Edited by: Kimmo Jensen, Aalborg University Hospital, Denmark
Reviewed by: Ove Wiborg, Aalborg University, Denmark; Marco Ledri, Lund University, Sweden
†These authors have contributed equally to this work
This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience
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Childhood absence epilepsy (CAE) is characterized by absence seizures, which are episodes of lack of consciousness accompanied by electrographic spike-wave discharges. About 60% of children and adolescents with absence seizures are affected by major neuropsychological comorbidities, including anxiety. Endocannabinoids and monoamines are likely involved in the pathophysiology of these CAE psychiatric comorbidities. Here, we show that the synthetic cannabinoid receptor type 1/2 (CB1/2R) agonist WIN 55,212-2 (2 mg/kg) has a strain-dependent effect on anxiety-like and motor behavior when assess in the hole board test and cerebral monoaminergic levels in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and their non-epileptic control (NEC) rat strain. Using quantitative and Temporal pattern (T-pattern) analyses, we found that WIN 55,212-2 did not affect the emotional status of GAERS, but it was anxiolytic in NEC. Conversely, WIN 55,212-2 had a sedative effect in GAERS but was ineffective in NEC. Moreover, vehicle-treated GAERS more motivated to explore by implementing more complex and articulated strategies. These behavioral changes correlate with the reduction of 5-HT in the hippocampus and substantia nigra (SN) and noradrenaline (NA) in the entopeduncular nucleus (EPN) in vehicle-treated GAERS compared to NEC rats, which could contribute to their low anxiety status and hypermotility, respectively. On the other hand, the increased level of NA in the EPN and 5-HT in the SN is consistent with an activation of the basal ganglia output-mediated motor suppression observed in WIN 55,212-2-treated GAERS rats. These data support the view of a strain-dependent alteration of the endocannabinoid system in absence epilepsy by adding evidence of a lower emotional responsiveness and a basal ganglia hypersensitivity to cannabinoids in GAERS compared to NEC rats.
Nearly 30% of people with convulsive epilepsy suffer from comorbid neuropsychiatric disorders, with the predominance of anxiety and depression (
Therefore, a major goal of translational research for children and teenagers with absence seizures (ASs) is to identify new treatments for their psychiatric comorbidities that do not exacerbate the seizure burden, thus, improving patients’ overall health and wellbeing. To achieve this goal, we need to further our understanding of the bidirectional link between anxiety and epilepsy (
To shed some light on CAE anxiety, we used the Genetic Absence Epilepsy Rats from Strasburg (GAERS), a very well-validated polygenic model of absence epilepsy presenting both recurrent generalized non-convulsive seizures and neuropsychiatric comorbidities (
To untangle CB1R-induced hypomotility (
The experimental approach of the
Graphical abstract summarizing the main aspects of the study. Adult male GAERS and NEC received an IP administration of 2 mg/kg of the CB1/2 receptor agonist WIN 55,212-2 and 30 min after were placed into the arena of the hole-board test to study the effect of the cannabinoid receptor activation on the neophilia and emotional behavior of the two inbred strains. We measured General Exploration, Focused Exploration, Grooming activity, and Immobility. We analyzed the data by quantitative and T-Pattern analysis. Different groups of animals received the same pharmacological treatment and after the 30 min from the injection their brains were removed and frozen for HPLC analysis of the monoamines dopamine (DA), noradrenaline and serotonin (5-HT) and some of their metabolites (3,4-Dihydroxyphenylacetic acid, DOPAC; 5-hydroxyindoleacetic acid, 5-HIAA). WIN 55,212-2 reduced motor behavior but did not affect emotionality of the epileptic GAERS rats and produced neurochemical changes in the hippocampus and basal ganglia.
Male GAERS and NEC rats (3–5 months old) were obtained from a colony bred at the University of Malta. Animals were housed in a 12:12 light cycle (lights on at 7:00 a.m. and off at 7:00 p.m.). All animal procedures were approved and carried out following University of Malta ethical guidelines and in conformity with Maltese and international laws and policies (EU Directive, 2010/63/EU for animal experiments, ARRIVE guidelines, and the Basel declaration including the 3R concept). All efforts were made to minimize animal suffering and to reduce the number of animals used.
The (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmetha- none mesylate, (R)-(+)-WIN 55,212-2, was purchased from Tocris Cookson Ltd. (Bristol, United Kingdom). WIN 55,212-2 was freshly dissolved in a vehicle solution (2 ml/kg) made of 5% PEG-400, 5% Tween 80 in saline, and was interpersonally (IP) administered.
The hole-board apparatus used in the present study consisted of a square (50 × 50 cm) arena made of white opaque Plexiglas with a raised floor, containing four equidistant holes 4 cm in diameter. Each hole’s center has a 10-cm distance from the two adjacent Plexiglas walls and all the four holes were equidistant. The floor of the apparatus was positioned 5 cm above a white opaque Plexiglas sub-floor. The square arena was surrounded by three white opaque Plexiglas walls (50 × 50 cm) and a front transparent wall (50 × 50 cm). A digital video camera (model: Toshiba HDDV P10) was placed in front of the transparent wall to record the behavior of each rat. The behavior of each subject was recorded on the cam’s SD card. At the end of each day’s recording sessions, video files from the SD card were transferred and stored on a personal computer for the following analyses.
On the day of the experiment, rats were allowed to acclimatize to the testing room for 30 min. Before the start of a new test or the use of a new subject, the apparatus was carefully cleaned with ethyl alcohol to remove possible scent cues left by the preceding animal. Thirty mins before the beginning of the test, WIN 55,212-2 (2 mg/kg) or its vehicle were IP-injected. Each subject, naïve to the test, was placed in the central area of the apparatus, was allowed to explore for 10 min and its behavior was recorded using a digital camera.
A list of all the components of the behavioral repertoire and their formal description is shown in
Concerning mean occurrences and mean durations, possible significant results were assessed using Two-Way ANOVA (strain x treatment) for independent samples, followed by Fisher’s Least Significant Difference (LSD)
We followed the previously described methodology (
The tissue concentrations of monoamines were measured by HPLC, coupled to a coulometric detection system (
The calibration curves were adapted according to the brain areas investigated, because the quantities of monoamines and their corresponding metabolites are heterogeneous, requiring different gains set at the level of the detector using a timeline method. NA, 5-HT, and 5-HIAA contents were observed in all sampled regions. The overall sensitivity for the compounds ranged from 3 pg/10 μl for DA to 13 pg/10 μl for HVA with a signal/noise ratio of 3:1.
The tissue levels of each molecule in the 14 brain structures were expressed in pg/mg of tissue. The ratio of DOPAC/DA and 5-HIAA/5-HT were also calculated whenever it was possible. The data are presented as the mean ± standard error of the mean (SEM).
Outlier data were discarded if they were larger than two standard deviations (SD). We addressed whether vehicle-treated NEC and GAERS had different levels of monoamines and whether the treatment of WIN 55,212-2 had distinct effects on GAERS vs. on NEC by performing a two-way ANOVA (strain x treatment). This was systematically associated with a one-way ANOVA using the group as the main factor, which was followed by the Fisher’s PLSD
Thirty NEC and thirty GAERS rats were randomly assigned to 4 groups: NEC treated with vehicle
With regard to mean occurrence of different behaviors (
Effect of WIN 55,212-2 and its vehicle on the occurrence of different behaviors of Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg,
The WIN 55,212-2 significantly decreased the occurrence of Walking (
As to the mean duration of the behavioral events (
Effect of WIN 55,212-2 and its vehicle on the duration of different behaviors of GAERS and NEC rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg, IP) and its vehicle affected the mean duration ± SEM of several behaviors of GAERS and NEC rats detected during the 10 min of the hole-board test. Mean durations (sec) ± SEM of all the behavioral components of the behavioral repertoire. § = significant (
Results of T-pattern detection in terms of terminal strings, length, and overall occurrences are presented in
Effect of WIN 55,212-2 and its vehicle on the temporal patterns (T-patterns) in GAERS and NEC rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg,
Effect of WIN 55,212-2 and its vehicle on the number of T-patterns of different lengths in GAERS and NEC rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg, IP) and its vehicle affected the overall number of different T-patterns detected based on their length for each group during the 10 min of the hole-board test. Filled bars = number of patterns detected in the real data; white bars = average number + 1xSD of patterns detected in randomized data.
The mean length of T-patterns and their mean occurrence are illustrated in
Effect of WIN 55,212-2 and its vehicle on the length and occurrences of T-patterns in GAERS and NEC rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg, IP) and its vehicle affected the mean length ± SEM
Finally, the percent distributions of T-patterns encompassing behavioral components of hole-exploration predictive of the animal anxiety level (
Effect of WIN 55,212-2 and its vehicle on the percent distribution of T-patterns containing Head-Dip and Edge-Sniff in GAERS and NEC rats. Administration of WIN 55,212-2 (WIN; 2 mg/kg, IP) and its vehicle affected the percent distribution of T-patterns containing Head-Dip
Compelling evidence shows that ECS interaction with monoaminergic systems is strongly involved in many psychiatric disorders including anxiety (
The analysis revealed both strain and drug effects, which are selective for each monoamine.
A significant strain x treatment interaction on NA tissue levels was observed only in the EPN (
Effect of WIN 55,212-2 and its vehicle on Noradrenaline (NA) levels in selected brain areas of Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) rats.
NA | Vehicle |
WIN 55,212-2 |
ANOVA | ||
NEC | GAERS | NEC | GAERS | ||
M1 | 177 ± 26 | 157 ± 11 | 148 ± 18 | 229 ± 54 | |
S1 | 208 ± 28 | 175 ± 9 | 206 ± 33 | 156 ± 6 | |
V1 | 143 ± 17 | 192 ± 6 | 216 ± 40 | 247 ± 38 | |
AD | 133 ± 67 | 177 ± 38 | 199 ± 54 | 301 ± 77 | |
dLGN | 198 ± 25 | 223 ± 51 | 138 ± 12 | 147 ± 29 | |
NRT | 141 ± 38 | 259 ± 59 | 257 ± 51 | 280 ± 31 | |
Po | 375 ± 128 | 324 ± 52 | 320 ± 43 | 387 ± 54 | |
VB | 136 ± 16 | 150 ± 15 | 131 ± 27 | 114 ± 24 | |
NAc (core) | 102 ± 11 | 88 ± 15 | 112 ± 41 | 200 ± 49 | |
mSTR | 61 ± 11 | 60 ± 19 | 58 ± 18 | 69 ± 19 | |
lSTR | 72 ± 4 | 57 ± 4 | 62 ± 8 | 66 ± 5 | |
EPN | 301 ± 15 | 147 ± 16 |
230 ± 58 | 293 ± 34 |
|
SN | 154 ± 26 | 126 ± 30 | 259 ± 47 | 224 ± 34 | |
dH | 256 ± 20 | 164 ± 36 | 212 ± 30 | 192 ± 27 |
A significant strain x treatment interaction on DA tissue content was observed in the dLGN only (
Effect of WIN 55,212-2 and its vehicle on Dopamine (DA) tissue content and the ratio DOPAC/DA in selected brain regions of GAERS and NEC rats.
A. DA | Vehicle |
WIN 55,212-2 |
ANOVA | ||
NEC | GAERS | NEC | GAERS | ||
M1 | 10 ± 2 | 11 ± 5 | 16 ± 5 | 66 ± 34 | |
S1 (PSC) | 14 ± 3 | 20 ± 7 | 16 ± 4 | 16 ± 2 | |
V1 | 9 ± 1.5 | 16 ± 1.7 | 9 ± 1.6 | 18 ± 3.1 | |
AD | 22 ± 5.5 | 22 ± 3 | 31 ± 3.7 | 32 ± 6.8 | |
dLGN | 34 ± 7.5 | 68 ± 17 |
55 ± 9.5 | 36 ± 6.5 |
|
Rt | 56 ± 12 | 33 ± 7 | 34 ± 8 | 23 ± 2 | |
Po | 82 ± 19 | 59 ± 16 | 74 ± 12 | 37 ± 16 | |
VB | 42 ± 7 | 23 ± 2 | 29 ± 9 | 34 ± 13 | |
NAc (core) | 2834 ± 566 | 2684 ± 473 | 2105 ± 203 | 2939 ± 728 | |
Medial STR | 2268 ± 446 | 2775 ± 647 | 2678 ± 388 | 4261 ± 1153 | |
Lateral STR | 5025 ± 414 | 4959 ± 1042 | 4763 ± 1021 | 5534 ± 935 | |
EPN | 100 ± 29 | 100 ± 17 | 144 ± 41 | 118 ± 29 | |
SN | 280 ± 91 | 243 ± 81 | 354 ± 44 | 384 ± 64 | |
dH | 4.1 ± 1.6 | 3.3 ± 0.9 | 2.2 ± 0.3 | 2.2 ± 0.8 | |
M1 | |||||
S1 | 0.64 ± 0.08 | 1.2 ± 0.47 | 0.73 ± 0.18 | 1.06 ± 0.28 | |
V1 | 0.76 ± 0.23 | 0.94 ± 0.13 | 1.28 ± 0.54 | 0.63 ± 0.18 | |
AD | 1.08 ± 0.5 | 0.58 ± 0.15 | 0.86 ± 0.24 | 0.36 ± 0.06 | |
dLGN | 0.91 ± 0.17 | 0.89 ± 0.22 | 0.38 ± 0.05 | 0.96 ± 0.09 | |
NRT | 0.41 ± 0.16 | 1.03 ± 0.44 | 1.12 ± 0.15 | 0.83 ± 0.18 | |
Po | |||||
VB | 0.35 ± 0.13 | 0.32 ± 0.08 | 0.41 ± 0.23 | 0.15 ± 0.04 | |
NAc (core) | 0.39 ± 0.02 | 0.56 ± 0.05 |
0.45 ± 0.05 | 0.4 ± 0.03 |
|
mSTR | 0.2 ± 0.02 | 0.26 ± 0.03 | 0.2 ± 0.02 | 0.26 ± 0.07 | |
lSTR | 0.28 ± 0.06 | 0.22 ± 0.03 | 0.22 ± 0.01 | 0.21 ± 0.01 | |
EPN | 0.34 ± 0.08 | 0.27 ± 0.06 | 0.33 ± 0.09 | 0.49 ± 0.08 | |
SN | 1.37 ± 0.17 | 1.42 ± 0.17 | 1.33 ± 0.14 | 1.3 ± 0.25 | |
dH |
The effect of treatments on 5-HT levels is reported in
Effect of WIN 55,212-2 and its vehicle on 5-HT tissue content and the ratio 5-HIAA/5-HT in selected brain regions of GAERS and NEC rats.
A. 5-HT | Vehicle |
WIN 55,212-2 |
ANOVA | ||
NEC | GAERS | NEC | GAERS | ||
M1 | 214 ± 60 | 95 ± 12 |
99 ± 11 | 215 ± 54 |
|
S1 (PSC) | 80 ± 14 | 55 ± 6 | 80 ± 11 | 66 ± 11 | |
V1 | 46 ± 12 | 62 ± 11 | 71 ± 17 | 63 ± 15 | |
AD | 76 ± 14 | 75 ± 11 | 82 ± 12 | 144 ± 34 | |
dLGN | 197 ± 41 | 180 ± 48 | 125 ± 10 | 134 ± 19 | |
NRT | 114 ± 31 | 77 ± 24 | 142 ± 39 | 150 ± 31 | |
Po | 278 ± 71 | 282 ± 53 | 284 ± 39 | 449 ± 135 | |
VB | 66 ± 16 | 103 ± 17 | 74 ± 39 | 115 ± 44 | |
NAc (core) | 363 ± 60 | 282 ± 47 | 328 ± 78 | 369 ± 60 | |
Medial STR | 244 ± 57 | 226 ± 58 | 166 ± 34 | 180 ± 72 | |
Lateral STR | 249 ± 19 | 207 ± 47 | 211 ± 41 | 276 ± 57 | |
EPN | 203 ± 21 | 200 ± 37 | 185 ± 35 | 240 ± 20 | |
SN | 966 ± 162 | 477 ± 125 |
1089 ± 112 | 1429 ± 267### | |
dHP | 170 ± 10 | 105 ± 13 |
117 ± 15 | 165 ± 35 | |
M1 | 1.67 ± 0.31 | 2.01 ± 0.27 | 1.74 ± 0.13 | 2.39 ± 0.28 | |
S1 (PSC) | 2.32 ± 0.35 | 2.29 ± 0.41 | 1.81 ± 0.28 | 2.06 ± 0.31 | |
V1 | 2.16 ± 0.25 | 1.79 ± 0.11 | 2.13 ± 0.24 | 3.37 ± 0.37### | |
AD | 3.26 ± 0.42 | 4.01 ± 0.63 | 2.25 ± 0.23 | 2.77 ± 0.46 | |
dLGN | 1.54 ± 0.36 | 1.07 ± 0.17 | 1.16 ± 0.26 | 0.88 ± 0.13 | |
NRT | 1.24 ± 0.38 | 2.08 ± 0.39 | 1.34 ± 0.2 | 1.4 ± 0.23 | |
Po | 1.15 ± 0.44 | 1.54 ± 0.51 | 0.62 ± 0.14 | 1.32 ± 0.41 | |
VB | 2.61 ± 0.62 | 1.84 ± 0.33 | 2.09 ± 0.29 | 1.75 ± 0.34 | |
NAc (core) | 0.9 ± 0.09 | 1.18 ± 0.14 | 1.48 ± 0.5 | 1.44 ± 0.32 | |
Medial STR | 0.86 ± 0.08 | 1.17 ± 0.11 | 1.04 ± 0.17 | 1.65 ± 0.23 | |
Lateral STR | 1.18 ± 0.17 | 1.53 ± 0.26 | 1.14 ± 0.17 | 1.26 ± 0.19 | |
EPN | 1.45 ± 0.26 | 1.72 ± 0.32 | 2.02 ± 0.22 | 1.95 ± 0.24 | |
SN | 0.64 ± 0.06 | 0.72 ± 0.09 | 0.62 ± 0.05 | 0.63 ± 0.08 | |
dHP | 3.26 ± 0.29 | 3.12 ± 0.33 | 3.2 ± 0.42 | 3.11 ± 0.51 |
In the present study, we show that vehicle-treated GAERS were more immobile and less anxious and had lower 5-HT levels in the cortex, dHP, and SN compared to vehicle-injected NEC. Moreover, acute systemic administration of 2 mg/kg of the CB1/2R agonist WIN 55,212-2 (
Mixed results have been reported on the anxiety levels of naïve GAERS rats from the original colony in Strasburg showing GAERS being less anxious than Wistars on the open field tests (OFT) (
This is the first time that GAERS are reported to be less anxious than NEC, but we need to underline that our animals were treated with the vehicle and were not untouched like those in previous studies (
In terms of monoamine levels in brain areas involved in the generation of SWDs, such as the thalamus (
Our findings in the basal ganglia are also intriguing. Basal ganglia play a pivotal role in ASs, classically considered modulatory (
In summary, the neurochemical changes that we found in GAERS and NEC rats, are similar to those described in WAG/Rij (
The WIN 55,212-2 caused strain-dependent effects for most of the behaviors examined in the hole-board test by standard quantitative analysis, with an increase and a reduction of the anxiety-like behavior in GAERS and NEC, respectively. Notably, the pan-T-type calcium channel antagonist Z944 was shown to reduce anxiety-like behavior in NEC and increased it in GAERS rats (
Here, we observed that GAERS rats showed a neophobic response and were much less prone to explore (i.e., reduction of both general and focused exploration), or self-groom and they were more immobile after administration of WIN 55,212-2, in comparison to NEC. On the other hand, WIN 55,212-2 administration failed to alter the duration of either general exploration (apart from reducing rearing occurrences) focused hole exploration, or grooming/licking activity, and instead reduced immobility frequency (but not its duration) in NEC. While the effect of WIN 55,212-2 on motor behavior is quite clear from the quantitative analysis, with clear sedation in GAERS and no effect in NEC, a more cautious interpretation of its effect on the anxiety status is necessary. Indeed, the decrease in Head-Dipping and edge-sniffing behaviors because of WIN 55,212-2 might have been confounded by the strong sedative effect of CB1R activation in GAERS. Indeed, it has been reported that Head-Dipping and locomotion are highly correlated (
Because of the strength of the multivariate T-pattern analysis (
The ECS plays a complex role in the regulation of emotional states and involves interaction with monoamines (
The effects induced by the activation of CB1Rs by WIN 55,212-2 on the anxiety status of the animals are likely to involve monoamines, such as NA, DA, and 5-HT (
Interestingly, we found that WIN 55,212-2 also reduced locomotor activity in a strain-dependent fashion, in agreement with previous observations in Wistar and Fischer 344 rats (
In conclusion, the higher level of anxiety observed here in vehicle-treated NEC compared to GAERS rats may be due to a maladaptive response to stress, a critical issue that deserves further investigation. Moreover, we revealed strain-dependent responsiveness to exogenous cannabinoid receptor activation consisting of changes in anxiety-like and motor behaviors, as well as in monoamine levels in a specific mood and motor brain areas, consistent with a sedative and anxiolytic effect in GAERS and NEC rats, respectively.
Furthermore, we show the importance of performing multivariate analysis of behavior
Finally, our data add important information to our current understanding of the mechanisms underlying anxiety-related traits in the GAERS model of CAE and their NEC control rats and the ECS abnormalities that are present in these two strains.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by the University Research and Ethics Committee (UREC) and Faculty Research Ethics Committee (FREC) of the University of Malta.
GD conceived the study. PD, MC, and GD designed the methodology, performed data analysis, and wrote the manuscript. MR, DC, EP, AC, and PD conducted laboratory-based research and performed data analysis. MC, PD, and AC prepared tables and figures. GD, PD, MC, AC, GC, and VC reviewed and edited the manuscript. All authors have agreed to this manuscript submission for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Our research was supported by the Project
The assistance provided by Dr. Massimo Pierucci was greatly appreciated.
The Supplementary Material for this article can be found online at:
anterodorsal nucleus of the thalamus
dopamine
dorsal part of the hippocampus
3,4-dihydroxyphenylacetic acid
entopeduncular nucleus
high-pressure liquid chromatography
homovanillic acid
dorsal lateral geniculate nucleus
lateral and medial striatum
motor cortex
noradrenaline
nucleus accumbens
nucleus reticularis thalami
posterior thalamic nucleus
5-hydroxy indole-3-acetic acid
5-hydroxytryptamine, serotonin
somatosensorial cortex
substantia nigra
substantia nigra par compacta
substantia nigra pars reticulata
ventrobasal complex of the thalamus
the visual cortex.