Non-genomic steroid signaling through the mineralocorticoid receptor: Involvement of a membrane-associated receptor?

Corticosteroid receptors in the mammalian brain mediate genomic as well as non-genomic actions. Although receptors mediating genomic actions were already cloned 35 years ago, it remains unclear whether the same molecules are responsible for the non-genomic actions or that the latter involve a separate class of receptors. Here we focus on one type of corticosteroid receptors, i.e. the mineralocorticoid receptor (MR). We summarize some of the known properties and the current insight in the localization of the MR in peripheral cells and neurons, especially in relation to non-genomic signaling. Previous studies from our own and other labs provided evidence that MRs mediating non-genomic actions are identical to the ones involved in genomic signaling, but may be translocated to the plasma cell membrane instead of the nucleus. With fixed cell imaging and live cell imaging techniques we tried to visualize these presumed membrane-associated MRs, using antibodies or overexpression of MR-GFP in COS7 and hippocampal cultured neurons. Despite the physiological evidence for MR location in or close to the cell membrane, we could not convincingly visualize membrane localization of endogenous MRs or GFP-MR molecules. However, we did find punctae of labeled antibodies intracellularly, which might indicate transactivating spots of MR near the membrane. We also found some evidence for trafficking of MR via beta-arrestins. In beta-arrestin knockout mice, we didn't observe metaplasticity in the basolateral amygdala anymore, indicating that internalization of MRs could play a role during corticosterone activation. Furthermore, we speculate that membrane-associated MRs could act indirectly via activating other membrane located structures like e.g. GPER and/or receptor tyrosine kinases.

Circadian and ultradian patterns of HPA-axis activity in rodents: Significance for brain functionality.

The hypothalamo-pituitary-adrenal (HPA) axis comprises interactions between the hypothalamus, the pituitary and the adrenal glands and its activation results in the release of corticosteroid hormones. Corticosteroids are secreted from the adrenal gland in a distinct 24-h circadian rhythm overarching an ultradian rhythm, which consists of hourly corticosteroid pulses exposing target tissues to rapidly changing steroid levels. On top of these rhythms surges can take place after stress. HPA-axis rhythms promote adaptation to predictable (i.e. the earth's rotation) and unpredictable (i.e. stressors) changes in environmental factors. Two steroid hormone receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), are activated by corticosteroids and mediate effects at fast and slow timescales on e.g. glucose availability, gene transcription and synaptic plasticity. The current review discusses the origin of the circadian and ultradian corticosteroid rhythms and their relevance for gene regulation, neuroendocrine and physiological responses to stress and the involvement in the maintenance of brain functionality in rodents.

Activation of type-1 cannabinoid receptor shifts the balance between excitation and inhibition towards excitation in layer II/III pyramidal neurons of the rat prelimbic cortex.

Activation of the endocannabinoid (eCB) system by exogenous cannabinoids (drug abuse) can alter the physiology of the brain circuits involved in higher-order cognitive functions such as the medial prefrontal cortex (mPFC). A proper balance between excitation and inhibition (E/I balance) is critical for neuronal network oscillations underlying cognitive functions. Since type-1 cannabinoid receptors (CB1Rs), expressed in many brain areas including the mPFC, can modulate excitatory and inhibitory neurotransmission, we aimed to determine whether CB1R activation results in modifications of the E/I balance. We first confirm the presence of functional presynaptic CB1Rs that can modulate both excitatory and inhibitory inputs to layer II/III pyramidal neurons of the prelimbic (PL) area of the mPFC. By decomposing the synaptic response evoked by layer I stimulation into its excitatory and inhibitory components, we show that in vitro CB1R activation with the cannabinoid receptor agonists WIN55,212-2 (WIN) and CP-55940 (CP) modulates the balance between excitation and inhibition (E/I balance) of layer II/III pyramidal neurons. This treatment caused a significant shift of the E/I balance towards excitation, from 18/82 % to 25/75 % (WIN) and from 17/83 to 30/70 % (CP). Finally, when animals were injected with a cannabinoid receptor agonist, we observed a shift of the E/I balance (measured in vitro) towards excitation 1 h after WIN (24/76 %) or after CP injection (30/70 %) when compared to vehicle-injected animals (18/82 %). This modulation of the E/I balance by CB1Rs may thus be fundamental in the regulation of local PL cortical network excitability and could be the mechanism through which excessive CB1R activation (cannabis abuse) affects cognitive functions.

Endocannabinoids produced upon action potential firing evoke a Cl(-) current via type-2 cannabinoid receptors in the medial prefrontal cortex.

The functional presence of type-2 cannabinoid receptors (CB2Rs) in layer II/III pyramidal neurons of the rat medial prefrontal cortex (mPFC) was recently demonstrated. In the present study, we show that the application of the endocannabinoids (eCBs) 2-arachidonoylglycerol (2-AG) and methanandamide [a stable analog of the eCB anandamide (AEA)] can activate CB2Rs of mPFC layer II/III pyramidal neurons, which subsequently induces a Cl(-) current. In addition, we show that action potential (AP) firing evoked by 20-Hz current injections results in an eCB-mediated opening of Cl(-) channels via CB2R activation. This AP-evoked synthesis of eCBs is dependent on the Ca(2+) influx through N-type voltage-gated calcium channels. Our results indicate that 2-AG is the main eCB involved in this process. Finally, we demonstrate that under physiologically relevant intracellular Cl(-) conditions, 20-Hz AP firing leads to a CB2R-dependent reduction in neuronal excitability. Altogether, our data indicate that eCBs released upon action potential firing can modulate, through CB2R activation, neuronal activity in the mPFC. We discuss how this may be a mechanism to prevent excessive neuronal firing.

Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors.

The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB(2)Rs) in the CNS. Here, we show that CB(2)Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP(3)R-dependent opening of Ca(2+)-activated Cl(-) channels. To investigate the functional role of CB(2)R activation, we induced neuronal firing and observed a CB(2)R-mediated reduction in firing frequency. The description of this unique CB(2)R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.

Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity.

The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB(1)) and cannabinoid 2 (CB(2)) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl substituent in 11-16. Furthermore, the 3-carbonyl tether was replaced with a carboxamide linker in 17-20 and the azaindole (pyrrolopyridine) nucleus was designed as indole bioisostere with improved physicochemical properties in 21-25. Through these SAR efforts, several high affinity CB(1)/CB(2) dual cannabinoid receptor ligands were identified. Indole-3-carboxamide 17 displayed single-digit nanomolar affinity and ~80 fold selectivity for CB(1) over the CB(2) receptor. The azaindoles displayed substantially improved physicochemical properties (lipophilicity; aqueous solubility). Azaindole 21 elicited potent cannabinoid activity. Cannabinoid receptor agonists 17 and 21 potently modulated excitatory synaptic transmission in an acute rat brain slice model of cannabinoid receptor-modulated neurotransmission.