Target cell-specific modulation of neuronal activity by astrocytes.

Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network.

Action potential propagation in dendrites of rat mitral cells in vivo.

Odors evoke beta-gamma frequency field potential oscillations in the olfactory systems of awake and anesthetized vertebrates. In the rat olfactory bulb, these oscillations reflect the synchronous discharges of mitral cells that result from both their intrinsic membrane properties and their dendrodendritic interactions with local inhibitory interneurons. Activation of dendrodendritic synapses is purportedly involved in odor memory and odor contrast enhancement. Here we investigate in vivo to what extent action potentials propagate to remote dendrodendritic sites in the entire dendritic tree and if this propagation is changed during discharges at 40 Hz. By combining intracellular recording and two-photon microscopy imaging of intracellular calcium ([Ca2+]i), we show that in remote branches of the apical tuft and basal dendrites, transient Ca2+ changes are triggered by single sodium action potentials. Neither the amplitude of these Ca2+ transients nor that of action potentials obtained from intradendritic recordings showed a significant attenuation as a function of the distance from the soma. Calcium channel density seemed homogeneous; however, propagating action potentials occasionally failed to trigger a Ca2+ transient at a site closer to the soma whereas it did farther. This suggests that measurements of calcium transients underestimate the occurrence of sodium action potentials. During 40 Hz bursts of action potentials, [Ca2+]i increases with the number of action potentials in all dendritic compartments. These results suggest that the presence of release sites in dendrites is accompanied by an "axonal-like behavior" of the entire dendritic tree of mitral cells, including their most distal dendritic branches.

Two-photon microscopy in brain tissue: parameters influencing the imaging depth.

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Dendritic glutamate autoreceptors modulate signal processing in rat mitral cells.

It has been shown recently that in mitral cells of the rat olfactory bulb, N-methyl-D-aspartate (NMDA) autoreceptors are activated during mitral cell firing. Here we consider in more details the mechanisms of mitral cell self-excitation and its physiological relevance. We show that both ionotropic NMDA and non-NMDA autoreceptors are activated by glutamate released from primary and secondary dendrites. In contrast to non-NMDA autoreceptors, NMDA autoreceptors are almost exclusively located on secondary dendrites and their activation generates a large and sustained self-excitation. Both intracellularly evoked and miniature NMDA-R mediated synaptic potentials are blocked by intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) and result from a calcium-dependent release of glutamate. Self-excitation can be produced by a single spike, and trains of spikes result in frequency facilitation. Thus activation of excitatory autoreceptors is a major function of action potentials backpropagating in mitral cell dendrites, which results in an immediate positive feedback counteracting recurrent inhibition and increasing the signal-to-noise ratio of olfactory inputs.

Odor-evoked calcium signals in dendrites of rat mitral cells.

Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca(2+)]) and define timing for dendritic release. Here we investigated in vivo, how odor controls calcium dynamics in mitral cell dendrites by combining intracellular recording and two-photon microscopy imaging of [Ca(2+)]. During odor stimulation, two types of [Ca(2+)] changes accompany membrane potential oscillations that are phase-locked with the respiratory cycle: (i) one is graded and parallels the membrane potential, even below the threshold for action potential firing; (ii) a second is transient, triggered by sodium action potentials that invade the entire dendritic tree. These results indicate that mitral cell dendritic compartments are synchronized by action potentials and suggest that the efficacy of dendritic synapses is finely tuned by odor-evoked graded changes in [Ca(2+)].

Coherent scattering in multi-harmonic light microscopy.

By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.

Target cell-specific modulation of transmitter release at terminals from a single axon.

In the hippocampus, a CA3 pyramidal cell forms excitatory synapses with thousands of other pyramidal cells and inhibitory interneurons. By using sequential paired recordings from three connected cells, we show that the presynaptic properties of CA3 pyramidal cell terminals, belonging to the same axon, differ according to the type of target cell. Activation of presynaptic group III metabotropic glutamate receptors decreases transmitter release only at terminals contacting CA1 interneurons but not CA1 pyramidal cells. Furthermore, terminals contacting distinct target cells show different frequency facilitation. On the basis of these results, we conclude that the pharmacological and physiological properties of presynaptic terminals are determined, at least in part, by the target cells.

Effect of bicuculline on thalamic activity: a direct blockade of IAHP in reticularis neurons.

The thalamic reticular nucleus (RTN) is the major source of inhibitory contacts in the thalamus and thus plays an important role in regulating the excitability of the thalamocortical network. Inhibition occurs through GABAergic synapses on relay cells as well as through GABAergic synapses between reticularis neurons themselves. Here we report that the role and mechanisms of this inhibition, which frequently have been studied using N-methyl derivatives of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline, should be revisited. Using the whole cell patch-clamp technique in thalamic slices from young rats, we observed an enhancement by bicuculline methiodide, methobromide, and methochloride (collectively referred to as bicuculline-M; 5-60 microM) of the low-threshold calcium spike burst in RTN neurons that persisted in the presence of tetrodotoxin (1 microM) and was not reproduced in picrotoxin (100-300 microM). The effect did not involve activation of any GABA receptor subtype. Voltage-clamp recordings showed that bicuculline-M blocked the current underlying the low-threshold spike burst afterhyperpolarization (AHP), an effect that was mimicked by apamin (100 nM). Recordings from nucleated patches extracted from reticularis neurons demonstrated that this effect was not mediated by modulation of the release of an unidentified neurotransmitter but that bicuculline-M directly blocks small conductance (SK) channels. The AHP-blocking effect also was observed in other brain regions, demonstrating that although bicuculline-M is a potent GABAA receptor antagonist, it is of limited value in assessing GABAergic network interactions, which should be studied using picrotoxin or bicuculline-free base. However, bicuculline-M may provide a useful tool for developing nonpeptide antagonists of SK channels.

Cardiac arrest in rodents: maximal duration compatible with a recovery of neuronal activity.

We report here that during a permanent cardiac arrest, rodent brain tissue is "physiologically" preserved in situ in a particular quiescent state. This state is characterized by the absence of electrical activity and by a critical period of 5-6 hr during which brain tissue can be reactivated upon restoration of a simple energy (glucose/oxygen) supply. In rat brain slices prepared 1-6 hr after cardiac arrest and maintained in vitro for several hours, cells with normal morphological features, intrinsic membrane properties, and spontaneous synaptic activity were recorded from various brain regions. In addition to functional membrane channels, these neurons expressed mRNA, as revealed by single-cell reverse transcription-PCR, and could synthesize proteins de novo. Slices prepared after longer delays did not recover. In a guinea pig isolated whole-brain preparation that was cannulated and perfused with oxygenated saline 1-2 hr after cardiac arrest, cell activity and functional long-range synaptic connections could be restored although the electroencephalogram remained isoelectric. Perfusion of the isolated brain with the gamma-aminobutyric acid A receptor antagonist picrotoxin, however, could induce self-sustained temporal lobe epilepsy. Thus, in rodents, the duration of cardiac arrest compatible with a short-term recovery of neuronal activity is much longer than previously expected. The analysis of the parameters that regulate this duration may bring new insights into the prevention of postischemic damages.

mu-Opioid peptides inhibit thalamic neurons.

Opioidergic inhibition of neurons in the centrolateral nucleus of the thalamus was investigated using an in vitro thalamic slice preparation from young rats. The mu-opioid receptor agonist D-Ala2,N-Me-Phe4,glycinol5-enkephalin (DAMGO) evoked a hyperpolarization and decrease in input resistance that was reversible, concentration-dependent, and persisted in the presence of tetrodotoxin. Application of the specific mu-receptor antagonist Cys2,Tyr3,Orn5,Pen7-amide blocked this response. The respective delta- and kappa-opioid receptor agonists, (D-Pen2, D-Pen5)-enkephalin and (+/-)-trans-U-50488 methanesulfonate had no effect. Voltage-clamp experiments showed that DAMGO activated an inwardly rectifying potassium conductance (GKIR) characterized by rectification at hyperpolarized potentials that increased in elevated extracellular potassium concentrations, a complete block by Ba2+ (1 mM), and a voltage-dependent block by Cs+. The extent of mu-opioid inhibition in other thalamic nuclei was then investigated. Widespread inhibition similar to that seen in the centrolateral nucleus was observed in a number of sensory, motor, intralaminar, and midline nuclei. Our results suggest that the net action of opioids would depend on their source: exogenous (systemically administered) opiates inhibiting the entire thalamus and favoring the shift of cell firing from tonic to bursting mode; and endogenously released opioids acting on specific thalamic nuclei, their release depending on the origin of the presynaptic input.

Heterogeneity of cell firing properties and opioid sensitivity in the thalamic reticular nucleus.

The thalamic reticular nucleus receives afferents from the dorsal thalamus, cortex and brainstem, and projects back onto most cortically projecting thalamic nuclei thus playing a key role in the synchronization of the thalamocortical network. Although this nucleus was initially thought to consist of a homogeneous population of cells using GABA as a transmitter, and sharing identical intrinsic membrane properties, some heterogeneity was subsequently reported. The morphological diversity is generally acknowledged, but only two studies have shown functional differences between two classes of cells which vary in their ability to discharge in bursts. However, the location of the non-bursting cells was not characterized with anatomical techniques. Our recent work on the action of mu-opioid agonists in the thalamus revealed a widespread K+-mediated inhibition of most, if not all, thalamic relay and diffuse projection neurons. However, in the reticular nucleus, preliminary experiments suggested that the opioid sensitivity was variable. Based on these results and on observations of a discrete localization of mu-opioid receptors in the reticular nucleus, we investigated cellular heterogeneity within the nucleus using opioid agonists as markers. Using the whole cell patch clamp technique in young rat thalamic slices, we tested the responses of 28 neurons to opioids, the intrinsic membrane properties of each cell, and their relative location within the nucleus. Two types of intrinsic membrane properties underlying distinct discharge behaviours were seen in neurobiotin-labelled cells clearly located in the reticular nucleus: type I with the typical bursting behaviour previously reported in reticularis neurons, and type II in which bursting was greatly reduced or absent. Each class of cell could be further divided into subpopulations based on their opioid sensitivity. About half of both bursting (20) and non-bursting or tonic (8) cells were strongly inhibited by the mu-opioid receptor agonist D-Ala2,N-Me-Phe4,glycinol5-enkephalin, an effect mediated by an increase in K+ conductance. At no time was inhibition by delta- or kappa-receptor agonists seen. Our work therefore further demonstrates that the reticular nucleus is functionally heterogeneous, although the role of such cell diversity has still to be determined.

The entorhinal cortex entrains fast CA1 hippocampal oscillations in the anaesthetized guinea-pig: role of the monosynaptic component of the perforant path.

Entorhinal inputs reach the hippocampal CA1 field through a trisynaptic circuit involving dentate granule cells and CA3 pyramidal neurons, as well as through a monosynaptic path ending on the distal apical dendrites of CA1 pyramidal cells. The influence of monosynaptic entorhinal inputs onto CA1 operations is poorly understood. In this study, we characterized the involvement of the monosynaptic pathway in the generation of the fast CA1 oscillation bursts (30-60 Hz) that occur in the dorsal hippocampus of anaesthetized guinea-pigs after partial cortex removal. Using multiple-site extracellular and intracellular recording, we found that in this particular preparation, devoid of theta rhythm, fast oscillations are temporally coherent over a large portion of the CA1 region along the hippocampal septotemporal axis. Current source density analysis revealed that fast CA1 oscillations involve two dipoles reflecting synchronous synaptic activities in the stratum lacunosum-moleculare of the hippocampus proper and in the stratum moleculare of the dentate gyrus. These layers constitute the two major termination zones of entorhinal afferents, suggesting that the entorhinal cortex entrains fast CA1 oscillations. This hypothesis was corroborated by the concomitant occurrence of fast oscillation bursts in the entorhinal cortex and CA1 region. Furthermore, fast CA1 oscillations were abolished by lidocaine or tetrodotoxin injections in the entorhinal cortex. Finally, acute interruption of the hippocampal trisynaptic loop did not affect the stratum lacunosum-moleculare dipole recorded extracellularly, but also intracellularly, as high-frequency postsynaptic potentials in CA1 pyramidal cells. These results indicate that the monosynaptic pathway is involved in the genesis of fast CA1 oscillations.

Glutamate mediates a slow synaptic response in hippocampal slice cultures.

Glutamate (GLU) mediates its 'fast' excitatory transmitter action in the brain by directly gating cation-selective ion channels ('ionotropic' receptors). However, GLU can also activate another type of receptor, coupled to phospholipase C ('metabotropic' receptor). In hippocampal cells, stimulation of this metabotropic receptor by GLU, or by a racemic mixture of (1S-3R and 1R-3S) 1-aminocyclopentyl-1,3-dicarboxylate (ACPD), induces a slower excitation mediated by inhibition of K+ currents. We have assessed whether this slow form of metabotropic receptor excitation can contribute to the effects of synaptically released GLU in hippocampal slice cultures, by recording the responses of CA3 pyramidal cells to afferent mossy fibre stimulation. When the fast ionotropic response was blocked pharmacologically, mossy fibre stimulation produced a slow depolarizing postsynaptic potential associated with a decrease in membrane conductance, a depression of the slow after-hyperpolarization following a train of action potentials, and reduced accommodation during the action potential train. Under voltage-clamp, mossy fibre stimulation produced a slow voltage-dependent inward current which resembled that produced by application of exogenous ACPD or quisqualate (QUIS), and which was occluded by these metabotropic agonists. We therefore suggest that synaptically released GLU can induce two types of postsynaptic responses: a fast excitation through activation of ionotropic receptors and a slower excitation associated with inhibition of K+ conductances through activation of metabotropic receptors. This is analogous to the dual action of acetylcholine on ionotropic (nicotinic) and metabotropic (muscarinic) receptors.

Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters.

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.

Cytosolic free calcium in hippocampal CA3 pyramidal cells.

The dynamics of cytosolic free Ca2+ ([Ca2+]i) of single voltage-clamped CA3 pyramidal cells in hippocampal slice cultures is reviewed. [Ca2+]i amounts to about 30 nM at resting membrane potential and increases slowly when the membrane potential is clamped at more positive values (up to 500 nM at -30 mV). Short lasting depolarizations (40-100 ms) induce a transient rise in [Ca2+]i which activates a slow aftercurrent (IAHP). The muscarinic or beta-adrenergic depression of IAHP is not accompanied by any change in the dynamics of Ca2+ and appears, therefore, to result primarily from an inhibition of the K(+)-current itself or of the ability of Ca2+ to activate the current. At higher concentrations than those required to inhibit IAHP, muscarine produces a pronounced inward current and this is accompanied by a rise in resting [Ca2+]i concentration.

Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology.

The development of oxytocin (OT) receptors in the rat brain and spinal cord was studied by in vitro light microscopic autoradiography and by electrophysiology. OT receptors were labeled using a monoiodinated OT antagonist in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the response of ongoing spike activity to the addition of OT was assessed in neurons located in the dorsal motor nucleus of the vagus nerve of the neonate. Specific binding was detected first at E14 in a region that later differentiated into the dorsal motor nucleus of the vagus nerve. Many other regions were progressively labeled between E20 and PN5. From PN5 to PN16, the distribution of binding sites remained essentially unchanged but differed markedly from that characteristic of the adult. The change-over from the "infant pattern" to the "adult pattern" occurred in 2 stages: the first change took place between PN16 and PN22, a time corresponding to the preweaning period; the second change occurred after PN35 and thus coincided with the onset of puberty. During the first transition period, binding was reduced or disappeared in several areas intensely labeled at earlier stages, in particular, in the cingulate cortex and the dorsal hippocampus. At the same time, binding sites appeared in the ventral hippocampus. At puberty, high densities of OT binding sites appeared in the ventromedial hypothalamic nucleus and the olfactory tubercle. Electrophysiological activity was recorded from vagal neurons in slices obtained from animals sacrificed at PN1-PN12. OT and a selective OT agonist reversibly increased the firing rate of these neurons in a concentration-dependent manner. The neuronal responsiveness was similar to that reported previously in the adult. These results suggest that OT binding sites detected by autoradiography in the developing rat brain represent, at least in some areas, functional neuronal receptors.

Vasopressin excites neurones located in the dorsal cochlear nucleus of the guinea-pig brainstem.

The effect of arginine vasopressin (AVP) on neurones in the dorsal cochlear nucleus (DCN) of young guinea-pigs of either sex was investigated in brainstem slices. Most impaled neurones fired in a regular manner, either spontaneously or following a depolarizing current injection. AVP, at concentrations of 10-1000 nM, excited 19/19 neurones from male and 16/19 neurones from female animals. This effect of AVP was concentration-dependent and could be mimicked by the V1 agonist [Phe2,Orn8]VT. Oxytocin was less potent than AVP and a selective V2 agonist, deamino-DAVP, was without effect. Thus, a class of DCN neurones is probably endowed with functional V1 vasopressin receptors. By making use of an antibody raised against the vasopressin-related glycopeptide, dense AVP-like immunoreactivity was found in the DCN of young animals of either sex.

A role of central oxytocin in autonomic functions: its action in the motor nucleus of the vagus nerve.

Neurones located in the dorsal motor nucleus of the vagus nerve were shown, in slices from the rat brainstem, to respond to oxytocin by a concentration-dependent increase in rate of firing. A newly available oxytocin antagonist suppressed the excitatory effect of oxytocin on single neurones; this antagonism was partially reversible. Further evidence that neurones located in the dorsal motor nucleus of the vagus nerve possess oxytocin receptors was obtained from in vitro light microscopical autoradiography using [125I]-labelled oxytocin antagonist. In conjunction with data by others which showed that oxytocin antagonist microinjected into the dorsal motor nucleus of the vagus nerve blocks gastric and cardiac effects caused by stimulation of the hypothalamic paraventricular nucleus, our results suggest a role for central oxytocin in autonomic efferent activity.

Direct inhibition by opioid peptides of neurones located in the ventromedial nucleus of the guinea pig hypothalamus.

In slices of guinea pig brain, intracellular recordings were obtained from neurones of the ventromedial nucleus of the hypothalamus (VMH). [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), an agonist selective for mu-opioid receptors, caused an inhibition of spontaneous firing activity and a membrane hyperpolarization. This effect was reversible, concentration-dependent and could be blocked by naloxone. DAGO directly inhibited VMH neurones since its effect persisted when the slice was perifused with a solution that blocks synaptic transmission. The hyperpolarization induced by DAGO was associated with a marked decrease in membrane input resistance and it was reversed in polarity at membrane potentials 30-40 mV more negative than the resting potential. A chloride current did not contribute to the hyperpolarization brought about by DAGO. We conclude that DAGO inhibits VMH neurones, probably by opening membrane potassium channels.

Electrophysiological evidence for oxytocin receptors on neurones located in the dorsal motor nucleus of the vagus nerve in the rat brainstem.

Intracellular recordings were obtained from vagal neurones and their response to oxytocin was investigated in slices from the rat brainstem. Following recording, Lucifer Yellow was injected into the cells in order to verify their localization within the dorsal motor nucleus of the vagus nerve. Virtually all neurones throughout the rostro-caudal extent of the nucleus increased their rate of firing in the presence of 10-1000 nM oxytocin and their membrane depolarized in a reversible, concentration-dependent manner. This excitation was probably exerted directly on the impaled cells rather than being synaptically mediated, since it persisted in a low calcium-high magnesium medium or in the presence of tetrodotoxin. These data provide evidence for a direct membrane effect of oxytocin on a defined population of neurones in the rat brain.