Cannabinoid Receptor Activation Reverses Kainate-Induced Synchronized Population Burst Firing in Rat Hippocampus

Cannabinoids have been shown to possess anticonvulsant properties in whole animal models of epilepsy. The present investigation sought to examine the effects of cannabinoid receptor activation on kainic acid (KA)-induced epileptiform neuronal excitability. Under urethane anesthesia, acute KA treatment (10 mg kg−1, i.p.) entrained the spiking mode of simultaneously recorded neurons from random firing to synchronous bursting (% change in burst rate). Injection of the high-affinity cannabinoid agonist (-)-11-hydroxy-8-tetrahydrocannabinol-dimethyl-heptyl (HU210, 100 μg kg−1, i.p.) following KA markedly reduced the burst frequency (% decrease in burst frequency) and reversed synchronized firing patterns back to baseline levels. Pre-treatment with the central cannabinoid receptor (CB1) antagonist N-piperidino-5-(4-clorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant, SR141716A 3 mg kg−1, i.p.) completely prevented the actions of HU210. The present results indicate that cannabinoids exert their antiepileptic effects by impeding pathological synchronization of neuronal networks in the hippocampus.


INTRODUCTION
The appearance of epileptic seizures is commonly associated with aberrant hypersynchronization of large neuronal aggregates (Walker and Kullmann, 1999). One of the most commonly affected brain areas is the hippocampus, where several mechanisms underlying pathological synchronization have been identifi ed (Miles and Wong, 1983). Such mechanisms include alterations in gap junctional signaling (Jefferys, 1995), imbalances of excitatory/inhibitory currents (Cohen et al., 2002) and an enhancement of inhibitory GABAergic feedback (Haas et al., 1996). In the present study, hippocampal epileptiform activity was induced pharmacologically with kainic acid (KA). This excitotoxin produces an episode of status epilepticus (Sokal and Large, 2001) and complex epileptiform EEG patterns (Ben-Ari, 1985;Medvedev and Willoughby, 1999) when administered acutely in vivo.
Available evidence from human and animal seizure models suggests that various cannabinoid compounds (derived from the marijuana plant, Cannabis sativa) have antiepileptic properties (Cunha et al., 1980;Wallace et al., 2002). Furthermore, anandamide has been shown to modulate seizure threshold and severity through CB1 receptors implicating the endogenous cannabinoid system in epilepsy (Karanian et al., 2007). An earlier study by Hàjos et al. (2000) provided a mechanistic explanation for the effects of the endogenous cannabinoid receptor ligand anandamide. Using hippocampal slices, the authors showed that bath application of cannabinoid receptor agonists reduce the power of KA-induced network oscillations, these actions were prevented by pre-treatment with the CB1 receptor antagonist rimonabant.
Population studies provide valuable information in terms of temporal associations among recorded neurons and of specifi c features of single-neuronal fi ring. As functional synaptic correlations and Watson (1986). The temperature of the animal was maintained at 37.7°C with the aid of a rectal probe connected to a homeothermic control unit (Harvard Apparatus, MA, USA). The second level of amplifi cation occurred at a 16-channel differential preamplifi er (fi xed gain ×100; Plexon Inc, TX, USA) after which the signal was transferred to a Multi-Channel Acquisition Processor (Plexon Inc, TX, USA). This allowed for computer-controlled amplifi cation, fi ltering, switching and digital signal processing of the signals originating from the microwires. The impedance of the electrodes was ∼300 kΩ measured at 1 kHz. Commercially available software (RASPUTIN; Plexon Inc, TX, USA) allowed on-line isolation and discrimination (using dual voltage-time widows or principal component analysis) of neuronal spikes from background noise. Typically, one to four action potential waveforms were observed and discriminated per microwire during any given experiment. Activity was also displayed on a Tektronix D11 5000 series dualbeam oscilloscope and corresponding action potential waveforms were isolated with the aid of a Gould 1425 Digital Oscilloscope and a Gould Type 125 waveform processor. Firing rate was also monitored aurally with the aid of a loudspeaker. Signals were fi ltered using a second order low cut-off (500 Hz) and high cut-off (5 kHz), with a 50-Hz notch fi lter. The gain block was a programmable gain multiplier with gain steps 1-30 (total gain: ×1000 to ×32,000); typically the gain was set at ×10,000 for the experiments.

PHARMACOLOGICAL MANIPULATIONS
HU210 was obtained from Tocris Cookson (UK, USA), KA was obtained from Sigma Chemical (UK, USA) and rimonabant (SR141716A) was a generous gift from Sanofi -Synthelabo. HU210 and rimonabant were resuspended to stock concentration (10 −2 M) in ethanol and refrigerated at −20°C. Drugs were dissolved in a physiological 1.0-ml saline (0.9% NaCl)/10-µl Tween 80 (1%) vehicle solution for injection on the day of the experiment. KA was resuspended to stock concentration (10 −2 M) in distilled water and frozen (−20°C) in 200-µl aliquots. When required, the aliquots were thawed and diluted in 0.2-ml saline and for each experiment an aliquot was thawed and diluted in 0.2-ml saline to the desired dose. All drugs were injected intraperitoneally (i.p.).

ELECTROPHYSIOLOGICAL ANALYSES
Discriminated single-unit data was initially visualized off-line as spike rasters; the spike raster representing action potential spikes, viewed as vertical ticks, corresponding to the temporal (time stamped) occurrence of the spikes. Data was subsequently analyzed as integrated fi ring rate histograms (accumulated over 5 or 10 s epochs); burst analyses were calculated for epochs of 15 s during periods of bursting activity and cross-correlation histograms (CCHs; Gerstein, 1970), between pairs of hippocampal neurons, were computed using NeuroExplorer (Plexon Inc, TX, USA). Basal activity was calculated as the mean ± SEM of fi ring during a selected 10-min pre-drug period for overall fi ring analyses and over 15 s for burst analyses. Visual evaluation of bursting discharge activity, induced by KA injection, in the spike raster displays revealed two types of neuronal discharge -"micro-bursts" and "macrobursts". Micro-bursts were empirically defi ned as follows: maximum burst start interval of 170 ms, maximum interspike interval within the burst 300 ms; minimum inter-burst interval 200 ms; minimum duration for a burst 100 ms and minimum number of spikes within a burst, minimum of three spikes. Macro-bursts were much slower oscillations in overall fi ring (clearly visible over longer time frames -see Figure 3A) with a periodicity of 180-220 s. CCH analysis ( Figure 1B) shows the conditional probability of the occurrence of a spike in the spike train at time t on the condition that there is a reference event (or reference spike; in this case spikes from neuron 1) at time 0 (Gerstein, 1970). Therefore, if the graph shows a fl at profi le, neurons are not fi ring synchronously with respect to a given reference neuron. Correlated discharge activity (i.e., functional fi ring synchronicity) between pairs of neurons were revealed in CCHs as a central peak (or trough) centered on the zero time bin, while a fl at cross-correlation indicated non-correlated discharge activity between neuronal pairs (Eblen-Zajjur and Sandkuhler, 1997). Conventional analyses of variance (ANOVA) were used to study treatment-response differences. The Scheffé's post-hoc test was used to assess differences between and within treatments. The criterion of signifi cance for the ANOVAs and post-hoc tests was set at p < 0.05. All data are expressed mean ± SEM and statistical analyses were computed using Statistica (Statsoft Inc, OK, USA).
On completion of experiments, the location of the recording electrode tips was marked by passing a 60-µA current for 30 s through electrode pairs in the array. Animals were then transcardially perfused with a 4% paraformaldehyde/5% potassium ferrocyanide solution. Ferrocyanide creates a blue-green stain by reacting with deposited iron ions at the mark site. The brains were then removed and stored at 4°C in the same fi xative solution overnight. Vibratome sections were then cut (80 µm) and transferred to slides with the aid of mounting medium (Vectashield). Photographic images were made using a low power objective without further counter-staining of the sections.

RESULTS
A total of 58 neurons (n = 6 rats) were recorded showing action potential signal-to-noise amplitude ratios of at least a 3:1. KA (10 mg kg −1 ) produced a signifi cant increase in the number of bursts over pre-drug activity in 46 of 58 units from 4.1 ± 1.06 to 10.5 ± 1.18 bursts (Figures 1A and 2; F (1,57) = 15.74, p < 0.0008). Every neuron studied was signifi cantly (p < 0.05) excited from predrug basal fi ring following KA injection (Figure 3; 6.89 ± 1.18 to 13.05 ± 2.04 Hz, for the entire population). The onset latency for KA observable effects was ∼6 min after injection.
Inspection of fi ring rate histograms showed that KA treatment elicited two types of bursts: micro-bursts (as defi ned in "Materials and methods") and macro-bursts that lasted ∼202 ± 21.2 s in duration ( Figure 3A). The number of KA-induced micro-burst events was signifi cantly reduced by the high-affi nity cannabinoid agonist HU210 from 10.5 ± 1.18 to 3.55 ± 2.07 bursts during each analysis period (Figures 1 and 2; F (1,57) = 8.89, p < 0.05). The KA-induced elevation in fi ring rate was only marginally reduced by concurrent administration of HU210 (100 µg kg −1 ), decreasing the fi ring rate of simultaneously recorded neurons from 13.05 ± 2.04 to 10.74 ± 1.56 Hz on this short time scale (F (2,85) = 1.1, p < 0.38, data not shown). The frequency of macro-bursts was similarly inhibited (Figure 3) by HU210 from 3.7 ± 1.1 to 1.1 ± 0.3 bursts (F (1,57) = 3.2, p < 0.05, data not shown); however on this longer time scale, overall fi ring rate was signifi cantly reduced 11.15 ± 3.04 to 4.84 ± 2.26 (F (2,85) = 8.4, p < 0.05). Cross-correlation histogram analyses confi rmed the visual inspection of the raster displays that there is limited or no synchronous activity between hippocampal neurons during pre-drug basal activity, at least under the recording conditions of the present experiments ( Figure 1B). Following KA injection, corresponding with the period of KA-induced micro-burst and macro-burst activity, there was development of synchronous activity, revealed by peaks in the CCHs around time bin 0 ( Figure 1B). This was reversed by injection of HU210, resulting in CCHs showing a fl at profi le similar to that expressed under basal conditions (Figure 1B), indicative of non-synchronous activity.

DISCUSSION
The present in vivo multiple single-unit study has generated two main fi ndings. First, functionally cross-correlated epileptiform-like population activity can be evoked by the non-NMDA receptor agonist KA in the hippocampal CA1 pyramidal layer of urethane-anesthetized rats, in a way reminiscent of epileptiformlike activity observed in hippocampal dissociated and organotypic cultures (Jagger et al., 2001(Jagger et al., , 2002Roe and Mason, 2002;Sokal et al., 2000). The excitotoxin injection increased the fi ring rate of all the recorded neurons and also increased the burst frequency in the majority of simultaneously recorded cells whilst entraining them to a synchronized fi ring mode (no KA-induced inhibitions of fi ring were observed). Secondly, when injected in the presence of KA, the potent CB1 receptor agonist HU210 signifi cantly reduced the elevation in burst rate (accompanied by a modest reduction in overall fi ring rate) elicited by KA treatment accompanied by a reversal of synchronized burst fi ring to random activity.
The hippocampal formation is one of the most seizure-prone structures in the brain due to its defi ning characteristic; the presence of a tri-synaptic circuit of fi bers. The reverberation of impulse activity in the hippocampal loop is suggested to cause and maintain epileptiform-like activity (Stringer and Lothman, 1992). Hippocampal cells are endowed with several voltage and ligand-gated conductances, which play a major role in the cells' excitability. Even under normal basal conditions cells produce burst discharges that are qualitatively similar to epileptiform-like activity. Blockade of GABAergic transmission has been consistently proven to precipitate seizures. It has thus been assumed that abnormal GABAergic neurotransmission may be related to epilepsy (Prince, 1978). Accordingly, the most common pharmacological manipulation to cause neuronal disinhibition is the use of the convulsant alkaloid bicuculline; a GABA A receptor antagonist (Margineanu and Wülfert, 1999). However, in vivo epilepsy can also be induced with exposure to toxins or direct brain electrical stimulation, in a manner that may not be directly related to GABAergic mechanisms. One such agent is the excitotoxin KA, an AMPA/kainate receptor agonist. KA-induced seizures are a well-established model of temporal FIGURE 2 | Incidence of mean burst rate (bursts per analysis epoch ± SEM) as a function of drug treatment. KA signifi cantly increased the burst frequency from pre-drug basal activity and entrained the network to burst synchronously. Co-application of the CB1 receptor agonist HU210 (HU) signifi cantly decreased burst rate and disrupted the synchronicity in fi ring induced by KA. When injected in the presence of the CB1 receptor antagonist rimonabant (SR), HU210 did not alter KA-induced synchronous bursting activity (**p < 0.01; ***p < 0.005 vs baseline and && p < 0.01 vs KA-induced bursting).

Mason and Cheer
Cannabinoids decrease epileptiform activity lobe epilepsy and epileptic seizures reliably occur at the chosen systemic dose of 10 mg kg −1 used in this study, a non-toxic dose (Sperk, 1994). Interestingly, Khazipov and Holmes (2003) elegantly implicated GABA inhibitory mechanisms in the emergence of synchronized epileptiform-like activity elicited by KA. In their in vivo superfused hippocampus preparation, GABA A receptor-mediated currents coincided with the inhibition of neuronal fi ring as assessed by the cross-correlogram of spikes vs inhibitory postsynaptic currents. The authors found that when inhibitory currents ceased, the probability of fi ring of pyramidal cells increased. Mediation of GABA A receptors was further confi rmed by the fi nding that bicuculline perfusion suppressed KA-induced oscillations.
The hippocampus is one of the areas with the highest cannabinoid receptor binding densities in the rat brain (Herkenham et al., 1991). This structure also exhibits the highest concentrations of both anandamide (Felder et al., 1996) and 2-arachidonylglycerol (Stella et al., 1997), the two most abundant endogenous cannabinoid ligands. It is now well accepted that presynaptic CB1 receptors are located on the terminals of cholecystokinin (CCK+)containing GABAergic interneurons to inhibit Ca 2+ dependent GABA release via N-type channels (for review see Freund et al., 2003). Cannabinoid receptors are also reported to be situated on glutamatergic presynaptic terminals inhibiting glutamate release via terminal N-type and P/Q type Ca 2+ channels (Twitchell et al., 1997) in autaptic circuits as well as in hippocampal slices (Kawamura et al., 2006). Thus, at least in the hippocampus, CB1 receptors are located on (CCK+) basket and dendritically projecting cells (Freund, 2003;Hàjos et al., 2000) as well as on excitatory afferents to pyramidal cells (Takahashi and Castillo, 2006). We are therefore left with the view that mixed actions of HU210 on inhibitory and excitatory neurotransmission are responsible for reversing synchronized burst fi ring. Since intact GABAergic inhibition is necessary for the maintenance of KA-induced bursting, the activation of CB1 receptors by HU210 on the terminals of different populations of GABAergic neurons in the hippocampus and the corresponding inhibition of GABA A synaptic inhibition (Hoffman and Lupica, 2000) could be suffi cient to shunt synchronous population bursting. However, direct effects of CB1 agonists in suppressing glutamatergic excitatory neurotransmission have been observed in a KA model of seizure (Monory et al., 2006). This suggests CB1 receptors are also critically involved in the control of excitatory glutamatergic neurotransmission. Thus, activation of CB1 receptors may therefore confer inhibitory effects via suppression of excitatory synapses under hyperexcitable conditions. Nevertheless, because of the systemic nature of the dosing regimen employed in our study, contributions from outside the hippocampal circuitry cannot be ruled out (microinjection studies are currently ongoing in our laboratory to address this issue).
The fi nding that the CB1 receptor antagonist rimonabant was devoid of effect on KA-induced synchronized bursting is puzzling (but see Robbe et al., 2006) since the present KA manipulations represent the ideal conditions for the on-demand release of endocannabinoids hippocampal slices. This may indicate that the basal level of endocannabinoid binding to CB1 receptors is not suffi cient to effectively modulate epileptiform-like activity in anesthetized animals by virtue of achieving a ceiling effect wit the dose of KA used here. It would be interesting to determine whether an effect of rimonabant would be uncovered when recording are performed in awake, freely-moving rats. It would also be important to determine whether injection of rimonabant prevents the induction of epileptiform-like activity by KA, since it does not reverse KA effects on its own. Nevertheless, the present study replicates and builds upon previous fi ndings that KA-induced increases in fi ring and burst fi ring rates are attenuated by inhibition of anandamide (an endocannabinoid) hydrolysis (Coomber et al., 2008). However, under those conditions rimonabant only partly diminished the effects of anandamide hydrolysis blockade (in that particular study functional connectivity was not assessed). The appearance of "macro-bursts" composed of "micro-bursts" in the present report could be associated with metabolic recovery following intense increases in fi ring rate. Alternatively, the extensive calcium infl ux during periods of sustained elevated activity may sequentially trigger downregulation of inhibitory and/or excitatory processing (see Hàjos and Freund, 2002) and the generation of the subsequent volley of intense fi ring.
These previously unseen fi ndings suggest that the reduction in spike timing coordination and the associated decrease in hippocampal population activity are important in the well-documented antiepileptic actions of cannabinoid compounds (Smith, 2005). A major implication of these data is that the synchrony of spike timing in neuronal assemblies is a critical component of epileptiform-like activity in vivo. The effect of cannabinoids on network dynamics is noteworthy because neurons form assemblies defi ned by synchronous action potential fi ring and these changes in population synchronicity are thought to accompany status epilepticus .
In conclusion we have shown that cannabinoid receptors modulate the rhythmicity and synchronization generated by inhibitory neuronal networks in the rat hippocampus. Further investigations are required to determine the clinical relevance and the site of action of the cannabinoid agonist, since drugs that reduce synchronization may represent novel anticonvulsants.