Ion R. Popescu

Activity‐dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus

Exogenous cannabinoids have been shown to significantly alter neuroendocrine output, presaging the emergence of endogenous cannabinoids as important signalling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation. We showed recently that magnocellular and parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus and supraoptic nucleus (SON) respond to glucocorticoids by releasing endocannabinoids as retrograde messengers to modulate the synaptic release of glutamate. Here we show directly for the first time that both of the main endocannabinoids, anandamide (AEA) and 2‐arachidonoyl glycerol (2‐AG), are released in an activity‐dependent fashion from the soma/dendrites of SON magnocellular neurones and suppress synaptic glutamate release and postsynaptic spiking. Cannabinoid reuptake blockade increases activity‐dependent endocannabinoid levels in the region of the SON, and results in the inhibition of synaptically driven spiking activity in magnocellular neurones. Together, these findings demonstrate an activity‐dependent release of AEA and 2‐AG that leads to the suppression of glutamate release and that is capable of shaping spiking activity in magnocellular neurones. This activity‐dependent regulation of excitatory synaptic input by endocannabinoids may play a role in determining spiking patterns characteristic of magnocellular neurones under stimulated conditions.

GABA Is Excitatory in Adult Vasopressinergic Neuroendocrine Cells

Neuronal excitability in the adult brain is controlled by a balance between synaptic excitation and inhibition mediated by glutamate and GABA, respectively. While generally inhibitory in the adult brain, GABAA receptor activation is excitatory under certain conditions in which the GABA reversal potential is shifted positive due to intracellular Cl− accumulation, such as during early postnatal development and brain injury. However, the conditions under which GABA is excitatory are generally either transitory or pathological. Here, we reveal GABAergic synaptic inputs to be uniformly excitatory in vasopressin (VP)-secreting magnocellular neurons in the adult hypothalamus under normal conditions. The GABA reversal potential (EGABA) was positive to resting potential and spike threshold in VP neurons, but not in oxytocin (OT)-secreting neurons. The VP neurons lacked expression of the K+-Cl− cotransporter 2 (KCC2), the predominant Cl− exporter in the adult brain. The EGABA was unaffected by inhibition of KCC2 in VP neurons, but was shifted positive in OT neurons, which express KCC2. Alternatively, inhibition of the Na+-K+-Cl− cotransporter 1 (NKCC1), a Cl− importer expressed in most cell types mainly during postnatal development, caused a negative shift in EGABA in VP neurons, but had no effect on GABA currents in OT neurons. GABAA receptor blockade caused a decrease in the firing rate of VP neurons, but an increase in firing in OT neurons. Our findings demonstrate that GABA is excitatory in adult VP neurons, suggesting that the classical excitation/inhibition paradigm of synaptic glutamate and GABA control of neuronal excitability does not apply to VP neurons.

Synchronized bursts of miniature inhibitory postsynaptic currents

Spike‐independent miniature postsynaptic currents are generally stochastic and are therefore not thought to mediate information relay in neuronal circuits. However, we recorded endogenous bursts of IPSCs in hypothalamic magnocellular neurones in the presence of TTX, which implicated a coordinated mechanism of spike‐independent GABA release. IPSC bursts were identical in the absence of TTX, although the burst incidence increased 5‐fold, indicating that IPSC bursts were composed of miniature IPSCs (mIPSCs), and that the probability of burst generation increased with action potential activity. IPSC bursts required extracellular calcium, although they were not dependent on calcium influx through voltage‐gated calcium channels or on calcium mobilization from intracellular stores. Current injections simulating IPSC bursts were capable of triggering and terminating action potential trains. In 25% of dual recordings, a subset of IPSC bursts were highly synchronized in onset in pairs of magnocellular neurones. Synchronized IPSC bursts displayed properties that were consistent with simultaneous release at GABA synapses shared between pairs of postsynaptic magnocellular neurones. Synchronized bursts of inhibitory synaptic inputs represent a novel mechanism that may contribute to the action potential burst generation, termination and synchronization responsible for pulsatile hormone release from neuroendocrine cells.

Glial Control of Endocannabinoid Heterosynaptic Modulation in Hypothalamic Magnocellular Neuroendocrine Cells

Cannabinoid receptors are functionally operant at both glutamate and GABA synapses on hypothalamic magnocellular neuroendocrine cells; however, retrograde endocannabinoid actions are evoked at only glutamate synapses. We tested whether the functional targeting of evoked retrograde endocannabinoid actions to glutamate, and not GABA, synapses on magnocellular neurons is the result of the spatial restriction of extracellular endocannabinoids by astrocytes. Whole-cell GABA synaptic currents were recorded in magnocellular neurons in rat hypothalamic slices following manipulations to reduce glial buffering of extracellular signals. Depolarization- and glucocorticoid-evoked retrograde endocannabinoid suppression of synaptic GABA release was not detected under normal conditions, but occurred in both oxytocin and vasopressin neurons under conditions of attenuated glial coverage and depressed glial metabolic function, suggesting an emergent endocannabinoid modulation of GABA synapses with the loss of astrocyte function. Tonic endocannabinoid suppression of GABA release was insensitive to glial manipulation. Blocking cannabinoid transport mimicked, and increasing the extracellular viscosity reversed, the effect of suppressed glial buffering on the endocannabinoid modulation of GABA release. Evoked, but not tonic, endocannabinoid modulation of GABA synapses was mediated by 2-arachidonoylglycerol. Therefore, depolarization- and glucocorticoid-evoked 2-arachidonoylglycerol release from magnocellular neurons is spatially restricted to glutamate synapses by astrocytes, but spills over onto GABA synapses under conditions of reduced astrocyte buffering; tonic endocannabinoid modulation of GABA release, in contrast, is likely mediated by anandamide and is insensitive to astrocytic buffering. Astrocytes, therefore, provide dynamic control of stimulus-evoked 2-arachidonoylglycerol, but not tonic anandamide, regulation of GABA synaptic inputs to magnocellular neuroendocrine cells under different physiological conditions.

Short‐term potentiation of GABAergic synaptic inputs to vasopressin and oxytocin neurones

Hypothalamic neurones that release the hormones vasopressin and oxytocin from the posterior pituitary are controlled, in part, by synaptic inputs mediated by the neurotransmitter GABA. GABA synapses on the vasopressin neurones, but not the oxytocin neurones, have been shown to elicit excitation. We show that brief (1 s) electrical stimulation of hypothalamus slices causes release of GABA lasting several minutes at synapses on vasopressin and oxytocin neurones. Consistent with GABA being excitatory in vasopressin neurones, brief electrical stimulation of GABA synaptic inputs elicited activation of vasopressin neurones, but not oxytocin neurones, that lasted for several minutes. Prolonged GABA release following a brief stimulus together with GABA excitation of vasopressin neurones constitute a basic synaptic mechanism for controlling vasopressin release.

Marked bias towards spontaneous synaptic inhibition distinguishes non-adapting from adapting layer 5 pyramidal neurons in the barrel cortex

Pyramidal neuron subtypes differ in intrinsic electrophysiology properties and dendritic morphology. However, do different pyramidal neuron subtypes also receive synaptic inputs that are dissimilar in frequency and in excitation/inhibition balance? Unsupervised clustering of three intrinsic parameters that vary by cell subtype – the slow afterhyperpolarization, the sag, and the spike frequency adaptation – split layer 5 barrel cortex pyramidal neurons into two clusters: one of adapting cells and one of non-adapting cells, corresponding to previously described thin- and thick-tufted pyramidal neurons, respectively. Non-adapting neurons presented frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) three- and two-fold higher, respectively, than those of adapting neurons. The IPSC difference between pyramidal subtypes was activity independent. A subset of neurons were thy1-GFP positive, presented characteristics of non-adapting pyramidal neurons, and also had higher IPSC and EPSC frequencies than adapting neurons. The sEPSC/sIPSC frequency ratio was higher in adapting than in non-adapting cells, suggesting a higher excitatory drive in adapting neurons. Therefore, our study on spontaneous synaptic inputs suggests a different extent of synaptic information processing in adapting and non-adapting barrel cortex neurons, and that eventual deficits in inhibition may have differential effects on the excitation/inhibition balance in adapting and non-adapting neurons.

Publisher Correction: Marked bias towards spontaneous synaptic inhibition distinguishes non-adapting from adapting layer 5 pyramidal neurons in the barrel cortex

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Firing pattern regulation in hypothalamic vasopressin neurons: roles of synaptic inputs and retrograde signaling

Magnocellular neurosecretory cells (MNCs) of the hypothalamus release the hormones oxytocin (OT) and vasopressin (VP) into the blood. These cells demonstrate enhancement of hormone release with bursting patterns of electrical activity. OT neurons fire synchronized bursts at long intervals during parturition and milk ejection; VP neurons generate an asynchronous phasic bursting in response to osmotic and cardiovascular stimuli. The mechanisms of bursting activity in VP are not known completely and are believed to be different in vitro and in vivo. Whereas in vitro, phasic bursting in VP neurons appears to be governed by intrinsic deterministic mechanisms, in vivo burst generation and termination significantly depends on synaptic activity. Mounting evidences suggest that retrograde signaling via endocannabinoids (eCBs) plays a prominent role in modulating MNC synaptic activity [1]. Our recent experiments suggest that bursts of action potentials are capable of suppressing glutamatergic input in VP neurons. We also found that blocking eCB receptors increased burst duration and intra-burst action potential frequency, consistent with a potential role in burst termination. To investigate theoretically the role of synaptic inputs in the phasic bursting activity in VP neurons, we used an updated multicompartmental model of the MNC [2]. The model takes into account MNC morphology and electrotonic properties and includes a set of realistic voltage-gated and Ca 2+ -activated ion currents, compartmental Ca 2+ dynamics and reproduces several of the hallmark characteristics of MNC electrophysiological properties. Phasic bursting in the model is controlled by both intrinsic and synaptic mechanisms: bursts of action potentials arise from the summation of slow depolarizing afterpotentials superimposed on a tonic background activation of glutamatergic synaptic inputs; activitydependent release of a retrograde messenger (eCB) from the dendrites of VP neurons attenuates tonic glutamate release and leads to burst termination. Background synaptic activity was simulated as independent excitatory and inhibitory inputs mediated by AMPA and GABAA conductances. Our computational studies also suggest that GABAA receptor activation promotes burst firing patterns, and stochastic synaptic inputs play an important role in the modulation of phasic activity in VP neurons.