Calcium Imaging and Amperometric Recording in Cultured Chromaffin Cells and Adrenal Slices from Normotensive, Wistar Kyoto Rats and Spontaneously Hypertensive Rats.

The spontaneously hypertensive rat (SHR) is a model widely used to investigate the causal mechanisms of essential hypertension. The enhanced catecholamine (CA) release reported in adrenal glands from adult SHRs raised considerable interest for its possible implication in the genesis of hypertension. The use of powerful techniques such as calcium imaging, electrophysiology, and single-cell amperometry to monitor in real time the key steps in CA secretion has allowed a better understanding of the role of chromaffin cells (CC) in the pathophysiology of hypertension, although several questions remain. Additionally, the implementation of these techniques in preparations in situ, such as the acute adrenal gland slice, which maintains the microenvironment, cell-to-cell communication, and anatomical structure similar to that of the intact adrenal gland, yields data that may have even greater physiological relevance. Here, we describe the procedures to measure the blood pressure of rats in a noninvasive manner, how to obtain primary cultures of adrenal chromaffin cells and acute adrenal slices, and how to perform amperometric recordings and intracellular calcium imaging in these preparations.

Intrinsic excitability mechanisms of neuronal ensemble formation.

Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals of both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we found that, after optogenetic or electrical stimulation, coactivated neurons increased their correlated activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Optogenetic and electrical stimulation also induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed unexpected strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reductions in spike threshold. A pharmacological agent that blocks changes in membrane resistance reverted this effect. These significant increases in excitability can explain the observed biphasic synaptic plasticity. We conclude that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an 'iceberg' model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.

In the brain, groups of neurons that are activated together ­ also known as neuronal ensembles ­ are the basic units that underpin perception and behavior. Yet, exactly how these coactive circuits are established remains under investigation. In 1949, Canadian psychologist Donald Hebb proposed that, when brains learn something new, the neurons which are activated together connect to form ensembles, and their connections become stronger each time this specific piece of knowledge is recalled. This idea that 'neurons that fire together, wire together' can explain how memories are acquired and recalled, by strengthening their wiring. However, recent studies have questioned whether strengthening connections is the only mechanism by which neural ensembles can be created. Changes in the excitability of neurons (how easily they are to fire and become activated) may also play a role. In other words, ensembles could emerge because certain neurons become more excitable and fire more readily. To solve this conundrum, Alejandre-García et al. examined both hypotheses in the same system. Neurons in slices of the mouse visual cortex were stimulated electrically or optically, via a technique that controls neural activity with light. The activity of individual neurons and their connections was then measured with electrodes. Spontaneous activity among connected neurons increased after stimulation, indicative of the formation of neuronal ensembles. Connected neurons also showed small changes in the strength of their connections, which first decreased and then rebounded after an initial recovery period. Intriguingly, cells also showed unexpected strong and persistent increases in neuronal excitability after stimulation, such that neurons fired more readily to the same stimulus. In other words, neurons maintained a cellular memory of having been stimulated. The authors conclude that ensembles form because connected neurons become more excitable, which in turn, may strengthen connections of the circuit at a later stage. These results provide fresh insights about the neural circuits underpinning learning and memory. In time, the findings could also help to understand disorders such as Alzheimer's disease and schizophrenia, which are characterised by memory impairments and disordered thinking.

Long-term stability of cortical ensembles.

Neuronal ensembles, coactive groups of neurons found in spontaneous and evoked cortical activity, are causally related to memories and perception, but it is still unknown how stable or flexible they are over time. We used two-photon multiplane calcium imaging to track over weeks the activity of the same pyramidal neurons in layer 2/3 of the visual cortex from awake mice and recorded their spontaneous and visually evoked responses. Less than half of the neurons remained active across any two imaging sessions. These stable neurons formed ensembles that lasted weeks, but some ensembles were also transient and appeared only in one single session. Stable ensembles preserved most of their neurons for up to 46 days, our longest imaged period, and these 'core' cells had stronger functional connectivity. Our results demonstrate that neuronal ensembles can last for weeks and could, in principle, serve as a substrate for long-lasting representation of perceptual states or memories.

GABAA receptor: a unique modulator of excitability, Ca2+ signaling, and catecholamine release of rat chromaffin cells.

The role of gamma-aminobutyric acid (GABA) in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and is presumably released together with CA, ATP, and opioids in response to physiological stimuli, playing an autocrine-paracrine role on CCs. The reported paradoxical "dual action" of GABAA-R activation (enhancement of CA secretion and inhibition of synaptically evoked CA release) is only one aspect of GABA's multifaceted actions. In this review, we discuss recent physiological experiments on rat CCs in situ which suggest that GABA regulation of CC function may depend on the physiological context: During non-stressful conditions, GABAA-R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca2+ fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca2+ influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Ca2+channels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABAA-R is depolarizing in about 50% of CCs, and thus GABA, acting as an autocrine/paracrine mediator, could help to maintain CA exocytosis under stress. GABAA-R activation is not excitatory in about half of CCs' population because it hyperpolarizes them or elicits no response. This percentage possibly varies, depending on functional demands, since GABAA-R-mediated actions are determined by the intracellular chloride concentration ([Cl-] i ) and therefore on the activity of cation-chloride co transporters, which is functionally regulated. These findings underscore a potential importance of a novel and complex GABA-mediated regulation of CC function and of CA secretion.

Comparison of Ca2+ currents of chromaffin cells from normotensive Wistar Kyoto and spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are widely used as model to investigate the pathophysiological mechanisms of essential hypertension. Catecholamine plasma levels are elevated in SHR, suggesting alterations of the sympathoadrenal axis. The residual hypertension in sympathectomized SHR is reduced after demedullation, suggesting a dysfunction of the adrenal medulla. Intact adrenal glands exposed to acetylcholine or high K+ release more catecholamine in SHR than in normotensive Wistar Kyoto (WKY) rats, and adrenal chromaffin cells (CCs) from SHR secrete more catecholamines than CCs from WKY rats. Since Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers exocytosis, alterations in the functional properties of these channels might underlie the enhanced catecholamine release in SHR. This study compares the electrophysiological properties of VGCC from CCs in acute adrenal slices from WKY rats and SHR at an early stage of hypertension. No significant differences were found in the macroscopic Ca2+ currents (current density, I­V curve, voltage dependence of activation and inactivation, kinetics) between CCs of SHR and WKY rats, suggesting that Ca2+ entry through VGCC is not significantly different between these strains, at least at early stages of hypertension. Ca2+ buffering, sequestration and extrusion mechanisms, as well as Ca2+ release from intracellular stores, must now be evaluated to determine if alterations in their function can explain the enhanced catecholamine secretion reported in CCs from SHR.