Submitted 18 June 2013
Accepted 30 October 2013
Published 21 November 2013
Additional Information and
Declarations can be found on
2013 Amada et al.
Creative Commons CC-BY 3.0
Cannabidivarin (CBDV) suppresses
increases in epilepsy-related gene
Naoki Amada1,2,3 , Yuki Yamasaki1,2 ,3, Claire M. Williams2and
Benjamin J. Whalley1
1School of Chemistry, Food and Nutritional Sciences and Pharmacy, The University of Reading,
Reading, Berkshire, UK
2School of Psychology and Clinical Language Sciences, The University of Reading, Reading,
3Qs’ Research Institute, Otsuka Pharmaceutical, Co. Ltd., Kagasuno, Kawauchi-cho, Tokushima,
To date, anticonvulsant eﬀects of the plant cannabinoid, cannabidivarin (CBDV),
have been reported in several animal models of seizure. However, these behaviourally
observed anticonvulsant eﬀects have not been conﬁrmed at the molecular level. To
examine changes to epilepsy-related gene expression following chemical convulsant
treatment and their subsequent control by phytocannabinoid administration, we
behaviourally evaluated eﬀects of CBDV (400 mg/kg, p.o.) on acute, pentylenetetra-
zole (PTZ: 95 mg/kg, i.p.)-induced seizures, quantiﬁed expression levels of several
epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and
Egr1) by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples
before examining correlations between expression changes and seizure severity.
PTZ treatment alone produced generalised seizures (median: 5.00) and signiﬁcantly
increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous
ﬁndings, CBDV signiﬁcantly decreased PTZ-induced seizure severity (median: 3.25)
and increased latency to the ﬁrst sign of seizure. Furthermore, there were correlations
between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc,
Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group.
When CBDV treated animals were grouped into CBDV responders (criterion: seizure
severity ≤3.25) and non-responders (criterion: seizure severity >3.25), PTZ-induced
increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV re-
sponders. These results provide the ﬁrst molecular conﬁrmation of behaviourally
observed eﬀects of the non-psychoactive, anticonvulsant cannabinoid, CBDV,
upon chemically-induced seizures and serve to underscore its suitability for clinical
Subjects Molecular Biology, Neuroscience, Neurology, Pharmacology
Keywords Cannabinoid, Pentylenetetrazole, Cannabidivarin, qPCR, Seizure, Epilepsy
How to cite this article Amada et al. (2013), Cannabidivarin (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in
epilepsy-related gene expression. PeerJ 1:e214;DOI 10.7717/peerj.214
Epilepsy aﬀects ∼1% of individuals and is often characterized by recurrent seizures. Many
treatments are available but more eﬀective and better-tolerated antiepileptic drugs (AEDs)
with new mechanisms of actions are needed due to drug resistance (∼35%) and poor AED
side-eﬀect proﬁles (Kwan & Brodie, 2007).
Several cannabinoids (19-tetrahydrocannabinol: 19-THC, cannabidiol: CBD,
19-tetrahydrocannabivarin: 19-THCV and cannabidivarin: CBDV) are anticonvulsant
in a variety of animal models of seizure and epilepsy (Consroe & Wolkin, 1977;Hill et
al., 2012a;Hill et al., 2010;Jones et al., 2010). Whilst CB1cannabinoid receptor (CB1R)
agonism is anti-epileptiform and anticonvulsant (Chesher & Jackson, 1974;Deshpande et
al., 2007b;Wallace et al., 2003;Wallace et al., 2001), the notable psychoactivity associated
with CB1R activation hinders the prospective clinical utility of this target. However,
many plant cannabinoids do not act at CB1R and the most promising non-psychoactive
anticonvulsant phytocannabinoid studied thus far is CBD, which exerts eﬀects via, as
yet unknown, non-CB1R mechanisms in vitro,in vivo and in humans (Consroe et al.,
1982;Cunha et al., 1980;Jones et al., 2010;Wallace et al., 2001). Because CBD has low
aﬃnity for CB1 and CB2 receptors (Pertwee, 2008), CBD may exert its eﬀects through
diﬀerent mechanisms. For instance, it is known that CBD can, at a number of diﬀerent
concentrations in vitro, inhibit adenosine uptake, inhibit FAAH (the enzyme primarily
responsible for degradation of the endocannabinoid, anandamide), inhibit anandamide
reuptake, act as a TRPA1 receptor agonist, a TRPM8 receptor antagonist, a 5-HT1A
receptor agonist, a T-type calcium channel inhibitor and a regulator of intracellular
calcium (Izzo et al., 2009).
Here, we have used molecular methods to further investigate the anticonvulsant
potential of CBD’s propyl analogue, CBDV (Hill et al., 2012a). Although ﬁrst isolated
in 1969 (Vollner, Bieniek & Korte, 1969), little is known about CBDV’s pharmacological
properties (Izzo et al., 2009). Scutt and Williamson reported CBDV to act via CB2
cannabinoid receptor-dependent mechanisms but direct CB2 receptor eﬀects were not
shown (Scutt & Williamson, 2007). De Petrocellis reported diﬀerential CBDV eﬀects at
transient receptor potential (TRP) channels in vitro, noting potent human TRPA1, TRPV1
and TRPV2 agonism and TRPM8 antagonism (De Petrocellis et al., 2011;De Petrocellis
et al., 2012). CBDV has also been reported to inhibit diacylglycerol (DAG) lipase-α,
the primary synthetic enzyme of the endocannabinoid, 2-arachidonoylglycerol (2-AG)
(Bisogno et al., 2003), in vitro (De Petrocellis et al., 2011). However, 2-AG inhibits status
epilepticus-like activity in rat hippocampal neuronal cultures (Deshpande et al., 2007a)
such that diacylglycerol lipase-αinhibition is unlikely to be anticonvulsant. Furthermore,
inhibition of DAG lipase-αby CBDV occurs at high micromolar concentrations (IC50:
16.6 µM) in vitro which are unlikely to have relevance in vivo making it unlikely that CBDV
exerts anticonvulsant eﬀects via this route. Although the pharmacological relevance of
these eﬀects remains unconﬁrmed in vivo and the targets identiﬁed have not yet been
linked to epilepsy, they illustrate an emergent role for multiple, non-CB receptor targets
of phytocannabinoids (Hill et al., 2012b;Pertwee, 2010). Furthermore, unlike 19-THC,
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 2/18
anticonvulsant doses of CBDV exert no detectable eﬀects upon motor function (Hill et al.,
2012a) which further supports the assertion that its eﬀects are not CB1R-mediated.
Despite our earlier report showing signiﬁcant anticonvulsant eﬀects of CBDV in animal
models of acute seizure (Hill et al., 2012a), molecular validation of these eﬀects has not
yet been undertaken. Here, we evaluated CBDV’s eﬀect (p.o.) on pentylenetetrazole
(PTZ)-induced seizures and quantiﬁed expression levels of several epilepsy-related genes
in tissue from hippocampus, neocortex and prefrontal cortex. Genes of interest were
selected on the basis that: (i) their expression was signiﬁcantly changed in previously
published gene expression microarray results from people with epilepsy (PWE) (Helbig
et al., 2008;Jamali et al., 2006;van Gassen et al., 2008) and animal models of epilepsy
(Elliott, Miles & Lowenstein, 2003;Gorter et al., 2006;Gorter et al., 2007;Okamoto et al.,
2010) and (ii) published results (Johnson et al., 2011;Link et al., 1995;McCarthy et al., 1998;
Nanda & Mack, 2000;Saﬀen et al., 1988;Sola, Tusell & Serratosa, 1998;Zhu & Inturrisi,
1993) suggested that expression changes were acute (within a few hours of seizure), making
them suitable for study in a model of acute seizure. On this basis, Early growth response 1
(Egr1), Activity-regulated cytoskeleton-associated protein (Arc), Chemokine (C-C motif)
ligand 3 (Ccl3), Chemokine (C-C motif) ligand 4 (Ccl4), Brain derived neurotrophic
factor (Bdnf), Proenkephalin (Penk) and Neuropeptide Y (Npy) and the downregulated
gene, Calcium/calmodulin-dependent protein kinase II alpha (Camk2a) were chosen. FBJ
osteosarcoma oncogene (Fos) and Caspase 3 (Casp3) were also selected due to the former’s
increased expression in brain regions including hippocampus following experimentally
induced seizures (e.g., via PTZ) (Popovici et al., 1990;Saﬀen et al., 1988) and the latter
as a result of increased expression in resected neocortex from people with temporal lobe
epilepsy (Henshall et al., 2000).
MATERIAL AND METHODS
Experiments were conducted in accordance with UK Home Oﬃce regulations (Animals
(Scientiﬁc Procedures) Act, 1986). A total of 51 Wistar-Kyoto rats (Harlan, UK; 3–4 weeks
old) were used in this study and ARRIVE guidelines complied with. Animals were group
housed in cages of ﬁve with water and food supplied ad libitum. Temperature and humidity
were maintained at 21◦C and 55 ±10% respectively.
Seizures were induced using PTZ (Sigma, Poole, United Kingdom). After overnight
fasting, rats received either vehicle (20% solutol (Sigma) in 0.9%w/vNaCl) or CBDV
(400 mg kg−1; GW Pharmaceuticals Ltd., Salisbury, UK) in vehicle by oral gavage. Three
and a half hours after vehicle or CBDV administration, rats were challenged (i.p.) with
saline or PTZ (95 mg kg−1) and behaviour monitored for 1 h. Animals were euthanised
by CO2overdose and brains immediately removed. Whole hippocampi, neocortices and
prefrontal cortices were isolated, snap-frozen in liquid nitrogen and stored at −80◦C until
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 3/18
Analysis of seizure behaviours
Seizure behaviour was video recorded and responses coded exactly as described previously
(Hill et al., 2012a). Responses were coded using the following modiﬁed Racine seizure
severity scale: 0, normal behaviour; 1, isolated myoclonic jerks; 2, atypical clonic seizure;
3, fully developed bilateral forelimb clonus; 3.5, forelimb clonus with tonic component
and body twist; 4, tonic–clonic seizure with suppressed tonic phase; 5, fully developed
tonic–clonic seizure. Latency to the ﬁrst sign of seizure was also recorded.
Gene expression analysis
Gene expression was quantiﬁed in rat hippocampus, prefrontal cortex and neocortex for
four experimental groups: vehicle +saline treated (n=5), vehicle +PTZ treated (n=7),
CBDV +saline treated (n=5) and CBDV +PTZ treated (n=7). Total RNA was extracted
using an miRNeasy Mini kit (Qiagen, West Sussex, UK), following the manufacturer’s
protocol. RNA purity was assessed spectrometrically at 260/280 nm. RNA integrity was
determined by gel electrophoresis. A 28S:18S rRNA ratio of ∼2:1 was taken to indicate
Total RNA (0.5 µg) was reverse-transcribed into cDNA using High Capacity cDNA
Reverse Transcription Kits (Applied Biosystems). qPCR assays were carried out in a volume
of 14 µl, containing 5 µl cDNA, 2 µl 2.5 µM primer mix (forward and reverse primers) and
7µl QuantiTect SYBR Green QPCR 2×Master Mix (Qiagen, West Sussex, UK). Samples
were processed for 40 cycles on a StepOnePlusTM (Applied Biosystems, Foster City, CA,
USA) as follows: denaturation at 95◦C for 15 min (one cycle), 40 cycles of denaturation
at 95◦C for 15 s and annealing at 60◦C for 1 min. All samples were analysed in the same
plate in a single PCR run and quantiﬁcation was based on the standard curve method.
Standard curves were constructed using cDNA solution diluted ﬁvefold in series for a total
of ﬁve dilutions and consisted of a mixture of cDNA equally from hippocampus, prefrontal
cortex and neocortex of all animals. Sample cDNA concentrations were expressed relative
to the concentration of the standard curves. Normalisation of quantitative data was
based on a housekeeping gene, β-actin. Values are expressed as a percentage of control
(mean of the vehicle +saline group). The following primers were used (parenthesised
values are forward and reverse sequence and amplicon length respectively): Ccl3
(50-TGCCCTTGCTGTTCTTCTCTGC-30, 50-TAGGAGAAGCAGCAGGCAGTCG-30, 96),
Ccl4 (50-CGCCTTCTGCGATTCAGTGC-30, 50-AAGGCTGCTGGTCTCATAGTAATCC-
30, 127), Npy (50-TCGTGTGTTTGGGCATTCTGGC-30, 50-TGTAGTGTCGCAGAGCGG
AGTAG-30, 111), Arc (50-AGGCACTCACGCCTGCTCTTAC-30, 50-TCAGCCCCAGCTC
AATCAAGTCC-30, 146), Bdnf (50-AGCCTCCTCTGCTCTTTCTGCTG-30, 50-TATCTGC
CGCTGTGACCCACTC-30, 150), Egr1 (50-AGCCTTCGCTCACTCCACTATCC-30, 50-GC
GGCTGGGTTTGATGAGTTGG-30, 113), Penk (50-CCAACTCCTCCGACCTGCTGAAA
G-30, 50-AAGCCCCCATACCTCTTGCTCGTG-30, 121) and Camk2a (50-TGAGAGCACC
AACACCACCATCG-30, 50-TGTCATTCCAGGGTCGCACATCTTC-30, 142), Fos (50-TGC
GTTGCAGACCGAGATTGC-30, 50-AGCCCAGGTCATTGGGGATCTTG-30, 104), Casp3
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 4/18
111) and β-Actin (50-CTCTATCCTGGCCTCACTGTCCACC-30, 50-AAACGCAGCTC
AGTAACAGTCCGC-30, 124). Primers were designed using NCBI/Primer-BLAST
CBDV eﬀects upon seizure severity and onset latency were assessed by comparing
vehicle +PTZ treated and CBDV +PTZ treated groups using a two-tailed Mann-Whitney
test and a two-tailed t-test, respectively. Subsequently, animals in the CBDV +PTZ treated
group were divided according to median seizure severity score into CBDV ‘responders’
(criterion: seizure severity ≤median) and ‘non-responders’ (criterion: seizure severity
>median) to permit a preliminary subgroup analysis of CBDV eﬀects in these two
groups without statistical analysis on subgroups. In qPCR analysis, diﬀerences of mRNA
expressions between treatment groups were analysed in each brain region using one-way
analysis of variance (one-way ANOVA) followed by Tukey’s test. Correlations between
seizure severity and mRNA expression in the CBDV +PTZ treated group were analysed
using Spearman’s rank correlation coeﬃcient. A preliminary assessment of gene expression
changes for CBDV ‘responders’ and ‘non-responders’ was performed, in which diﬀerences
of mRNA expressions between the vehicle +PTZ treated and the CBDV responder or
non-responder subgroups were analysed in each brain region by two-tailed t-test. Since
samples from each brain region were analysed on physically separate PCR plates, no
comparisons of seizure or drug eﬀects between brain areas were made. Diﬀerences were
considered statistically signiﬁcant when the P≤0.05.
Anticonvulsant effects of CBDV on PTZ-induced acute seizures
400 mg kg−1CBDV signiﬁcantly decreased seizure severity (vehicle: 5; CBDV: 3.25;
P<0.05) and increased latency to the ﬁrst seizure sign (vehicle: 60 s; CBDV: 272 s;
P<0.05; Figs. 1A and 1B). Responses of CBDV +PTZ animals sub-grouped into CBDV
responders (criterion: seizure severity ≤3.25; n=10) and non-responders (criterion:
seizure severity >3.25; n=10) showed clear behavioural diﬀerences (Figs. 1C and 1D)
where CBDV responders exhibit lower seizure severity and increased onset latency.
Effects of PTZ treatment on mRNA expression of epilepsy-related
genes in the hippocampus, neocortex and prefrontal cortex
PTZ treatment signiﬁcantly upregulated Fos mRNA expression in neocortex (P=0.0001)
and prefrontal cortex (P=0.0003; Table 1) whilst hippocampal Fos mRNA expression
only showed a trend to increase (P=0.1089). Egr1 mRNA expression was signiﬁcantly
upregulated by PTZ treatment in the hippocampus (P=0.0244), neocortex (P=0.0001)
and prefrontal cortex (P<0.0001) whilst Arc mRNA expression was also signiﬁcantly up-
regulated by PTZ treatment in the hippocampus (P=0.0374), neocortex (P=0.0039) and
prefrontal cortex (P=0.0038). Expression of Ccl4 mRNA was signiﬁcantly upregulated
only in the prefrontal cortex (P=0.0220) by PTZ treatment. Trends toward an increase
of Ccl4 mRNA expression in the hippocampus (P=0.1720) and neocortex (P=0.1093)
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 5/18
Figure 1 Anticonvulsant eﬀects of CBDV on PTZ-induced acute seizures. (A) Plot showing median
seizure severity in the vehicle- and CBDV-treated groups following PTZ administration. (B) Plot showing
latency (seconds) to the ﬁrst seizure sign in the vehicle- and CBDV-treated groups. (C) Seizure severity
after sub-grouping CBDV treated group animals into CBDV non-responders and CBDV responders.
(D) Latency (seconds) to the ﬁrst seizure sign after subgrouping CBDV treated group animals into CBDV
non-responders and CBDV responders. In seizure severity plots, median seizure severity is represented
by a thick horizontal line, the 25th and the 75th percentiles are represented by the box and maxima and
minima are represented by ‘whiskers’. Latency to the ﬁrst seizure sign was presented as mean ±SEM.
∗,P<0.05 by Mann-Whitney Test vs vehicle group; #, P<0.05 by t-test vs vehicle group.
by PTZ treatment were seen. Expression of Bdnf mRNA was signiﬁcantly upregulated in
the neocortex (P=0.0308) and prefrontal cortex (P=0.0345) but only a trend towards
increased expression in the hippocampus was seen (P=0.0564). mRNA expression of
Casp3, Npy, Penk, Ccl3 and Camk2a were not signiﬁcantly changed by any treatment.
Effects of CBDV upon PTZ-induced mRNA expression of epilepsy-
related genes in the hippocampus, neocortex and prefrontal
Fos and Egr1 mRNA expression were signiﬁcantly upregulated in the neocortex
(P=0.0201 and P=0.0033, respectively) and the prefrontal cortex (P=0.0156 and
P=0.0023, respectively) in the CBDV +PTZ treated group. Although there were no
statistically signiﬁcant changes in the expression levels of any other genes between the
vehicle +saline and CBDV +PTZ treated groups which suggests an inhibitory eﬀect
of CBDV on PTZ-induced upregulation of gene expression, neither were statistically
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 6/18
Table 1 Relative mRNA expression levels of epilepsy-related genes in the hippocampus (HIP), neocortex (Nctx) and prefrontal cortex (PFC). Ex-
pressions of Fos, Egr1, Arc, Ccl4 and Bdnf were upregulated by PTZ treatment. mRNA levels are presented as a fold change vs mean level of vehicle
+saline treated group (data are expressed as mean ±s.e.m.). Diﬀerences between individual groups were assessed by 1-way ANOVA (followed by a
Tukey’s post-hoc test if warranted).
HIP 1.0 ±0.2 55.6 ±22.2 0.8 ±0.1 25.4 ±15.0
Nctx 1.0 ±0.3 21.5 ±3.5** 0.7 ±0.1 13.2 ±2.8*
FBJ osteosarcoma oncogene Fos
Cellular response to calcium ion,
cellular response to extracellular
stimulus, inﬂammatory response,
nervous system development PFC 1.0 ±0.1 20.0 ±3.8** 0.8 ±0.1 13.5 ±2.3*
HIP 1.0 ±0.1 0.9 ±0.1 0.9 ±0.1 0.9 ±0.1
Nctx 1.0 ±0.1 1.1 ±0.1 1.0 ±0.1 1.1 ±0.1
Caspase 3 Casp3 Apoptosis, intracellular signal
PFC 1.0 ±0.0 1.1 ±0.1 0.9 ±0.1 0.9 ±0.1
HIP 1.0 ±0.0 6.1±1.5*0.8 ±0.1 3.6 ±1.1
Nctx 1.0 ±0.1 3.0 ±0.4** 0.7 ±0.1 2.5 ±0.2**
Early growth response 1 Egr1
Cellular response to drug, cellular
response to growth factor stimulus,
cellular response to steroid
hormone stimulus, circadian
PFC 1.0 ±0.1 2.7 ±0.3** 0.8 ±0.1 2.2 ±0.2**
HIP 1.0 ±0.1 8.6 ±2.5*0.8 ±0.1 4.2 ±1.7
Nctx 1.0 ±0.2 5.0 ±1.1** 0.6 ±0.1 3.4 ±0.5
Arc Regulation of neuronal synaptic
PFC 1.0 ±0.1 4.4 ±0.9** 0.7 ±0.1 3.0 ±0.4
HIP 1.0 ±0.1 0.9 ±0.1 1.0 ±0.1 1.0 ±0.1
Nctx 1.0 ±0.1 1.0 ±0.1 1.0 ±0.1 1.1 ±0.1
Neuropeptide Y Npy
Feeding behavior, negative
regulation of blood pressure,
synaptic transmission PFC 1.0 ±0.1 0.9 ±0.0 1.0 ±0.1 0.9 ±0.0
HIP 1.0 ±0.1 16.7 ±5.9 0.7 ±0.2 7.9 ±6.3
Nctx 1.0 ±0.3 36.0 ±14.8 1.4 ±0.3 15.4 ±8.8
Chemokine (C-C motif)
ligand 4 Ccl4 Chemotaxis, inﬂammatory
PFC 1.0 ±0.2 13.3 ±3.4*1.0 ±0.2 7.9 ±3.0
HIP 1.0 ±0.2 8.8 ±3.7 1.1 ±0.2 5.3 ±3.7
Nctx 1.0 ±0.2 21.1 ±10.5 1.6 ±0.2 13.0 ±6.2
Chemokine (C-C motif)
ligand 3 Ccl3
Chemotaxis, elevation of cytosolic
calcium ion concentration,
inﬂammatory response PFC 1.0 ±0.1 16.4 ±6.3 1.5 ±0.1 13.5 ±5.9
HIP 1.0 ±0.1 2.6 ±0.6 0.9 ±0.1 1.7 ±0.3
Nctx 1.0 ±0.0 2.5 ±0.4*0.9 ±0.1 2.1 ±0.4
neurotrophic factor Bdnf
positive regulation of long-term
neuronal synaptic plasticity,
glutamate secretion PFC 1.0 ±0.1 2.1 ±0.4*1.1 ±0.2 1.9 ±0.2
HIP 1.0 ±0.1 1.2 ±0.2 1.1 ±0.1 1.1 ±0.1
Nctx 1.0 ±0.2 1.1 ±0.2 0.8 ±0.1 1.1 ±0.1
Proenkephalin Penk Behavioral fear response,
sensory perception of pain
PFC 1.0 ±0.2 0.9 ±0.2 1.1 ±0.2 0.9 ±0.2
HIP 1.0 ±0.1 0.9 ±0.0 0.9 ±0.1 0.9 ±0.1
Nctx 1.0 ±0.1 0.9 ±0.1 1.0 ±0.1 1.0 ±0.1
kinase II alpha
Calcium ion transport, ionotropic
glutamate receptor signaling
pathway, protein phosphorylation,
regulation of neuronal synaptic
plasticity, regulation of
neurotransmitter secretion PFC 1.0 ±0.1 1.0 ±0.1 1.1 ±0.1 1.0 ±0.1
*P<0.05 vs vehicle +saline group.
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 7/18
signiﬁcant diﬀerences in gene expression levels between the vehicle +PTZ and
CBDV +PTZ treated groups found. However, when potential correlations between the
behavioural measure of seizure severity and mRNA expression levels of Fos, Egr1, Arc,
Bdnf and Ccl4 in the CBDV +PTZ treated group were examined using Spearman’s rank
correlation coeﬃcient, mRNA expression levels of these genes were highly correlated with
seizure severity in the majority of brain regions examined (Fig. 2: hippocampus, Fig. 3:
neocortex and Fig. 4: prefrontal cortex). Fos mRNA expression correlated with seizure
severity in the hippocampus (R2=0.91, P=0.0008), neocortex (R2=0.91, P=0.0008)
and prefrontal cortex (R2=0.91, P=0.0008) of the CBDV +PTZ treated group.
Egr1 mRNA expression was correlated with seizure severity only in the hippocampus
(R2=0.91, P=0.0008) whilst Arc mRNA expression was correlated with seizure severity
in the hippocampus (R2=0.91, P=0.0008), neocortex (R2=0.91, P=0.0008) and
prefrontal cortex (R2=0.71, P=0.0175). Bdnf mRNA expression was correlated with
seizure severity in the hippocampus (R2=0.71, P=0.0175) and neocortex (R2=0.65,
P=0.0291) whilst Ccl4 mRNA expression was correlated with seizure severity in the
hippocampus (R2=0.91, P=0.0008), neocortex (R2=0.71, P=0.0175) and prefrontal
cortex (R2=0.71, P=0.0175). Together, these suggest a possible contribution of the
anti-convulsant eﬀects of CBDV in reduction of mRNA expression of Fos, Egr1, Arc, Bdnf
Effects of CBDV treatment on the PTZ-induced increases of the
epilepsy-related genes in CBDV responders
Consistent with diﬀering behavioural patterns observed between CBDV responder and
non-responder subgroups, alterations in gene expression were also seen. Importantly,
changes in gene expression levels between the vehicle +PTZ and the CBDV responder
subgroups were most obvious, with few changes seen in gene expression levels between
vehicle +PTZ and the CBDV +PTZ non-responder subgroups. Importantly, PTZ-
induced increases in gene expression were most reliably suppressed in the hippocampus
of CBDV responders, with less obvious suppression in prefrontal cortex and neocortex.
The PTZ-induced increase of Fos mRNA expression in CBDV responders was suppressed
in the neocortex (P=0.0274) and the prefrontal cortex (P=0.0337), and there was
a strong trend towards a decrease in the hippocampus (P=0.0579; Fig. 5A). The
PTZ-induced increase of Egr1 mRNA expression was suppressed in the hippocampus
(P=0.0234) of CBDV responders, but less obviously so in the neocortex (P=0.1837)
and the prefrontal cortex (P=0.1038; Fig. 5B). The increase in Arc mRNA expression
induced by PTZ treatment was also suppressed in the hippocampus (P=0.0221) of CBDV
responders, and there were strong trends towards decreases in the neocortex (P=0.0643)
and the prefrontal cortex (P=0.0879; Fig. 5C). The increase of Bdnf mRNA expression
following PTZ treatment was most suppressed in the hippocampus (P=0.0441) of CBDV
responders whilst less decreases were seen in the neocortex (P=0.1099) and prefrontal
cortex (P=0.4128; Fig. 5D). Finally the PTZ-induced increase of Ccl4 mRNA expression
was suppressed in the hippocampus (P=0.0323) and the prefrontal cortex (P=0.0459),
and there was a strong trend towards a decrease in the neocortex (P=0.0942; Fig. 5E). On
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 8/18
Figure 2 Correlation analysis between seizure severity and mRNA expression levels in the hippocam-
pus. Correlations between mRNA expression of Fos (A), Egr1 (B), Arc (C), Bdnf (D) and Ccl4 (E) and
seizure severity were analysed using Spearman’s rank correlation coeﬃcient.
the other hand, neither statistically signiﬁcant decreases nor trends towards decreases in
the gene expressions were found in the CBDV non-responder subgroup.
PTZ treatment upregulated (signiﬁcant increase or statistically strong trend to increase)
mRNA expression coding for Fos, Egr1, Arc, Ccl4 and Bdnf in all brain regions tested. Clear
correlations between seizure severity and mRNA expression were observed for these genes
in the majority of brain regions of CBDV +PTZ treated animals and mRNA expression
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 9/18
Figure 3 Correlation analysis between seizure severity and mRNA expression levels in the neocor-
tex. Correlations between mRNA expression of Fos (A), Egr1 (B), Arc (C), Bdnf (D) and Ccl4 (E) and
seizure severity were analysed using Spearman’s rank correlation coeﬃcient.
of these genes was suppressed in the majority of brain regions examined from the CBDV
responder subgroup. Upregulation of Fos and Egr1 mRNA expression following PTZ
treatment has previously been reported in rat hippocampi (Saﬀen et al., 1988) and both Fos
and Egr1 are transcription factors belonging to IEG (immediate early gene) family which
is transiently and rapidly activated following a variety of cellular stimuli. IEGs can identify
activated neurons and brain circuits since seizure activity, and other excitatory stimuli, can
induce rapid and transient Fos expression increases (Herrera & Robertson, 1996), making
it a useful metabolic marker for brain activity (Dragunow & Faull, 1989). Fos expression
level in the brain is typically low under basal conditions and is induced in response to
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 10/18
Figure 4 Correlation analysis between seizure severity and mRNA expression levels in the prefrontal
cortex. Correlations between mRNA expression of Fos (A), Egr1 (B), Arc (C), Bdnf (D) and Ccl4 (E) and
seizure severity were analysed using Spearman’s rank correlation coeﬃcient.
extracellular signals such as ions, neurotransmitters, growth factors and drugs and is
closely linked to the induction of transcription of other genes (Kovacs, 2008). Fos induction
also correlates with the mossy ﬁbre sprouting (Kiessling & Gass, 1993;Popovici et al., 1990)
that occurs during epileptogenesis and may play a role in the subsequent manifestation of
seizure symptoms. Like Fos, Egr1 also activates transcription of other genes (Beckmann
et al., 1997;Christy & Nathans, 1989) and is considered to play an important role in
neuronal plasticity (Knapska & Kaczmarek, 2004). Furthermore, the expression of Fos
and Egr1 in seizure onset regions in PWE strongly correlates with interictal spiking
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 11/18
Figure 5 Subgroup-analysis of mRNA levels of epilepsy-related genes in CBDV responders and nonresponders. Subgrouping CBDV +PTZ
treated animals into responders (criterion: seizure severity ≤3.25) and non-responders (criterion: seizure severity >3.25) revealed that the
PTZ-induced increases of mRNA expression of Fos (A), Egr1 (B), Arc (C), Bdnf (D) and Ccl4 (E) were signiﬁcantly suppressed in brain regions
examined from the CBDV responder subgroup. mRNA levels are presented as a fold change vs mean level of vehicle +saline treated group (data are
expressed as mean ±s.e.m.). ∗,P<0.05 by t-test vs vehicle +PTZ group.
(Rakhade et al., 2007). Thus, suppression of Fos and Egr1 mRNA expression are consistent
with ameliorative drug eﬀects on seizures, epileptogenesis and/or epilepsy. In addition,
increased Arc mRNA expression in rat hippocampus (0.5–4 h) and cortex (0.5–1 h) after
PTZ treatment has also been reported (Link et al., 1995). It has been reported that newly
synthesised Arc mRNA is selectively localised in active dendritic segments and that Arc
plays a role in activity-dependent plasticity of dendrites (Lyford et al., 1995;Steward et al.,
1998). Arc is induced by hippocampal seizures, and glutamatergic neurons increase Arc
expression in response to increased synaptic activity (Korb & Finkbeiner, 2011), implying
a relationship between seizure activity and Arc expression. Ccl4 is a proinﬂammatory
chemokine that is known as a chemo-attractant for monocytes and T cells and has been
suggested to play a part in various nervous system pathologies such as inﬂammation,
trauma, ischemia and multiple sclerosis (Semple, Kossmann & Morganti-Kossmann, 2010).
Although a relationship between CCL4 and epilepsy is unclear, a relationship between
epilepsy and immune response has been suggested (Vezzani & Granata, 2005). Moreover,
increased Ccl4 mRNA expression has been reported in rat hippocampi and temporal
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 12/18
lobe tissue following status epilepticus events triggered by electrical stimulation of the
amygdala (Guzik-Kornacka et al., 2011). In the present study, PTZ-induced increase
of Ccl4 expression was suppressed in CBDV responders, although whether this is a
direct anti-inﬂammatory eﬀect of CBDV or an indirect eﬀect of reduced seizure severity
remains unknown. Increased expression of mRNA coding for Bdnf was conﬁrmed in
rat hippocampus after PTZ treatment (Nanda & Mack, 2000). BDNF is one of many
neurotrophic factors and is known to promote survival and growth of a variety of neurons
in addition to strengthening excitatory (glutamatergic) synapses (Binder & Scharfman,
2004). BDNF is involved in the control of hippocampal plasticity and is thought to play
an important role in epileptogenesis and in temporal lobe epilepsy (Binder et al., 2001;
Scharfman, 2002), suggesting therapeutic importance for control of Bdnf expression.
We have conﬁrmed upregulation of mRNA expression coding for Fos, Egr1, Arc, Ccl4
and Bdnf in the brains of rats treated with PTZ and shown that PTZ-induced increases of
mRNA expression for these genes were suppressed in CBDV responders, and not animals
that failed to respond to CBDV treatment. Overall, we provide molecular evidence that
directly supports behavioural evidence that CBDV exerts signiﬁcant anticonvulsant
eﬀects via oral and other routes of administration (Hill et al., 2012a). Whether gene
expression changes demonstrated here also underlie cellular and molecular mechanisms by
which CBDV exerts its anticonvulsant eﬀect presently remains unknown. However, these
results provide important acute biomarkers for additional investigation in models of the
progressive disorder and following longer term CBDV treatment.
We thank GW Pharmaceuticals Plc. for providing CBDV.
ADDITIONAL INFORMATION AND DECLARATIONS
This work was supported by GW Pharmaceuticals Plc. and Otsuka Pharmaceutical Co,
Ltd. The funders gave ﬁnal approval to submit the prepared manuscript to peer review for
The following grant information was disclosed by the authors:
GW Pharmaceuticals Plc.
Otsuka Pharmaceutical Co, Ltd.
GW Pharmaceuticals Plc. and Otsuka Pharmaceutical Co, Ltd. are collaborators on an
epilepsy research project. As an employee of Otsuka Pharmaceutical Co, Ltd., Naoki
Amada is participating in this collaborative project, and Naoki Amada’s PhD project
is funded by this research collaboration. GW Pharmaceuticals Plc. provided materials
Amada et al. (2013), PeerJ, DOI 10.7717/peerj.214 13/18
(CBDV). Yuki Yamasaki is an employee of Otsuka Pharmaceutical Co, Ltd., and holds
stocks in the company. Benjamin Whalley is an Academic Editor for PeerJ.
•Naoki Amada and Yuki Yamasaki conceived and designed the experiments, performed
the experiments, analyzed the data, wrote the paper.
•Claire M. Williams and Benjamin J. Whalley conceived and designed the experiments,
wrote the paper.
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
All work involving the use of animals was reviewed by the University of Reading
Local Ethical Review Panel in addition to being conducted under the authority of UK
Home Oﬃce Project Licence 30/2538 issued to Dr Benjamin Whalley under the Animals
(Sceintiﬁc Procedures) Act, 1986.
The following information was supplied regarding the deposition of related data:
CentAUR (Central Archive at the University of Reading)
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