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PRoline-Rich Transmembrane Protein 2 deficiency in primary neuronal
cultures increases excitability and promotes network instability
Alessandra Romei, PhD Student
Center for Synaptic Neuroscience and Technology, IIT
Supervisor: Prof. Fabio Benfenati
Curriculum Neuroscience and Brain Technologies
Cycle XXXI
1st Year Report
This project has received funding from the European Union’s Horizon 2020 research and innovation
programme under the Marie Sklodowska-Curie grant agreement No 642881
Background
Paroxysmal kinesigenic dyskinesia (PKD), previously called paroxysmal kinesigenic
choreoathetosis, is the most common type of paroxysmal movement disorder of familial
inheritance. PKD was first reported in 1892 by the Japanese doctor Shuzo Kure in a 23year-old man, who displayed brief attacks of purposeless, involuntary movements of
kinesigenic trigger.
PKD patients present recurrent episodes of abnormal unintentional movements, commonly
lasting from few seconds to a minute, which are precipitated by sudden voluntary
movements from rest (such as starting to walk or rising from a seated position) or by an
alteration of ongoing actions. The current diagnostic criteria, proposed by Bruno and
colleagues (2004), comprehend brief attacks of kinesigenic trigger, occurring without pain
nor alteration of consciousness, normal neurologic examination and age at onset between
1 and 20 years. These attacks can be generalized, focal or multifocal, and can engage the
limbs, trunk and/or the orofacial area; they can be reduced or suppressed with a therapy
based on low doses of antiepileptic drugs, especially those that modulate voltage-gated
sodium channels.
The genetics underlying this pathology have been elusive until recently, when next–
generation sequencing combined with linkage analysis enabled the recognition of the
proline-rich transmembrane protein 2 (PRRT2) as causative gene for PKD (Chen et al. 2011;
Wang et al. 2011). More recently, mutations in the PRRT2 gene have been found to provoke
infantile convulsions and choreoathetosis (ICCA) (Heron et al. 2012, Lee et al. 2012) and
benign familial infantile epilepsy (BFIE) (Heron et al. 2012, Ono et al. 2012) and to a lesser
extent/sporadically in patients affected with hemiplegic migraine, episodic ataxia,
paroxysmal hypnogenic dyskinesia, paroxysmal torticollis and migraine. To date over 70
different heterozygous mutations (including frameshift, nonsense and missense) in PRRT2
gene have been documented (Ebrahimi Fakhari et al. 2015). Most of them lead to the
production of an unstable mRNA or a truncated protein that undergoes nonsense-mediated
decay (Chen et al. 2011, Lee et al. 2012, Wu et al. 2014). There is no evidence for a clear
genotype-phenotype relationship between PRRT2 mutations and the major phenotypes with
which they relate. For instance, about 80% of families with mutations in PRRT2 carry the
same frameshift mutation c.649dupC, yet different individuals (even belonging to the same
family) display paroxysmal syndromes of diverse nature (Brueckner et al. 2014; Heron and
Dibbens 2013). This considerable pleiotropy suggests the involvement of additional genetic
or environmental factors in determining the expression of a specific disease, which have not
been elucidated yet.
PRRT2 is a small gene located in the chromosome 16, containing four exons the first of
which is noncoding. Among the three human isoforms identified, the most common is a type
II
transmembrane
protein
with
a
C-terminal
anchor,
resembling
the
proteins
VAMP/synaptobrevin and syntaxin of the SNARE family (Rossi et al. 2016). This topology
indicate a possible involvement for PRRT2 in intracellular processes. Recent studies have
demonstrated that in human and rodents PRRT2 is a neuron-specific protein whose
expression is developmentally regulated and parallels the period of synapse formation and
rearrangement. It is only detected in brain and spinal cord, with the highest levels in
neocortex, basal ganglia and cerebellum (Chen et al. 2011; Heron et al. 2012; Valente et al.
2016). In neurons PRRT2 localizes at synapses, low levels are detected in fractions
enriched in post-synaptic densities while the largest protein levels co-distribute with
presynaptic proteins (Lee et al. 2012; Liu et al. 2016; Valente et al. 2016 ). Acute silencing
of PRRT2 via RNA interference reduces the number of synaptic contacts in primary neurons
(Valente et al. 2016) and in vivo, where a delayed migration of cortical neurons during
embryonic development is also observed (Liu et al. 2016). The decreased sensitivity of
spontaneous and evoked neurotransmission upon acute PRRT2 knockdown in primary
neurons also indicates a defective Ca2+-secretion coupling. The molecular mechanism
underlying this phenotype is most likely to be ascribed to the high affinity interaction between
PRRT2 and the fast Ca2+ sensors synaptotagmin 1/2 mediating fast synchronous release
(Valente et al. 2016).
Taking into account the haploinsufficient state associated to PRRT2-related paroxysmal
disorders supports the idea of a loss-of-function mechanism, constitutively inactivating this
gene may be a pertinent experimental model to reproduce and study the pathogenesis of
these diseases. Within this context, our laboratory has recently characterized the first
genetically altered mouse in which carry a constitutive inactivation of the PRRT2 gene.
Despite not exhibiting gross brain abnormalities nor being affected by spontaneous
seizures, these animals display motor disturbances and paroxysmal backwalking behavior
in infancy and adulthood that well summarize the major clinical symptoms of human PRRT2related disorders. PRRT2 KO mice also show an increased sensitivity to the convulsive
effect of pentylentetrazol PTZ and are highly prone to audiogenic paroxysms (Michetti et al.
submitted).
Despite recent findings, the pathophysiological mechanisms by which a chronic deficit in
PRRT2 lead to paroxysmal manifestations remain unidentified. The clinical symptoms of
PKD and their frequent link with migraine and epilepsy drop a hint on hyperexcitability at the
level of the cortex, basal ganglia and the cerebellum. The transition between normal and
paroxysmal states may be facilitated by network instability or the occurrence of activitydependent, abnormal network disinhibition.
Since they were first developed in the early seventies as a platform for studying cultured
cardiomyocytes (Thomas et al. 1972), planar multi-electrode arrays (MEAs) have raised
growing interest as a tool for extracellular electrophysiology studies and have subsequently
found application on neuronal cultures (Pine, 1980) and brain slices (Boppart et al. 1992).
This methodology has been extensively applied to neuronal in vitro models for investigating
complex signaling behaviors at different developmental stages (Chiappalone et al. 2006;
Wagenaar et al. 2006), learning mechanisms (Marom and Eytan, 2005) and even evaluate
the effects of neuroactive compounds (Colombi et al. 2013; Morefield et al. 2000). MEA
A
B
C
PRD
Figure 1. Structure of the PRRT2 gene and protein (A) The PRRT2 gene consists of 4 exons, of whom exons
2-4 (shown in dark blue color) encode a 340 amminoacid protein. (B) Domain structure of PRRT2 with
mutations previously reported to cause paroxysmal neurological disorders. PRRT2 hold an N-terminal
domain containing a proline-rich sequence (PRD), two presumed transmembrane helices (M1 and M2)
separated by an intracellular loop (CYT) and a C-terminal dipeptide. Nonsense/frameshift mutations are
shown in green, missense mutations in red. (C) Membrane topology of the PRRT2 protein. PRRT2 has been
assigned to the type II transmembrane proteins group, having a long cytosolic N-terminal domain and an
extracellular C-terminal anchor. M2 is the only true transmembrane domain, being M1 folded in two sections
thus not completely crossing the cell membrane. Modified from Valtorta et al. 2016.
chips can contain dozens of electrodes and thus allow simultaneous recording from multiple
sites of a neuronal circuit. In addition, they form non-invasive neuro-electronic interfaces,
well adapted for the stable and long-term recordings that are required for cultured neuronal
networks.
By performing MEA recordings on primary cortical neurons obtained from WT and PRRT2 KO
E17 embryos, the experiments here described aim at:
(i) elucidating the role of PRRT2 in the maintenance and stability of synaptic transmission
in high-density neuronal networks;
(ii)verify whether an imbalance between excitatory and inhibitory transmission occurs in
networks which chronically lack PRRT2;
(iii)
determine the responsiveness of these networks to external focal stimuli.
Methods
Primary cortical cultures on MEAs
The day before cell culture preparation, planar multi-well MultiElectrode Arrays (MEAs) were
coated by depositing a 20µl drop of poly-L-lysine (0.1mg/ml, Sigma-Aldrich) over each
recording area and subsequently incubated O/N. After thorough washing, dissociated
cortical neurons prepared from E17 WT and PRRT2 KO embryos were plated at a final
density of 80,000 neurons per well. After plating, the cells were incubated with 1% Glutamax,
2% B27, 1% Penicillin-streptomycin supplemented Neurobasal medium. Every week halfvolume replacement of the culture medium was performed.
MEA recording system
The MEA plates employed (768-GL1-30Au200 from Axion BioSystems, Atlanta, GA, USA)
are composed of 12 wells, each containing a square grid of 64 nano-porous gold electrodes
(30µm electrode diameter; 200µm center-to-center spacing) that create a 1.43 x 1.43 mm
recording area. Four grounds (2 stimulation and 2 recording grounds) are also integrated in
every well.
Spiking activity from cortical networks grown onto MEAs was recorded and monitored using
Axion BioSystems hardware (Maestro amplifier and Middle-man data acquisition interface)
and the software Axion’s Integrated Studio (AxIS 2.1). After 1200x amplification, raw data
were digitized at 12.5KHz/channel and stored on a hard disk for subsequent off-line analysis.
A
C
B
50µV
15ms
25µV
50ms
50µV
2ms
Figure 2. High-density cortical cultures grown in 12-well MEAs form active neuronal networks. (A) Our MEA
chip is composed by 12 independent chambers each containing 64 electrodes disposed in an 8 x 8 layout. In
the picture a representative WT culture aged 21 DIVs. (B) Sample trace of the signal recorded from a single
electrode (top panel). The spontaneous activity developed by primary cortical neurons plated onto MEAs is
characterized by the presence of isolated spikes (lower inset, red trace), high frequency single-channel bursts
(upper inset, orange trace) and phases of coherent spiking engaging the vast majority of active electrodes,
called network bursts. (C) Rasterplot of array-wide spontaneous activity over a 5 ms time window. Tick marks
indicate the time at which spikes occur (in blue those contained within a burst) and each horizontal line shows the
spikes recorded from a single electrode (1 to 64). Magenta rectangles specify network bursts. Legend bar:
500ms.
Experimental protocol
Experiments were performed at various developmental stages (ongoing synaptogenesis,
DIV 12-14; mature synaptic connections, DIV 19-21), in culture medium maintained at 37°.
MEA plates were set on the Maestro apparatus and their spontaneous activity recorded for
15 minutes, starting 10 minutes after the transfer to let the culture reach a stable level of
activity. On the last day of recording, the spontaneous activity was monitored for 20-30 min
before applying electrical stimulation. For pulse stimulation, the electrodes were chosen
among 64 possible stimulation sites based on visual inspection of the evoked responses to
ensure maximal activation of the network. Only the electrodes recording spiking activity were
chosen as stimulation sites. Test stimuli were sent sequentially to each selected electrode
at 0.2-Hz frequency. Pulse stimulation was biphasic with 1.5-V peak-to-peak amplitude, 500-
μs duration, and 50% duty cycle (Wagenaar et al. 2004). After the electrical stimulation
session, networks were exposed to the GABAA receptor antagonist bicuculline (BIC, Tocris,
Bristol, UK) added at the final concentration of 30 M (BIC, Tocris Bioscience, Bristol, UK)
(Keefer and Gramowski 2001; Lin et al. 2002; Gramowski et al. 2004) and recording was
carried on for further 20 min.
Signal processing for primary cortical neurons cultured on MEAs
Primary neuronal networks show both random spiking activity and bursting behavior
(Robinson et al. 1993; Opitz et al. 2002). Spike detection and spike train data analysis were
both computed using the Axion BioSystems software. Single extracellular action potentials
were detected running a voltage‐threshold‐based algorithm over 200 Hz high‐pass‐filtered
traces; the voltage threshold was 7x the standard deviation of the rms-noise on each
channel. Bursts within single channels were identified by applying an ISI threshold algorithm
(Chiappalone et al. 2005), which defines bursts as collections of a minimum number of
spikes (Nmin = 5) separated by a maximum inter-spike interval (ISImax = 100 ms). Network
bursts represent the peculiar activity pattern of mature neuronal cultures, consisting of
periods of array-wide coherent spiking typically lasting several hundreds of milliseconds and
separated by windows of lesser activity (Maeda et al. 1995; Penn et al. 2016). Network
activity within WT and mutant cultures was detected by running an ISI algorithm similar to
that applied for burst detection: it identifies a network burst only when a minimum number
of spikes (Nmin =10) occurs on at least 25% of the electrodes and it is set to automatically
estimate the ISImax (Bakkum et al. 2014). Thereafter, from the identified bursts and network
bursts several parameters (e.g. duration, firing rate within burst, average number of spikes
per burst, burst frequency) were extracted by means of the Axion Biosystems software
NeuralMetric Tool 2.0.4. Instantaneous firing rate and relative parameters (i.e. mean peak
firing rate and latency to the peak) where calculated with custom-made algorithm created in
Python (Nieus T.).
When stimulating currents/voltages are delivered through one electrode of the array at a
constant frequency, the network responds by generating a rich repertoire of reverberating
electrical activities, lasting 100-200 ms (Shahaf and Marom 2001). To investigate the
neuronal activity evoked by stimulation, the post-stimulus time histogram (i.e., PSTH) was
computed. The PSTH is used to visualize the impulse response of each site of the
experimental preparation to the electrical stimulation. The PSTHs for each electrode were
calculated by averaging the spiking activity recorded over 50 consecutive stimulations, over
a 200-ms period after each stimulus. Then, the number of spikes occurring in each 5-msec
bin was calculated to generate a cumulative histogram that was subsequently normalized
by the total number of stimuli and the bin size (Rieke et al. 1997). Evoked activity analysis
was computed using Neuroexplorer (Nex Technologies, Littleton, MA, USA).
Statistical analysis
Data are expressed as means ± SEM. Normal distribution of the experimental data was
assessed using the D’Agostino-Pearson test. As the data were not normally distributed,
nonparametric statistical tests including Mann Whitney U-test and Kruskal-Wallis multiple
comparison tests were used. P-values <0.05 were considered significant. Statistical analysis
was carried out using GraphPad Prism.
Results
PRRT2 KO networks hyperactivity parallels in vitro development
Primary cultures obtained from the cortices of E17 PRRT2 KO and WT embryos were plated
at high density onto MEA chips (Fig.1A) and their spontaneous activity monitored. The
analysis was focused on two different developmental windows: the first (12-14 DIV)
corresponded to a period of intense synaptogenesis, while in the second (19-21DIV) mature
connections are well established.
Figure 2A shows the rasterplots of the spiking activity recorded over 60s from representative
WT (left panel) and KO (right panel) cultures, aged 21DIV. For both genotypes, basal activity
was marked by the occurrence of isolated spikes, single-channel bursts and periodic
collective events called “network bursts”, in which single-channel activity was highly
synchronized over a period which could last from few hundreds of ms up to 1s (Van Pelt et
al. 2004; Vajda et al. 2008).
A total of 90 cultures was recorded, 46 for PRRT2 KO and 44 for WT and coming from 4
independent preparations for each genotype. All of them exhibited an activity pattern which
varied consistently in an age-specific manner. Similarly to previous work (Muramoto et al.
1993; Van Pelt et al. 2005; Chiappalone et al. 2006; Wagenaar et al. 2006) in WT cultures
the main activity parameters (firing rate, bursting rate, burst duration, intraburst firing rate)
varied consistently along development: firing rate and intraburst firing rate augmented with
age (firing rate: from 1.43 ± 0.09 to 2.47 ± 0.20 Hz; intraburst firing rate: from 33.24 ± 1.22
to 92.35 ± 3.08 Hz), conversely burst duration was diminished (from 178.4 ± 4.6 to 140 ±
5.8 ms). The only exception was represented by bursting rate, which had already reached
a plateau after 2 weeks in vitro (from 6.72 ± 0.61 to 8.13 ± 0.64 bursts/min).
In PRRT2 KO networks although network maturation being also paralleled by an increase
in the mean firing rate (from 1.86 ± 0.15 to 3.55 ± 0.24 Hz) and intraburst firing rate (from
38.29 ± 2.05 to 81.02 ± 4.33), under basal conditions some genotype-specific differences
were present. First, firing rate that in WT cultures had a 60% increase, was almost doubled
in KO at late stages of network development. Bursting rate (which was unaltered for WT)
further increased with age (from 8.35 ± 1.01 to 13.38 ± 1.23 bursts/min) resulting significantly
higher than that recorded in WT cultures. Furthermore early in development PRRT2 KO
networks fired more intense bursts than WT. Conversely, at a mature stage this genotype
effect on intraburst firing rate disappeared, likely due to the differential changes in burst
duration displayed by the two groups during maturation (significant decrease in WT cultures,
no change in KO group: from 166.5 ± 7.1 to 164.5 ± 7.2 ms)
GABAA receptor block with bicuculline has a comparable effect on the two genotypes
The pathophysiology of the paroxysmal disturbances associated with PRRT2 loss-offunction mutations is still fundamentally unknown. However, they have been suggested to
result from hyperexcitable cortical, basal ganglia and cerebellar networks (Zhou et al. 2010;
Luo et al. 2013; Valtorta et al. 2016 ). Besides, the strong comorbidity of motor paroxysmal
disorders to migraine and epilepsy points toward an imbalance between excitatory and
inhibitory transmission in these brain areas, which is also known to play a fundamental role
in epileptogenesis and epilepsy.
To test whether an impairment of inhibitory transmission, an increase of excitatory
transmission or both gave rise to the diffuse hyperactivity of mutant networks under basal
conditions, all cultures were challenged with an acute treatment with the GABAA receptor
blocker BIC. This approach for instance has allowed to to define the physiological impact of
mutated or deleted forms of synapsins, presynaptic phosphoproteins implicated in synaptic
vesicle trafficking and acting in the post-docking steps of exocytosis (Fassio et al. 2011), on
hippocampal (Terada et al. 1999; Baldelli et al. 2007; Lignani et al. 2013) and cortical
(Chiappalone et al. 2009) primary neurons.
In case of a sole impairment of GABAergic transmission, the differences in activity between
WT and PRRT2 KO networks under BIC should be attenuated. Conversely, if changes in
excitatory transmission are also engaged, the hyperactivity of mutant networks should be
preserved under BIC.
A
B
C
D
E
Figure 3. Cortical networks from PRRT2 KO mice display spontaneous hyperactivity during in vitro
development, while GABAA block with 30µM BIC has a comparable effect on the two genotypes.
Spontaneous activity from WT and KO cultures was recorded between the second and the third week in
vitro (12-21 DIV). On the last day of the experiments (DIV 21), the networks were exposed to the GABAA
receptor blocker Bicuculline (BIC) at a final concentration of 30µM and the recording continued for further
20 minutes. (A) A WT network cultured over a MEA chip (calibration bars: respectively 200µm and 50µm
in the left and right panels) at 21 DIV. (B) Raster plots of the spiking activity recorded from WT (upper
panel) and PRRT2 KO (lower panel) in a 60s time window, under basal conditions (left) and following bath
application of 30µM BIC (right panels). (C-F top panels) Firing rate (C), bursting rate (D), burst duration(E)
and intraburst firing rate (F) were measured for each experimental group at two developmental stages: 1214DIV and 19-21DIV. n=4 preparations for each genotype and developmental stage; 44 independent
experiments for WT and 46 for PRRT2 KO. (C-F bottom panels) Effect of BIC treatment on the firing rate,
bursting rate, burst duration and intraburst firing rate. Data are shown as ratios between the single values
of each network parameter under BIC and basal conditions (BIC/base ratio). n=4 preparations for each
genotype; 31 independent experiments for WT and 36 for PRRT2 KO. Data are plotted as mean ± SEM
for WT (gray bars) and KO (orange bars) cultures Statistical analysis was carried out by using the MannWhitney U test for independent samples (*P<0.05, **P<0.01, ***P<0.001: KO vs WT within the same age
range; °P<0.05, °°°P<0.001: 19-21 DIV vs 12-14 DIV within genotype).
Acute treatment with 30uM BIC changed considerably the activity dynamics of both WT and
KO networks. For WT, bursting activity sharply increased, while was random spiking was
almost completely abolished (rasterplots in figure 2A, left panels). Similarly in PRRT2 KO
networks bursting activity, already high under basal conditions, was further synchronized
and intensified (rasterplots in figure 2A, right panels). To directly evaluate and compare the
impact of BIC treatment on the two networks, the values for each activity parameter (i.e.
mean firing rate, bursting rate, burst duration and intraburst firing rate) were expressed as
ratios between BIC and control conditions. Pharmacological removal of GABAergic
transmission interestingly upscaled the values of all these parameters by the same order of
magnitude in WT and PRRT2 KO cultures (mean firing rate: +54 ± 15% for WT, +57 ± 9%
in KO; bursting rate: +66 ± 22% in WT, 59 ± 21% in KO; burst duration: +32 ± 13% in WT;
+53 ± 19% in KO; +20 ± 14% in WT, +40 ± 19% in KO), thus the differences between the
two genotypes were maintained under BIC (Fig. 2B, lower panels).
PRRT2 KO networks display prominent synchronization in collective bursting events
Preliminary data obtained in our team by immunocytochemistry have evidenced a reduction
in the number of excitatory synapses (-50%) on mutant primary cultures compared to WT,
while inhibitory synapses are ultimately preserved. Patch-clamp experiments performed on
autaptic neurons also revealed a decreased frequency of spontaneous miniature excitatory
events (-50%) and an enhancement of paired-pulse facilitation for excitatory synapses;
meanwhile, the frequency and amplitude of spontaneous inhibitory currents was unaffected,
and the paired-pulse depression decreased.
Despite the decrease in spontaneous excitatory transmission observed on hippocampal
autapsis, we hypothesized that the hyperactivity observed in mutant networks grown onto
MEAs may be accounted for by a long-lasting synaptic facilitation and Ca2+ build-up in the
pre-synapse. This may be particularly evident when neurons are brought into play at very
high frequencies as it is the case during network bursts.
Network bursting activity of WT and PRRT2 KO networks measured at mature stages (2021 DIV) was analyzed by taking into account four major parameters: network bursting rate,
network burst duration, mean number of spikes per network burst, intraburst firing rate (Fig.
4A). Interestingly, the number of spikes per network burst being equal, the duration of such
events was consistently lower for mutant compared to WT cultures (858.6 ± 107 for WT,
609.8 ± 92.65 ms for KO, this resulting in a higher intraburst firing rate for PRRT2 KO (909.6
± 137.4 for WT; 1221 ± 126.6 spikes/s for KO). At this stage of in vitro development the
average network bursting rate was higher but not statistically significant in KO than in WT
networks (14.56 ± 1.1 for WT, 18.28 ± 1.69 bursts/min for KO).
To better appreciate differences between mutant and WT collective behavior, instantaneous
frequency in the network bursts was measured over a 200ms time frame from the beginning
of each burst (bin size = 5ms), then averaged to calculate the mean peak firing rate and the
latency to the firing peak (Fig.4C). Noteworthy PRRT2 KO networks reached a significantly
higher firing rate compared to WT, while the latency to the firing peak was similar between
the two genotypes. These data are in compliance with the excitability measures performed
by our group on primary hippocampal neurons. In particular PRRT2 KO excitatory neurons
were able to follow trains of high frequency stimulation (80-100-120 Hz) with much higher
fidelity compared to WT ones.
These data together support the idea of an enhanced and long-lasting synaptic facilitation
in PRRT2 KO networks during collective high-frequency events. Because mutant cultures
can sustain network activity at higher frequencies, the shorter network bursts observed in
mutant cultures could be explained as a consequence of a faster recruitment of the inhibitory
mechanisms and of the enhancement of the inhibitory strength observed in patch clamp
experiments.
PRRT2 KO networks display a prolonged spiking probability in response to low-frequency
stimulation
The response of 23 and 28 independent cultures for WT and PRRT2 KO genotypes
respectively to trains of low-frequency, localized extracellular stimulation was analyzed.
The rasterplots in Figure 5a show the spiking response of two representative WT (top panel)
and KO (lower panel) cultures to a single stimulation within a time window of 200ms following
the stimulus delivery. Compared to WT cultures, mutant networks exhibited higher and longlasting firing probability after the electrical stimulation.
To evaluate the changes of firing probability as a function of time after the pulse, the
instantaneous firing frequency resulting from all the 50 stimuli in the train for the entire
datasets was summated over time (bin size = 5ms) and used to calculate the corresponding
PSTHs. The WT network-wide response to the stimulation was characterized by the
presence of a short latency (0-20 ms) response abruptly decreasing and followed by a
secondary weaker response that lasted for about 50-100ms (Fig. 5b, gray trace). PRRT2
*
*
Figure 4. Enhanced network synchronization in PRRT2 KO primary cultures. Network bursts are a
commonplace feature of neuronal networks both in vitro and in vivo. In in vitro cultures on MEAs, these are
array-wide bursting events whose expression is strongly dependent on connectivity and excitability. (A)
Network bursting activity of WT and PRRT2 KO networks measured at mature stages (20-21 DIV) was
analyzed by taking into account four major parameters: network bursting rate, network burst duration, mean
number of spikes per network burst, intraburst firing rate. Interestingly, PRRT2 KO networks fired shorter but
significantly more intense bursts. (B) Superimposed instantaneous frequencies for 50 consecutive network
bursts generated by representative WT (left panel, black trace) and PRRT2 KO (right panel, red trace)
cultures, plotted over the first 200ms of each burst (bin size = 5ms). (C) Instantaneous frequency in the
network bursts measured over a 200ms time frame from the beginning of each burst was averaged to
calculate the mean peak firing rate and the latency to the peak. During network-wide events, PRRT2 KO
networks reached a considerably higher firing rate, whereas the latency to the firing peak did not differ
between the 2 genotypes. Statistical analysis was performed using the Mann-Whitney U test for independent
samples (* P<0.05, KO vs WT).
KO cultures responded to focal electrical stimulation with a similar spiking profile, yet firing
probability remained much higher and long-lasting compared to WT (Fig. 5b, orange trace).
The frequency distribution of the areas under the PSTHs generated from each channel of
the entire dataset of the electrical stimulation experiments revealed a skewed profile for WT
network, whereas the frequency profile for KO was shifted towards larger values. Further
emphasizing the diffuse hyperexcitability of the mutant networks, the mean PSTH area for
mutant networks proved to be significantly larger than that of WT (513.9 ±14.06 for WT,
705.5 ± 19.93 spikes for PRRT2 KO).
Conclusions and future experiments
All PRRT2-associated diseases share paroxysmal manifestations which suggest a common
pathophysiology for these that might involve PRRT2 function in synaptic vesicle release and
neuronal excitability, in accordance with previous work. In this context I have investigated
the basis of paroxysms propensity of PRRT2 KO mice (Michetti et al. submitted) by studying
the electrical activity of dissociated cortical cultures on multi-electrode arrays.
In WT mouse brain and dissociated cultures PRRT2 expression increases in conjuction with
the process of synaptogenesis. Interestingly, under basal conditions, PRRT2 KO primary
cultures on MEAs display developmentally regulated hyperexcitability. Despite showing
some genotype specific differences after 2 weeks of in vitro development, the largest
dissimilarities between the groups in terms of firing and bursting rate become evident at the
third week in vitro, when mature connections are established. This suggests that for KO
neurons a gradual buildup of hyperexcitability occurs during this time window. However,
mutant and WT cultures exhibit a similar developmental pattern, and the hyperexcitable
phenotype is mild when compared for instance to that of networks in which proteins involved
in crucial presynaptic processes have been deleted (Chiappalone et al. 2006). In the case
of PRRT2, we may speculate that in its absence some compensatory effects come into play.
The hyperexcitability exhibited by PRRT2 KO networks under basal conditions can’t be
ascribed to a sole impairment of GABAergic transmission. In fact when the influence of
GABAAR-mediated inhibition was pharmacologically blocked, spiking and bursting rate,
burst duration and intraburst firing rate for the two groups were upscaled by the same
magnitude. Therefore PRRT2 KO networks maintained a significant hyperactivity with
respect to WT.
The enhanced synchronization and responsiveness to external stimuli displayed by cortical
cultures chronically lacking PRRT2 also points towards a role for this protein in the correct
development and stability of neuronal networks. The instability of networks in which the
A
C
B
D
Figure 5. Enhanced firing probability in PRRT2 KO cortical networks in response to electrical
stimulation at low frequency. (A) Representative raster plots of evoked spiking activity recorded from WT
(top panel) and PRRT2 KO (bottom panel) cultures for a 200ms time window after stimulus delivery. Each (B)
Average PSTH for a single stimulation session (i.e. 50 stimuli delivered at 0.2Hz), for all the active channels
in WT (gray trace) and KO (orange trace) cultures. Bin size: 5 ms. (C) Histogram of PSTH areas for all the
active channels of the entire dataset (gray trace for WT, orange trace for PRRT2 KO). n=4 preparations for
each genotype; 23 and 28 independent cultures for WT and PRRT2 KO respectively. Mean number of active
channels: 56.43 ± 9.06 for WT; 52.50 ± 7.20 for KO networks. (D) Mean (± SEM) area of the PSTH for WT
(gray bar) and KO (orange bar) networks. Statistical analysis was carried out by using the Mann-Whitney U
test for independent samples (***P<0.001).
expression of PRRT2 has been constitutively inactivated could have a significance in the
pathophysiology of PRRT2-releated diseases. Indeed it may render the brain areas involved
in sensory information processing and subsequent selection and execution of the motor
program more prone to generate aberrant and undesired movements (i.e. cortico-basal
ganglia-thalamic loop and cortico-cerebellar system).
The ongoing/future steps of this work are the following:
1. With external collaborators (Lonardoni D., Nieus. T.), we are conducting an in silico study
on the basis of the experimental data obtained in vitro. The aim is to develop a model that
is able to merge the observations on primary cortical neurons by MEA recordings and the
results obtained on hippocampal autapsis by patch-clamp experiments.
2. Perform MEA recordings on primary hippocampal cultures to check whether spontaneous
hyperactivity and hyperexcitability is also verified in this preparation.
3. Check whether synaptic transmission in acute cerebellar slices is altered by chronic
deletion of PRRT2, by performing electrophysiological recordings with high-resolution
CMOS-MEAs. In this brain area, that is typically implicated in motor coordination and
balance via sensory feedbacks processing, a very strong expression of PRRT2 is present
in the granule cell layer; furthermore an aberrant plasticity at the level of the parallel fiberPurkinje cell synapse has been described (Michetti et al. submitted).
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