Final Report Summary - CANNABISTARG (Regulation of normal and pathological activity of cortical networks by cannabinoids: focus on direct modulation of inhibitory GABAA and glycine receptors)
Cannabinoids have widespread actions in the brain: they influence learning and memory, modulate locomotor activity and reward pathways. Cannabis has a long history of consumption both for recreational and medical uses. The therapeutic action of cannabinoid agonists and antagonists, or compounds which may modify endocannabinoid metabolism and activity indicates they may have promise as neuroprotectants, and may be of value in the treatment of certain types of pain, epilepsy, spasticity, and other disorders associated with overexcited neural circuits.
It is widely accepted that cannabinoids influence brain function by activating the specific cannabinoid CB1 receptor (CB1R). This G-protein-coupled receptor is expressed throughout the brain at high levels. Several endogenous lipids, including anandamide (AEA) and 2-arachidonylglycerol (2-AG) have been identified as CB1R ligands. Endocannabinoids, rapidly released from neurons upon depolarisation mediate some forms of activity-dependent short- and long-term presynaptic modulation of synaptic transmission. In line with these data, CB1Rs were suggested to account for most of all central effects of cannabinoids. However, there are critical areas of controversy, including those related to the actions of cannabinoids in epilepsy and neurotoxicity. While most of behavioural effects of cannabinoids are absent in CB1R -deficient mice, it was reported that AEA still induce the catalepsy, analgesia and decreasing spontaneous network activity in these mice. The latter indicates the existence of functionally important target(s) for brain cannabinoid signalling in addition to CB1R. Indeed, recently growing number of studies reports that cannabinoids at physiologically relevant concentrations can directly affect various voltage-gated ion channels, transient receptor potential vanilloid type 1 (TRPV1) channels; and also ligand-gated channels of the Cys-loop receptor superfamily, namely the nicotinic acetylcholine, the serotonin and the glycine (GlyR) receptors. Nevertheless, the functional implications of the direct effects of cannabinoids are poorly studied.
In the framework of Marie Curie grant we have obtained new data that shed light on the mechanisms of direct CB1-independent interaction of cannabinoids with two inhibitory receptors: GABAA receptor and glycine receptors, and the role of these interaction in the antiepileptic activity of endocannabinoids.
Our new findings uncover a hitherto hidden dimension in cannabinoid signaling in the brain that point to a novel CB1R -independent mechanism for tonic and activity-dependent regulation of synaptic inhibition in various brain areas. This newly discovered mechanism of interaction of cannabinoids with inhibitory receptors display fundamentally different features and may have important functional implications on neuronal network functioning especially at high frequency presynaptic stimulation: Thus,
1) Cannabinoids modulate both the amplitude and the decay kinetics of GABAARs-mediated IPSCs. Both parameters are important determinants of the temporal precision and synchrony within neuronal networks. Consequently, cannabinoids may reduce the influence of interneurons on pyramidal neuron firing;
2) CB1R-dependent inhibition affects only low frequency input, leaving high frequency signals unaffected (i.e. it operates as a high pass frequency filter). In contrast, direct suppression of inhibition still remains potent at high frequencies (i.e. operates at broad band of frequencies). Our data show that endocannabinoids (2-AG and anandamide) strongly facilitated the depression of GABA and glycine-induced currents during repetitive (10-20 Hz) application of short (2-ms duration) pulses of agonist to outside-out patches. We suggest that endocannabinoids induced a dramatic increase of the fraction of desensitised GlyRs and GABAARs; correspondingly, this leads to an enhancement of short-term depression (STD) in a condition of repetitive stimulation. Thus, endocannabinoids enhance the low-pass filtering ability of GABAAR and GlyR synapses for inhibitory signals and, thus, increase the probability of transfer of the high-frequency excitatory signals conveyed to neurons. Therefore, endocannabinoids may induce striking changes in the temporal information processing in inhibitory synapses by direct action on postsynaptic GABAARs and GlyRs. These observations indicate that endocannabinoids can be a powerful modulator of inhibitory receptors at high frequency activation.
3) Especially impressive is that direct modulation of inhibitory receptors, in contrast to the CB1R-mediated modulation, may influence tonic inhibitory currents mediated by extrasynaptic receptors, thus regulating excitability of neurons postsynaptically and therefore controlling overall network activity.
These phenomena may have significant functional implication in epileptic neuronal network. Indeed, our major finding in this project that anandamide effectively prevent epileptiform activity in a number of in vivo pharmacological models of epilepsy in CB1-independent manner.
Our understanding of mechanisms of epilepsy is limited by the lack of appropriate animal models. During last year new state-of-art genetic model of epilepsy associated with TSC1 has been installed on the postgenomic platform of INMED. In utero, electroporation has been used to knock out TSC1 gene in selected fluorescently labeled neuronal population at precise developmental time point, in mice heterozygous for mutant TSC1 allele. Novelty of this model relay on using of the double-hit-strategy and using of in utero electroporation that allow to reproduce patchy distribution and focal nature of the growths of the tubers and epileptical activity observed in human TSC. Our data show that pronounced spontaneous activity, recorded by intracrainial EEG in TSC1 animal model was effectively suppressed by anandamide. Thus our data show potent antiepileptic activity of endocannabinoids in different animal models of epilepsy.
Epilepsy is one of the most common neurological disorders affecting 2 % of the world's population, in which normal brain function is disrupted as a consequence of intensive and synchronous burst activity from neuron assemblies. It is estimated, that at present ~30 % of epileptics are not adequately controlled with conventional drugs due to development of tolerance and pharmacoresistance, therefore justifying the search for new antiepileptic targets. CBs, known as protective against neurodegeneration and exhibit anticonvulsant activity are promising pharmacological tools to treat epilepsies. Our understanding of cannabinoid signalling pathways, their modulation of inhibitory synaptic transmission and plasticity, the cellular targets of CBs in different CNS regions and, will lead to important new insights into neuronal function. This will likely result in the development of new therapeutic strategies for the treatment of a number of key CNS disorder including epilepsy.
It is widely accepted that cannabinoids influence brain function by activating the specific cannabinoid CB1 receptor (CB1R). This G-protein-coupled receptor is expressed throughout the brain at high levels. Several endogenous lipids, including anandamide (AEA) and 2-arachidonylglycerol (2-AG) have been identified as CB1R ligands. Endocannabinoids, rapidly released from neurons upon depolarisation mediate some forms of activity-dependent short- and long-term presynaptic modulation of synaptic transmission. In line with these data, CB1Rs were suggested to account for most of all central effects of cannabinoids. However, there are critical areas of controversy, including those related to the actions of cannabinoids in epilepsy and neurotoxicity. While most of behavioural effects of cannabinoids are absent in CB1R -deficient mice, it was reported that AEA still induce the catalepsy, analgesia and decreasing spontaneous network activity in these mice. The latter indicates the existence of functionally important target(s) for brain cannabinoid signalling in addition to CB1R. Indeed, recently growing number of studies reports that cannabinoids at physiologically relevant concentrations can directly affect various voltage-gated ion channels, transient receptor potential vanilloid type 1 (TRPV1) channels; and also ligand-gated channels of the Cys-loop receptor superfamily, namely the nicotinic acetylcholine, the serotonin and the glycine (GlyR) receptors. Nevertheless, the functional implications of the direct effects of cannabinoids are poorly studied.
In the framework of Marie Curie grant we have obtained new data that shed light on the mechanisms of direct CB1-independent interaction of cannabinoids with two inhibitory receptors: GABAA receptor and glycine receptors, and the role of these interaction in the antiepileptic activity of endocannabinoids.
Our new findings uncover a hitherto hidden dimension in cannabinoid signaling in the brain that point to a novel CB1R -independent mechanism for tonic and activity-dependent regulation of synaptic inhibition in various brain areas. This newly discovered mechanism of interaction of cannabinoids with inhibitory receptors display fundamentally different features and may have important functional implications on neuronal network functioning especially at high frequency presynaptic stimulation: Thus,
1) Cannabinoids modulate both the amplitude and the decay kinetics of GABAARs-mediated IPSCs. Both parameters are important determinants of the temporal precision and synchrony within neuronal networks. Consequently, cannabinoids may reduce the influence of interneurons on pyramidal neuron firing;
2) CB1R-dependent inhibition affects only low frequency input, leaving high frequency signals unaffected (i.e. it operates as a high pass frequency filter). In contrast, direct suppression of inhibition still remains potent at high frequencies (i.e. operates at broad band of frequencies). Our data show that endocannabinoids (2-AG and anandamide) strongly facilitated the depression of GABA and glycine-induced currents during repetitive (10-20 Hz) application of short (2-ms duration) pulses of agonist to outside-out patches. We suggest that endocannabinoids induced a dramatic increase of the fraction of desensitised GlyRs and GABAARs; correspondingly, this leads to an enhancement of short-term depression (STD) in a condition of repetitive stimulation. Thus, endocannabinoids enhance the low-pass filtering ability of GABAAR and GlyR synapses for inhibitory signals and, thus, increase the probability of transfer of the high-frequency excitatory signals conveyed to neurons. Therefore, endocannabinoids may induce striking changes in the temporal information processing in inhibitory synapses by direct action on postsynaptic GABAARs and GlyRs. These observations indicate that endocannabinoids can be a powerful modulator of inhibitory receptors at high frequency activation.
3) Especially impressive is that direct modulation of inhibitory receptors, in contrast to the CB1R-mediated modulation, may influence tonic inhibitory currents mediated by extrasynaptic receptors, thus regulating excitability of neurons postsynaptically and therefore controlling overall network activity.
These phenomena may have significant functional implication in epileptic neuronal network. Indeed, our major finding in this project that anandamide effectively prevent epileptiform activity in a number of in vivo pharmacological models of epilepsy in CB1-independent manner.
Our understanding of mechanisms of epilepsy is limited by the lack of appropriate animal models. During last year new state-of-art genetic model of epilepsy associated with TSC1 has been installed on the postgenomic platform of INMED. In utero, electroporation has been used to knock out TSC1 gene in selected fluorescently labeled neuronal population at precise developmental time point, in mice heterozygous for mutant TSC1 allele. Novelty of this model relay on using of the double-hit-strategy and using of in utero electroporation that allow to reproduce patchy distribution and focal nature of the growths of the tubers and epileptical activity observed in human TSC. Our data show that pronounced spontaneous activity, recorded by intracrainial EEG in TSC1 animal model was effectively suppressed by anandamide. Thus our data show potent antiepileptic activity of endocannabinoids in different animal models of epilepsy.
Epilepsy is one of the most common neurological disorders affecting 2 % of the world's population, in which normal brain function is disrupted as a consequence of intensive and synchronous burst activity from neuron assemblies. It is estimated, that at present ~30 % of epileptics are not adequately controlled with conventional drugs due to development of tolerance and pharmacoresistance, therefore justifying the search for new antiepileptic targets. CBs, known as protective against neurodegeneration and exhibit anticonvulsant activity are promising pharmacological tools to treat epilepsies. Our understanding of cannabinoid signalling pathways, their modulation of inhibitory synaptic transmission and plasticity, the cellular targets of CBs in different CNS regions and, will lead to important new insights into neuronal function. This will likely result in the development of new therapeutic strategies for the treatment of a number of key CNS disorder including epilepsy.