Institute
of Theoretical and Experimental Biophysics Russian Academy of Sciences,
Pushchino, Moscow District, 142290 Russia.
Endocannabinoid
(eCB) system is a part of the complex group of natural auto-regulatory
processes. This system includes cannabinoid G protein-coupled CB1 and CB2
receptors, their endogenous ligands (eCBs) and the relevant enzymes
participating in their synthesis, transport and degradation. eCB synthesis in neurons from membrane
predecessors is carried out “on demand” depending on the current brain activity
and they emitted in specific local networks only. Thus, two known eCBs,
anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are greatly elevated in
response to a variety of pathological events in brain structures involved in
these events. It is known that eCBs mostly act as retrograde messengers and,
upon release from postsynaptic neurons, they modulate neurotransmitter release
by activating presynaptic cannabinoid receptors (for review,
see Freund et al., 2003; Degroot
and Nomikos, 2007; Kano, 2014). Thus, through the CB1 receptors
the endogenous and exogenous cannabinoids can regulate neuronal excitability
and prevent excitotoxicity and seizure activity (Katona and Freund,
2008; Khaspekov et al., 2004). It should be noted that seizure activity creates
ideal conditions for eCB synthesis. It was shown that genetical deletion of CB1 receptors in principal forebrain neurons increased seizure susceptibility and vulnerability of neurons during excitotoxicity (Marsicano et al., 2003; Monory et al., 2006). In the same way, pharmacological blockade of CB1 receptors was found to
facilitate seizure activity in certain animal models of epilepsy (Deshpande et al., 2007; van Rijn et al., 2011; Wallace et al., 2002). About two thirds of TLE patients are resistant to the existing therapy.
Therefore it is obvious that new approaches are required to treatment this
disease. One of the perspective disease-modifying approaches may be activation of eCB system.
In
previous work we have shown that synthetic agonist of CB1 receptors WIN55,212-2
produces an “antikindling” effect during the electrical stimulation of
the perforant path and decreased susceptibility to KA (Shubina and Kitchigina,
2012). The main aim of the present study was to shed light to question whether
enhance of endocannabinoid signaling would protect the brain against the
KA-induced excitotoxicity and subsequent epileptogenesis. Enhance of
endocannabinoid signaling was achieved via blockade of eCB reuptake by AM404 or
via inhibition of their enzymatic destruction by URB597. Instead, for
preventing the CB1 receptor function, antagonist AM251 was applied. We use
precondition approach (injection of eCB-related drugs before seizure onset and
during KA-evoked seizures) to understand how activation of eCB system can help
to prevent the action of excitotoxin, in particular, a death of neurons, subsequent network remodeling in the hippocampus and
changes in brain oscillations.
In the
present study we used enhanced cannabinoid signaling that is believe the most
adequate approach for activation of eCB system (“on demand”). It was achieved
by blocking eCB reuptake with AM404 and inhibiting AEA degradation enzyme fatty
acid amide hydrolase (FAAH) with URB597. For preventing the CB1 receptor
function, antagonist AM251 was applied. To provoke epileptogenesis KA-induced
excitotoxicity and evoked by its status epilepticus was used.
We
employed local field potential recordings in non-attentive guinea pigs to study
oscillatory activity of four brain structures (hippocampus, entorhinal cortex,
medial septum, and amigdala) in the health animals and within three months
after kainic acid introduction. Different frequency bands of spontaneous
rhythmic activity were analyzed: delta, theta, alpha, gamma and high frequency
(putative ripples) oscillations. Electrophysiological investigations revealed various disturbances of the oscillatory activity in all
frequency bands in the different structures during epileptogenesis. Cell loss
in the CA1 and CA3 fields and mossy fiber sprouting in the dentate gyrus were
observed in histochemical experiments. Intrabrain i.v.c. injection of AM404 or
URB597 softened alterations in electric activity and prevent changes in the
structure of hippocampus. Instead, infusion of AM251 as a rule did not exert
distinct effects on epileptogenesis, but in the some cases aggravated the excitotoxic influences of kainic acid.
The newest and
interesting data of the present our work concern the results of
electrophysiological experiments where modulation of eCB system activity was
used. Namely, in the animal groups where injections of AM404 or
URB597 performed (i.e., eCB system was activated)
we did not find the sharp changes of oscillations in anyone of recorded
structures, except Ent. In the hippocampus in the first month after KA
administration there was not found significant increase of the rhythms, which
was observed in KA group. Though in the animals with AM404 infusion the gradual
increasing of LFP power was revealed (except HFO), it was slow and less expressed.
Since it is known that substantial increase of HFO power in the hippocampus is
one of markers of epileptogenesis (Staba et al., 2014), we can state that in
our study infusion of AM404 or URB597 into the brain prevent
this pathological change. We also found that in URB597 group in the first month
after KA injection the power of oscillations even some decreased. Earlier it
was shown that the first month after toxic KA influence is a critical period
for development of pathological alteration in different hippocampal cells and
synapses (Best et al., 1994; Morin et al., 1999; Karanian et al., 2005; Kotaria et al., 2013), and we believed that
the decrease of excitability during this period, which could be achieved by the
blocking of eCB inactivation, interfered with
further epileptogenesis.
We believe that discovered here effects of
modulation of eCB metabolism point out the protective potential of endocannabinoid system in preventing epileptogenesis
during excitotoxic influences.