6 independent experiments, colorized shade for the SD across recordings).</figcaption></figure>
<figure><img src="image_9.png" /></figure>

maintained a high degree of population activity inde-
pendence in our experiments (DeMarse *et al* 2016).
In the future, this number may be tuned to produce
circuits with more or less structural–functional bias.

**3. Discussion**

Here, we set out to characterize the influence of
asymmetric microchannel designs in the structure
and function of two-compartment *in vitro* neuronal
circuits. Following the principles of edge guidance,
such designs exert blocking, delaying and re-routing
effects on axonal outgrowth, which we used to bias
connectivity between ‘Source’ and ‘Target’ neuronal
populations.

Even though Tesla valve-inspired designs have
gained some traction in the microfluidics field
(reviewed in (Purwidyantri and Prabowo 2023)), they
had not been tested in this context until very recently
(Winter-Hjelm et al 2023). Despite qualitative indic-
ations from the literature, our Tesla valve-inspired
designs performed relatively poorly in the re-routing
of axonal outgrowth and the establishment of direc-
tional functional connectivity. Similarly, we could not
replicate previously reported results with ‘Arrowhead’
(Holloway et al 2019), despite our efforts (e.g. more

motifs, longer microchannels) to increase the asym-
metry bias in the ‘Arrows’ design. Several factors
may account for these discrepancies, including exper-
imental conditions such as cell density, viability, or
species of origin; as well as subtle differences in fabric-
ation protocols or extra-motif choices. For example,
the increased travel distance between each motif in
the original ‘Arrowhead’ design may exert a blocking
effect which is not dependent on the motif’s structural
features *per se*. These findings emphasize the import-
ance of considering both design and contextual exper-
imental variables when replicating (or modifying)
such systems.

It is also worth noting that ‘Source’ axons can
serve as guiding lines to axons growing in the oppos-
ite direction (Renault *et al* 2016). Since our structural
experiments were performed with mono-cultures
(‘Target’-only, figure 2), as in most studies in the field,
the results may be already overestimating the motifs’
structural efficiency.

The ‘Rams’ motif, which combines the block-
ing capabilities from ‘Arrows’-like designs and re-
routing capabilities of ‘Tesla’ or ‘Arches’ designs
(Renault *et al* 2016), achieved the highest structural
and functional connectivity bias across all experi-
ments. This finding supports the idea that a combined
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 5.** (A) Example of a single putative network burst (NB) propagation from a 'Source' to a 'Target' population ('forward' direction). The activity was recorded at 12 d *in vitro* (DIV) from populations interconnected via asymmetric microchannels ('Rams'). Traces show the summed spike train data (1 ms time bins) of each population. Blue lines represent the time delay between NB onset in each population. (B) Tukey plots of the total number of detected NBs per population and per design (n = 12–20, Kruskal–Wallis test with Dunn's multiple comparisons, \*p < 0.05). (C) Event-trigger averaging of the spontaneous activity from a 'Target' population after NB onset on the 'Source' population. The dashed line represents the baseline activity of the 'Target' population. The activity was recorded at 12 DIV from populations interconnected via asymmetric microchannels ('Arrows'). (D) Bar plots comparing the fraction of NB events that propagate 'Source'-to-'Target' (forward direction) and 'Target'-to-'Source' (backward direction), per design (n = 7–12, Kruskal–Wallis test with Dunn's multiple comparisons, \*p < 0.05, mean ± SD).</figcaption></figure>

approach—diverting and then trapping or re-routing undesired axons—is more effective than using either strategy alone. Impressively, ~76% of the microchannels with this motif completely blocked 'Target' axons at 14 DIV, and >90% of the propagating events were in the forward direction until, at least, 21 DIV. This bias in information flow was also observed in the relative fraction of NB events that propagate from the 'Source' to the 'Target' population, a clear indication of directional functional connectivity. Overall, our results pose the 'Rams' motif as a strong candidate to be used in studies aiming to achieve a high degree of structural and functional directionality. Moreover, although we specifically designed the microfluidic devices to be compatible with standard 256-electrode MEAs, these could be readily used (or easily adapted to be compatible) with other readout methods, such as calcium imaging (Ming et al 2021) or high-density MEAs (Duru et al 2022).

State-of-the-art node-based designs have demonstrated that strict unidirectional connectivity can be achieved *in vitro*. For example, a stomach-like design was reported to achieve unidirectional structural connectivity between nodes with ~92% probability (Forró et al 2018). Nanochannels of ~150 nm have been shown to completely block axon growth while allowing dendritic spines to pass through, thus, in combination with microchannels that guide axon growth, can be used to establish pure feed-forward circuits (Mateus et al 2022). These studies' concepts can be used to form small-scale neuronal circuits (i.e. with dozens of neurons), but, intrinsically, cannot be scaled for implementation in standard Taylor-based microfluidics with hundreds of thousands of neurons. However, motif designs, such as the ones here proposed, can be easily implemented in Taylor-based microfluidics or micropatterning configurations (Yamamoto et al 2023). If needed, their
performance may be further enhanced by incorporating chemical cues (e.g. gradients of trophic factors) that attract axonal outgrowth and are commonly used in standard microfluidic devices (Rifes et al 2020).

Still, it is important to consider that strict unidirectional structural connectivity does not necessarily translate to function. We and others have previously shown that signal flow along axons can be spontaneously bidirectional (Mateus et al 2021, Liu et al 2022, Rózsa et al 2023). Future studies could incorporate complementary analyzes, such as fluorescent assays of subcellular calcium signaling (Moutaux et al 2018, Mateus et al 2022) and/or synaptic vesicle release and recycling (Jackson and Burrone 2016), to better understand the functional properties of the synaptic connections formed via asymmetric microchannels.

While further research is needed to fully dissect the structure–function relationships that emerge in these *in vitro* models, asymmetric microchannels, such as the ones proposed in this study, have immediate applicability across multiple research fields. For example, mechanistic studies, of physiology or disease, where certainty regarding axon directionality is crucial for accurate interpretation of the results (e.g. studies of ‘prion-like’ mechanisms; axonal transport) could benefit greatly from the implementation of these devices (Courte et al 2023).

Finally, in combination with induced-pluripotent stem cells, these asymmetric microchannels could be integrated into ‘Brain-on-a-chip’ platforms or high-throughput microphysiological systems dedicated to preclinical screening (Nikolakopoulou et al 2021, Habibey et al, 2022, Liu et al 2024). Such platforms could provide physiologically-relevant *in vitro* models with well-defined neuronal connectivity, serving as tools for studying diseases where synaptic connectivity plays a crucial role. Potential applications include modeling of neurodevelopmental disorders (Hommersom et al 2024) and neurodegenerative diseases (Holloway et al 2021, Miny et al 2022). Furthermore, by enabling biologically relevant and scalable experimentation, these platforms hold promise for reducing our reliance on animal models, in alignment with the 3Rs (Replacement, Reduction, and Refinement) principle (Nikolakopoulou et al 2021).

## 4. Conclusion

This study investigated the impact of asymmetric microchannel designs on *in vitro* neuronal circuits. While previously reported designs achieved limited success in directing axonal growth and signal flow, a novel motif, termed ‘Rams’, demonstrated strong structural and functional directionality bias, offering

a promising approach for engineering neuronal circuits. While challenges remain in achieving perfect unidirectional control at such a large scale, this study provides valuable insights for various research fields requiring controlled neuronal connectivity in both fundamental and preclinical research.

## 5. Methods

### 5.1. Microfluidic design and fabrication

The microfluidic chambers were designed to bias axonal outgrowth in a unidirectional manner. The ‘Arrows’ motif mimics the design proposed by (Holloway et al 2019), whereas the ‘Tesla’ motif drew inspiration from the original Tesla valves proposed by Nikola Tesla in the #US001329559 patent (Tesla 1920). The ‘Rams’ motif is original but emerged from the ‘Arrows’ motif.

Polydimethylsiloxane (PDMS) was chosen to fabricate the microfluidic chambers due to its widespread use in research and its advantages, including transparency, flexibility, and biocompatibility. However, the motif designs could be adapted for fabrication with alternative materials (e.g. thermoplastics) to address PDMS limitations, such as small molecule absorption, which is critical for standardization and drug testing applications (van Meer et al 2017).

In all microfluidic devices, the number of microchannels matched exactly the number of electrode columns of 256-electrode planar MEAs (16 microchannels in total). This allowed the detection of all activity propagation between the neuronal populations. Concisely, the microfluidic chambers were composed of two separate somal compartments interconnected by 16 microchannels with 1030 µm length (measured in a straight line) × 5 µm height × 10 µm width dimensions and interspaced by 200 µm. Control devices with straight microchannels with the same dimensions were used in the electrophysiology experiments.

The SU-8 (Kayaku Advanced Materials, Inc.) on silicon master molds was fabricated using a dual-thickness photolithography process (figure 1(A)) in a cleanroom using two photomasks (Renatech, LAAS, France). A first layer of SU-8 2005 (5 µm thick) was used to create the microchannels. The SU-8 photoresist was spin-coated at 3000 rpm on a 3 inch silicon wafer, then pre-baked at 95 °C, exposed to a dose of 110 mJ cm⁻², post-baked at 95 °C, developed in PGMEA SU-8 developer (Kayaku Advanced Materials, Inc.), rinsed in isopropyl alcohol, and hard-baked at 130 °C. After optical examination and profilometry, a second layer of SU-8 2100 (100 µm thick) was subsequently deposited on top of the first layer. The second photoresist layer was spin-coated at 3000 rpm for 30 s and pre-baked at 95 °C
with slow temperature ramps. The second mask was aligned (Karl Suss MJB4) with the first level using alignment marks and ultraviolet (UV)-exposed (i-line at 365 nm) to a dose of 250 mJ cm⁻², then post-baked at 95 °C again with slow temperature ramps and developed in PGMEA. Note that a short silicon over-etching was performed using plasma dry reactive ion etching with CHF3/O2 gases between the two steps so that the microchannel pattern of the 1st layer was visible for the alignment of the second mask. The final wafer was then hard baked at 150 °C for 10 min and carefully examined before PDMS molding.

From the microfabricated SU-8 mold, the PDMS microfluidic chambers were produced by replica molding. PDMS was prepared using a 10:1 mix of silicone elastomer and its curing agent (Sylgard 184, Dow Corning) and then degassed using a vacuum desiccator. The PDMS was cured at 70 °C for 3 h, after which the microfluidic chambers were unmolded and cut. The medium reservoirs were manually punched with a steel biopsy punch (Ø 6 mm).

## 5.2. Substrate preparation

Coverslips or planar MEAs (multi channel systems (MCSs), Germany) of 252 titanium nitride recording electrodes (30 µm in diameter; 200 µm spacing) and 4 internal reference electrodes (organized in a 16 by 16 square grid) were used as cell culture substrates for all experiments.

Cell culture substrates were prepared as previously detailed (Lopes *et al* 2018). Briefly, both the substrate (i.e. coverslip or MEA) and the microfluidic chambers were sprayed with 70% ethanol, allowed to air-dry inside a laminar flow hood, and sterilized by UV light exposure. In the case of MEA substrates, µEF assembly was guided by a stereomicroscope, to correctly align the microchannels with the electrode grid. Each microchannel encompassed 5 electrodes, 3 within the straight segment and 2 within the asymmetric segment. Moreover, this configuration allowed for each neuronal population to be probed with a minimum of 78 electrodes. A phase-contrast image of a culture within an assembled µEF is shown in figure 3(A). After alignment, the assembled devices were air plasma-cleaned for 1 min at 15 W and coated with poly-D-lysine (20 µg ml⁻¹) to promote cell adhesion. The excess coating was washed out thrice before cell seeding.

## 5.3. Cell culture

Experimental procedures involving animals were carried out following current Portuguese laws on Animal Care (DL 113/2013) and the European Union Directive (2010/63/EU) on the protection of animals used for experimental and other scientific purposes. The experimental protocol (reference 0421/000/000/2017) was approved by the ethics committee of the Portuguese official authority on animal

welfare and experimentation (Direção-Geral de Alimentação e Veterinária). All efforts were made to minimize the number of animals and their suffering.

Primary embryonic rat hippocampal neurons were isolated from Wistar rat embryos (E18), as in (Lopes *et al* 2018). Briefly, hippocampus tissues were dissected in Hank's balanced salt solution (HBSS) and enzymatically digested in 0.6% (w/v) trypsin (1:250) in HBSS for 15 min at 37 °C. Then, trypsin was inactivated with HBSS containing 10% fetal bovine serum and washed away. Tissue fragments were mechanically dissociated with a plastic pipette and the cells' suspension was filtered with a 40 µm strainer (Falcon). After manual cell counting, 100k viable cells suspended in 4 µl were seeded in each somal compartment of previously prepared µEFs. Cells were cultured in Neurobasal Plus medium supplemented with 0.5 mM GlutaMAX, 2% (v/v) B27 Plus, and 1% (v/v) penicillin/streptomycin for 21 d. Cultures were kept in a humidified incubator at 37 °C supplied with 5% CO₂.

Transductions with AAVs were performed to live-image neuronal morphology and axonal outgrowth. For neuronal morphology, scAAV-9/2-hSyn1-chI-loxP-EGFP-loxP-SV40p(A) (8.5 × 10¹² vg ml⁻¹ titer) or scAAV-DJ/2-hSyn1-chI-loxP-mRuby3-loxPSV40p(A) (7.2 × 10¹² vg ml⁻¹ titer) were added to each somal compartment (0.3 µl per somal compartment at 1–3 DIV). Viral vectors were produced by the Viral Vector Facility of the Neuroscience Center Zurich (Zentrum für Neurowissenschaften Zürich, ZNZ, Switzerland).

## 5.4. Imaging analysis

Fluorescence imaging experiments were performed with hippocampal neurons at 14 DIV. Images were acquired by a sCMOS camera Prime 95B, 22 mm (Teledyne Photometrics, UK), mounted on a Nikon Eclipse Ti2-E (Nikon, Japan) inverted microscope with a Nikon Pl Apo 20×/0.75NA objective. Image acquisition was performed using Micromanager (Version 1.4) and images were processed using ImageJ (Fiji).

Tiled images, such as the ones presented in figures 1(B) and 2(A), were produced via stitching of the original images. Importantly, due to their size and higher levels of protein expression, somata are brighter and thus more visible than axons on microscopy images. To enhance the signal intensity of the axons compared to the somata, the enhance local contrast (CLAHE) algorithm was applied to the tiled images with default parameters. For the analysis presented in figure 2(B), images were further saturated to emphasize axons with low signal. Microchannels were manually labeled according to the complete blockage of axonal outgrowth by the motifs.

For the analysis presented in figure 2(C), regions of interest of defined size (encompassing all 16
microchannels) were outlined. A background sub-
traction (100 pixel radius) was applied to the result-
ing 8-bit tiles. Then, the analysis proceeded in Matlab
using custom scripts. Pixel intensity profiles were
obtained for each image via column-wise averaging.
Then, pixel intensity was normalized against the first
5 pixel columns (corresponding to the entrance of the
microchannels), for each image. The profiles of 5-
6 independent experiments (i.e. images) per design
were averaged. Finally, a 20 pixel moving average was
applied to the traces before plotting and fitting the
exponential curve.

**5.5. Electrophysiological recordings**

Hippocampal neurons at 12–21 DIV were used in electrophysiological experiments. Recording sessions started after 10 min of adjustment to recording conditions. Electrophysiological recordings of spontaneous electrical activity were obtained at a sampling rate of 20 kHz for 10 min. All recordings were obtained using a commercial MEA2100-256 system (Multichannel Systems MCS, Germany) mounted on an inverted widefield microscope (Eclipse Ti2-E, Nikon) with controlled temperature and humidified 5% CO₂ (stage top incubator, ibidi, Germany). Temperature was maintained at 37 °C by external temperature controllers.

**5.6. AP detection and propagation characterization**

Raw signals were high-pass filtered (200 Hz) and APs
were detected by a threshold method set to 5× the
standard deviation (SD) of the peak-to-peak elec-
trode noise. An AP time was extracted at this surpass-
ing point and no detection was considered for the next
3 ms ('dead time'). Analysis was then performed off-
line using custom MATLAB scripts.

'Forward' ('Source'-to-'Target') and 'backward' ('Target'-to-'Source') APs propagating along the microchannels were identified based on the extracted spike times. Propagation sequence identifications were performed as previously reported (Costa et al 2020, Mateus et al 2021). The custom scripts are available on Github (https://github.com/elifesciences-publications/Calculate_APs Distances_in_MEAs). A propagating event had to fulfill the following requirements: event detected over the entire microchannel (5 electrodes; thus satisfying 4 inter-electrode propagations) and time delay between electrode pairs lower than or equal to 1 ms (minimum propagation velocity of 0.2 m s⁻¹).

The method's validity was confirmed with exper-
iments with tetrodotoxin (TTX) injection (1 µM) on
the 'Source' compartment for all asymmetric designs
(n = 4). TTX injection abolished forward spiking
activity (to a fraction of ~0.001% post-TTX) (figure
S2), strongly supporting that the events detected as

'forward' are a result of the neuronal activity in the
'Source' compartment.

**5.7. NB detection and propagation characterization**

For the detection of NBs, an inter-spike interval (ISI)-statistics-based adaptive threshold alternative, developed by Pasquale *et al* (Pasquale *et al* 2010), was implemented. After exhaustive analysis, we fine-tuned the parameters to comply with diverse network dynamics. As such, if the percentage of active electrodes was higher or equal to 50%, the values of *min-PercElec* (minimum percentage of electrodes involved in a NB), *maxISITh* (maximum ISI allowed for burst core spikes), *maxISImax* (maximum ISI allowed for burst boundaries spikes) were set to 15%, 250% and 600%, respectively, and lowered to 10%, 100% and 450%, otherwise. *minSpikes* (minimum number of consecutive spikes belonging to a burst), was set to 4, regardless.

After identifying each population's ('Source' and
'Target') NBs, we assessed the directionality of putat-
ive population-to-population communications. If
each population initiated concurrent NBs within a
(20, 240) ms causality window (as in (Winter-Hjelm
et al 2023)), we considered these events as related.
'Forward' events correspond to a 'Source' popula-
tion's NB driving a 'Target' population's NB and
'backward' events to vice versa. Each population's NB
could only be considered for a single propagation fol-
lowing sequential order.

**5.8. Inclusion criteria and statistical analyzes**

Electrodes with a MFR of at least 0.1 Hz were con-
sidered as ‘active’ electrodes. A minimum of 10%
of each of the populations’ electrodes (‘Source’ and
‘Target’) had to be active for the recording to be con-
sidered for downstream analysis. Likewise, to ensure
that the populations could communicate, a minimum
of 100 summed (forward + backward) propagating
events on the microchannels had to be detected. For
the comparison of the ‘forward’ and ‘backward’ NB
propagation fractions (figure 5(D)), a minimum of 3
propagations was considered.

All statistical data is presented as mean ± SD unless otherwise specified. The method of Shapiro-Wilk was used as a normality test and parametric or non-parametric tests were chosen as appropriate. Sample sizes and used tests are indicated in the figures' legends or the main text. Statistical significance was considered for *p* < 0.05.

**Data availability statement**

The data that support the findings of this study
are openly available at the following URL/DOI:
https://doi.org/10.5281/zenodo.14525181 (Mateus
and Aguiar 2024).
## Acknowledgment

All the members of the Neuroengineering and Computational Neuroscience (NCN) group for help and critical discussions. Luís Jacinto (FMUP, Portugal) for valuable comments on a previous version of the manuscript. This work was supported by la Caixa Foundation in scope of the grant CaixaResearch Health 2022 HR22-00189, and by national funds through Foundation for Science and Technology (FCT) under the projects PTDC/NAN-MAT/4093/2021 and 2023.11280.PEX (https://doi.org/10.54499/2023.11280.PEX). JM and MA were supported by FCT through the Scientific Employment Stimulus (2023.08291.CEECIND and CEECIND/03415/2017, https://10.54499/CEECIND/03415/2017/CP1392/CT0003, respectively).

## ORCID iDs

J C Mateus https://orcid.org/0000-0001-8058-5093

P Melo https://orcid.org/0009-0008-9275-6249

M Aroso https://orcid.org/0000-0002-3118-0185

B Charlot https://orcid.org/0000-0001-7178-1949

P Aguiar https://orcid.org/0000-0003-4164-5713

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