Cilia-based flow network in
the brain ventricles
Regina Faubel,1 Christian Westendorf,2 Eberhard Bodenschatz,2 Gregor Eichele1*
Cerebrospinal fluid conveys many physiologically important signaling factors through the
ventricular cavities of the brain. We investigated the transport of cerebrospinal fluid in the third
ventricle of the mouse brain and discovered a highly organized pattern of cilia modules, which
collectively give rise to a network of fluid flows that allows for precise transport within this
ventricle. We also discovered a cilia-based switch that reliably and periodically alters the flow
pattern so as to create a dynamic subdivision that may control substance distribution in the
third ventricle. Complex flow patterns were also present in the third ventricles of rats and pigs.
Our work suggests that ciliated epithelia can generate and maintain complex, spatiotemporally
regulated flow networks.
The ventricular system of the brain consists of four interconnected, cerebrospinal fluid (CSF)–filled cavities, lined with ependyma whose apical surface bears bundles of motile cilia (1). CSF directional flow through the
ventricles is driven by continuous CSF secretion
into the ventricles and by orchestrated beating of
the cilia. Motile cilia are eyelash-shaped cellular
protrusions containing a microtubule-based axo-neme that, by bending, confers motility. The direction of cilia beating depends on coupling of planar
cell polarity signaling with hydrodynamic forces
(2, 3). CSF flow toward the exit of each ventricle
affords an efficient washout of waste (4) such as
neurotoxins and protein aggregates. Additionally,
CSF flow delivers nutrients, signaling molecules,
microRNAs, and exosomes (5–12). How these CSF
components reach their target sites is an important question that prompted us to investigate the
CSF flow dynamics along the ventricular walls.
We conducted this analysis in mice in the ventral
part of the third ventricle (v3V; Fig. 1A) because
of its simple geometry and close juxtaposition to
potential targets, including the physiologically
important periventricular hypothalamic nuclei
that control circadian rhythms, hormone release,
thermoregulation, blood pressure, satiety, and
feeding (Fig. 1B).
Organotypic v3V wall explant cultures were
established, and cilia-generated flow was visualized
by adding 1-mm fluorescent beads. Global flow was
determined by particle tracking, and near-wall flows
were extracted, yielding a highly complex flow map
(Fig. 1C) that was consistent among animals (the
number of animals used is given in table S1). Flow
from the inflow duct fanned out after passing by
the anterior commissure. A first flow was directed
anteriorly toward the optic recess (Fig. 1C, flow 1).
A second stream of the fan moved ventrally (flow
2). A third stream advanced posterodorsally (flow 3)
and confronted, at Bregma level –0.9, an opposing
flow (flow 4). When 70-kDa fluorescein isothio-
cyanate (FITC)–dextran was applied locally to
the head of flow 6, it propagated as prescribed by
streamlines of the flow map (Fig. 1D). Movie S1
shows the flow of FITC-dextran when applied to
the heads of flows 1, 2, or 3. Injections to flows
1 and 2 resulted in an initially coherent flow that
then fanned out, thereby dispersing the tracer over
a sizable territory. Local application to the head of
flow 3 revealed the ability of ciliated ependyma to
produce a winding flow that replicates the shape
of the streamlines seen in the flow map. Thus, the
flow map may reflect the propagation of macro-
molecules in CSF.
To explore how the confrontation of flows 3
and 4 affects transport along the wall, beads were
applied to v3V whole mounts (movie S2). Flow 4
carried beads anteriorly; near the site of confrontation with flow 3, beads moved into two mirror-symmetrical streams that intimately followed the
ventricular wall and transported the beads down
into the v3V. This flow dynamic created a separatrix
that, together with flow 5, divided the v3V into two
volumes. This flow-induced boundary hindered
the passage of tracer. Flow 6 formed another intraventricular boundary that hindered flow 7 from
drawing tracer away from flow 4. The flow pattern
remained unchanged up to a distance of 120 mm
above the ependyma (fig. S1), and flows along the
two walls were related by mirror symmetry (fig. S2).
The velocity map showed that flow velocity at
21°C varied across the v3V wall within the range
of 150 to 500 mm/s (fig. S3, A and B). Thus, it takes
only a few seconds to move substances from the
entrance of the v3V to its floor. These velocities
are typical for cilia-driven flows (13).
We next analyzed cilia movement in the v3V
and found that the flow map matched the cilia
beating pattern. To show this, we captured movies
176 8 JULY 2016 • VOL 353 ISSUE 6295 sciencemag.org SCIENCE
1Max Planck Institute for Biophysical Chemistry, Am
Fassberg 11, 37077 Göttingen, Germany. 2Max Planck
Institute for Dynamics and Self-Organization, Am Fassberg
17, 37077 Göttingen, Germany.
*Corresponding author. Email: firstname.lastname@example.org
Fig. 1. Anatomy of the third ventricle in the mouse and flow map of the v3V. (A) Sagittal section
through the third ventricle (blue), consisting of a dorsal (d3V) and a ventral (v3V) part connected by two
ducts. As indicated by white arrows, CSF enters the v3V through the foramen interventriculare and exits
through the aqueduct. (B) The position of periventricular hypothalamic nuclei [reconstructed from (22)].
Blue and red outlines differentiate the d3V and v3V, and hatching indicates the choroid plexus (blue) and
median eminence (red). Medial preoptic nucleus, yellow; suprachiasmatic nucleus, gray; anterior hypothalamic
area, brown; paraventricular nucleus, pink; ventromedial nucleus, green; arcuate nucleus, orange; dorsomedial
nucleus, blue. (C) The flow map of the v3V, generated by particle tracking, shows that near-wall flow is subdivided into multiple flow domains. The associated eight major flow directions are indicated with turquoise
arrows. (D) FITC-dextran, applied at the head of flow 6 (asterisk) from a femtopipette (p), follows the streamlines of the flow map shown in the first panel (t, time). Scales in (B) to (D) are Bregma levels in millimeters.
a, anterior; ac, anterior commissure; aq, aqueduct; cx, cortex; d, dorsal; fiv, foramen interventriculare; mb,
midbrain; my, myelencephalon; p, posterior; S, separatrix; th, thalamus; tz, tanycyte zone; v, ventral.