A single psychotomimetic dose of ketamine decreases thalamocortical spindles and delta oscillations in the sedated rat.

BACKGROUND: In patients with psychotic disorders, sleep spindles are reduced, supporting the hypothesis that the thalamus and glutamate receptors play a crucial etio-pathophysiological role, whose underlying mechanisms remain unknown. We hypothesized that a reduced function of NMDA receptors is involved in the spindle deficit observed in schizophrenia. METHODS: An electrophysiological multisite cell-to-network exploration was used to investigate, in pentobarbital-sedated rats, the effects of a single psychotomimetic dose of the NMDA glutamate receptor antagonist ketamine in the sensorimotor and associative/cognitive thalamocortical (TC) systems. RESULTS: Under the control condition, spontaneously-occurring spindles (intra-frequency: 10-16 waves/s) and delta-frequency (1-4 Hz) oscillations were recorded in the frontoparietal cortical EEG, in thalamic extracellular recordings, in dual juxtacellularly recorded GABAergic thalamic reticular nucleus (TRN) and glutamatergic TC neurons, and in intracellularly recorded TC neurons. The TRN cells rhythmically exhibited robust high-frequency bursts of action potentials (7 to 15 APs at 200-700 Hz). A single administration of low-dose ketamine fleetingly reduced TC spindles and delta oscillations, amplified ongoing gamma-(30-80 Hz) and higher-frequency oscillations, and switched the firing pattern of both TC and TRN neurons from a burst mode to a single AP mode. Furthermore, ketamine strengthened the gamma-frequency band TRN-TC connectivity. The antipsychotic clozapine consistently prevented the ketamine effects on spindles, delta- and gamma-/higher-frequency TC oscillations. CONCLUSION: The present findings support the hypothesis that NMDA receptor hypofunction is involved in the reduction in sleep spindles and delta oscillations. The ketamine-induced swift conversion of ongoing TC-TRN activities may have involved at least both the ascending reticular activating system and the corticothalamic pathway.

Medium-voltage 5-9-Hz oscillations give rise to spike-and-wave discharges in a genetic model of absence epilepsy: in vivo dual extracellular recording of thalamic relay and reticular neurons.

In humans with absence epilepsy, spike-and-wave discharges develop in the thalamocortical system during quiet immobile wakefulness or drowsiness. The present study examined the initial stage of the spontaneous development of spike-and-wave discharges in Genetic Absence Epilepsy Rats from Strasbourg. Bilateral electrocorticograms were recorded in epileptic and non-epileptic rats under freely moving and undrugged conditions and under neuroleptanalgesia. Short-lasting episodes of medium-voltage 5-9-Hz (mean=6-Hz) oscillations usually emerged spontaneously from a desynchronized electrocorticogram and in bilateral synchrony in both rat strains. These oscillations were distinguishable from sleep spindles regarding their internal frequency, duration, morphology, and moment of occurrence. Spontaneous spike-and-wave discharges developed from such synchronized medium-voltage oscillations, the spike-and-wave complex occurring at the same frequency as the 5-9-Hz wave. Because the thalamus is thought to play a significant role in the generation of spike-and-wave discharges, dual extracellular recording and juxtacellular labelling of relay and reticular neurons were conducted to study the thalamic cellular mechanisms associated with the generation of spike-and-wave discharges. During medium-voltage 5-9-Hz oscillations, discharges of relay and reticular cells had identical patterns in epileptic and non-epileptic rats, consisting of occasional single action potentials and/or bursts (interburst frequency of up to 6-8 Hz) in relay cells, and of rhythmic bursts (up to 12-15 Hz) in reticular neurons, these discharging in the burst mode almost always before relay neurons. The discharge frequency of reticular bursts decelerated to 6 Hz by the beginning of the spike-and-wave discharges. During these, relay and reticular neurons usually fired in synchrony a single action potential or a high-frequency burst of two or three action potentials and a high-frequency burst, respectively, about 12 ms before the spike component of the spike-and-wave complexes. The frequency of these corresponded to the maximal frequency of the thalamocortical burst discharges associated with 5-9-Hz oscillations. The patterns of relay and reticular phasic cellular firings associated with spike-and-wave discharges had temporal characteristics similar to those associated with medium-voltage 5-9-Hz oscillations, suggesting that these normal and epileptic oscillations are underlain by similar thalamic cellular mechanisms. In conclusion, medium-voltage 5-9-Hz oscillations in the thalamocortical loop give rise to spike-and-wave discharges. Such oscillations are not themselves sufficient to initiate spike-and-wave discharges, meaning that genetic factors render thalamocortical networks prone to generate epileptic electrical activity, possibly by decreasing the excitability threshold in reticular cells. While these GABAergic neurons play a key role in the synchronization of glutamatergic relay neurons during seizures, relay cells may participate significantly in the regulation of the recurrence of the spike-and-wave complex. Furthermore, it is very likely that synchronization of relay and reticular cellular discharges during absence seizures is generated in part by corticothalamic inputs.

Anatomical evidence for a mechanism of lateral inhibition in the rat thalamus.

The aim of this study was to determine whether or not thalamic reticular nucleus (Rt) neurons form synaptic connections with the thalamocortical (TC) neurons from which they receive synaptic contacts. Therefore, we examined, in adult rats, the relationships between single TC and Rt neurons, which had been marked simultaneously with an anterograde/retrograde tracer (biocytin or Neurobiotin), using the extracellular or juxtacellular technique. (i) From 30 successful extracellular microapplications of marker into the Rt, 22 gave retrogradely marked TC somatodendritic arbors at the fringe of or clear outside the anterogradely darkly stained Rt axon terminal fields. Following biocytin application into the thalamus, few cells were retrogradely stained in the Rt at the periphery of the anterogradely labelled axon terminal field. (ii) The juxtacellular filling of a single Rt cell was accompanied by the back-filling of a single TC neuron (n = 4 pairs), which presumably formed synaptic contacts with the former cell. The somatodendritic complex of the back-filled TC neuron was located outside the Rt cell's axonal arbor. These anatomical data provide clear evidence that Rt and thalamic neurons predominantly form between themselves open rather than closed loop connections. Because TC neurons make glutamatergic synapses onto Rt cells, which are GABAergic, and are the first elements synaptically activated by prethalamic afferents into the TC-Rt network, the present results strongly support the hypothesis that Rt neurons principally generate a mechanism of lateral inhibition in the thalamus.

Intracellular recordings in thalamic neurones during spontaneous spike and wave discharges in rats with absence epilepsy.

1. In vivo extracellular and intracellular recordings were performed from thalamocortical (TC) neurones in a genetic model of absence epilepsy (genetic absence epilepsy rats from Strasbourg) during spontaneous spike and wave discharges (SWDs). 2. Extracellularly recorded single units (n = 14) fired either a single action potential or a high frequency burst of up to three action potentials, concomitantly with the spike component of the spike-wave complex. 3. Three main events characterized the intracellular activity of twenty-six out of twenty-eight TC neurones during SWDs: a small amplitude tonic hyperpolarization that was present throughout the SWD, rhythmic sequences of EPSP/IPSPs occurring concomitantly with the spike-wave complexes, and a small tonic depolarization at the end of the SWD. The rhythmic IPSPs, but not the tonic hyperpolarization, were mediated by activation of GABAA receptors since they reversed in polarity at -68 mV and appeared as depolarizing events when recording with KCl-filled electrodes. 4. The intracellular activity of the remaining two TC neurones consisted of rhythmic low threshold Ca2+ potentials, with a few EPSP/IPSP sequences present at the start of the SWD. 5. These results obtained in a well-established genetic model of absence epilepsy do not support the hypothesis that the intracellular activity of TC neurones during SWDs involves rhythmic sequences of GABAB IPSPs and low threshold Ca2+ potentials.

Projection and innervation patterns of individual thalamic reticular axons in the thalamus of the adult rat: a three-dimensional, graphic, and morphometric analysis.

The gamma-aminobutyric acid-ergic thalamic reticular nucleus (Rt), which carries matching topographical maps of both the thalamus and cortex and in which constituent cells can synaptically communicate between each other, is the major extrinsic source of thalamic inhibitions and disinhibitions. Whether all the Rt axonal projections into the thalamus are similarly organized and have common projection and innervation patterns are questions of great interest to further our knowledge of the functioning of the Rt. The present study provides architectural and morphometric data of individual, anterogradely labeled axonal arbors that arose from distinct parts of the Rt. One hundred twenty-seven Rt neurons from all regions of Rt were marked juxtacellularly with biocytin or Neurobiotin in urethane-anesthetized adult rats. Eighteen two-dimensional and 14 three-dimensional reconstructions of single tracer-filled Rt neurons were made from serial, frontal, horizontal, or sagittal sections. Both the somatodendritic and axonal fields of tracer-filled Rt cells were mapped in three dimensions and illustrated to provide a complementary stereotaxic reference for future studies. Most marked units projected to a single nucleus of the anterior, dorsal, intralaminar, posterior, or ventral thalamus. Axons emerging from cells in distinct sectors of the Rt projected to distinct nuclei. Within a sector, neurons with separate dendritic fields innervated separate regions either in a single nucleus or into different but functionally related thalamic nuclei. Neurons with an overlap of their dendritic fields gave rise either to overlapping axonal arborizations or, more rarely, to distinct axonal arbors within two different thalamic nuclei implicated in the same function. In rare instances, an Rt axon could project within these two nuclei. Thalamic reticular axons commonly displayed a single well-circumscribed arbor containing a total of about 4,000 +/- 1,000 boutons. Every arbor was composed of a dense central core, which encompassed a thalamic volume of 5-63 x 10(6) microm3 and was made up of patches of maximal innervation density (10 +/- 4 boutons/tissue cube of 25 microm each side), surrounded by a sparse component. The metric relationships between the Rt axonal arbors and the dendrites of their target thalamocortical neurons were determined. Both the size and maximal innervation density of the axonal patches were found to fit in with the somatodendritic architecture of the target cells. The Rt axonal projections of adult rats are thus characterized by their (1) well-focused terminal field with a patchy distribution of boutons and (2) parallel organization with a certain degree of divergence. The role of the Rt-mediated thalamic inhibition and disinhibition may be to contrast significant with nonrelevant ongoing thalamocortical information.

Dendrodendritic and axoaxonic synapses in the thalamic reticular nucleus of the adult rat.

Currently, it is believed that cell-cell communications occur in the thalamic reticular nucleus (RT) during thalamocortical operations, but the anatomical substrate underlying these intrinsic interactions has not been characterized fully in the rat yet. To further our knowledge on this issue, we stained juxtacellularly rat RT neurons with biocytin or Neurobiotin and examined their intrinsic axon collaterals and "axon-like processes" at both light and electron microscopic levels. Of 111 tracer-filled RT cells for which the axon could be followed from its origin up to the thalamus, 12 displayed short-range, poorly ramifying varicose local axon collaterals, which remained undistinguishable from parent distal dendrites, raising the question as to whether their varicosities were presynaptic terminals. Correlated light and electron microscopic observations of the proximal part of these intrinsic varicose axonal segments revealed that their varicosities and intervaricose segments were, in fact, postsynaptic structures contacted by a large number of boutons that, for the most, formed asymmetric synapses and were nonimmunoreactive for GABA. Similarly, the so-called "axon-like processes" stemming from the soma or dendrites also were identified as postsynaptic structures. Two unexpected observations were made in the course of this analysis. First, the hillock and initial segment of some RT axons were found to receive asymmetric synaptic inputs from GABA-negative terminals. Second, examination of serial ultrathin sections of dendritic bundles cut in their longitudinal plane revealed the existence of several short symmetric dendrodendritic synapses and numerous puncta adhaerentia between component dendrites. In conclusion, dendrodendritic junctions might be a prominent anatomical substrate underlying interneuronal communications in the RT of the adult rat. Furthermore, excitatory axoaxonic synapses on the axon hillock, initial segment, and local axon collaterals might represent a powerful synaptic drive for synchronizing the firing of RT neurons. Future studies are essential to verify whether excitatory axoaxonic synapses with the axon hillock are a general feature in the RT.

A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin.

We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: approximately 1 micron), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and 'tickled' by the juxtamembranous iontophoretic pulses. Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate ( > 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.

Single striatofugal axons arborizing in both pallidal segments and in the substantia nigra in primates.

The striatofugal fiber system in primates is believed to be composed of separate subsystems terminating in either the external (GPe) or internal (GPi) segment of the globus pallidus, or in the substantia nigra (SN). At variance with this concept is the present demonstration of single biocytin-labeled striatofugal axons that arborize in the three major target structures of the striatum in cynomolgus monkeys. Out of nine single-labeled axons that were analyzed in detail, one terminated exclusively in GPc, another in both GPc and GPi, whereas the rest arborized in GPe, GPi and SN. The axons that branched in the three sites had one preferential recipient structure where they arborized profusely and formed typical woolly fibers. These findings suggest that, in contrast to previous beliefs based on results of retrograde double-labeling studies, most striatofugal axons arborize within more than one striatal target structures in primates.

Backpropagation of action potentials generated at ectopic axonal loci: hypothesis that axon terminals integrate local environmental signals.

This review deals with the fascinating complexity of presynaptic axon terminals that are characterized by a high degree of functional distinctiveness. In vertebrate and invertebrate neurons, all-or-none APs can take off not only from the axon hillock, but also from ectopic axonal loci including terminals. Invertebrate neurons display EAPs, for instance alternating with somatic APs, during survival functions. In vertebrate, EAPs have been recorded in the peripheral and central nervous systems in time relationship with physiological or pathological neuronal activities. In motor or sensory axon, EAP generation may be the cause of motor dysfunctioning or sensory perceptions and pain respectively. Locomotion is associated with rhythmic depolarizations of the presynaptic axonal membrane of primary afferents, which are ridden by robust EAP bursts. In central axons lying within an epileptic tissue EAP discharges, coinciding with paroxysmal ECoG waves, get longer as somatic discharges get shorter during seizure progression. Once invaded by an orthodromic burst, an ectopic axonal locus can display an EAP after discharge. Such loci can also fire during hyperpolarization or the postinhibitory excitatory period of the parent somata, but not during their tonic excitation. Neurons are thus endowed with electrophysiological intrinsic properties making possible the alternate discharges of somatic APs and EAPs. In invertebrate and vertebrate neurons, ectopic axonal loci fire while the parent somata stop firing, further suggesting that axon terminal networks are unique and individual functional entities. The functional importance of EAPs in the nervous systems is, however, not yet well understood. Ectopically generated axonal APs propagate backwards and forwards along the axon, thus acting as a retrograde and anterograde signal. In invertebrate neurons, somatically and ectopically generated APs cannot have the same effect on the postsynaptic membrane. As suggested by studies related to the dorsal root reflex, EAPs may not only be implied in the presynaptic modulation of transmitter release but also contribute significantly during their backpropagation to a powerful control (collision process) of incoming volleys. From experimental data related to epileptiform activities it is proposed that EAPs, once orthodromically conducted, might potentiate synapses, initiate, spread or maintain epileptic cellular processes. For instance, paroxysmal discharges of EAPs would exert, like a booster-driver, a powerful synchronizing synaptic drive upon a large number of excitatory and inhibitory postsynaptic neurons. We have proposed that, once backpropagated, EAPs are likewise capable of initiating (and anticipating) threshold and low-threshold somatodendritic depolarizations. Interestingly, an antidromic EAP can modulate the excitability of the parent soma.(ABSTRACT TRUNCATED AT 400 WORDS)

The thalamic reticular nucleus does not send commissural projection to the contralateral parafascicular nucleus in the rat.

The reticular nucleus of the thalamus (NRT) projects to virtually all thalamic nuclei ipsilaterally. In addition, recent studies suggest that NRT sends contralateral projections through an intrathalamic commissural fiber system to several thalamic nuclei, including the NRT itself. In the present study we used retrograde cell labeling, multi-unit anterograde labeling and immunohistochemical methods to study both ipsi- and contralateral NRT projection to the parafascicular nucleus (Pf) in the rat. Injections of the fluorescent tracers true blue or fluorogold in Pf led to massive retrograde cell labeling in rostral and dorsal portions of the ipsilateral NRT, whereas the same sectors of the contralateral NRT were devoid of labeling. Some retrogradely labeled cells were nevertheless present on the contralateral side in the borderline region between NRT and the zona incerta (ZI). Retrograde cell labeling experiments with cholera toxin B subunit (CTb) combined to immunohistochemistry for parvalbumin (PV) and calbindin D-28k (CB) indicated that the few retrogradely labeled cells encountered at the border between NRT and ZI displayed immunoreactivity for CB but not for PV. Since PV and CB label neurons belonging to NRT and ZI, respectively, it is concluded that these contralateral retrogradely labeled cells belong to ZI and not to NRT. Multi-unit cell anterograde labeling experiments with biocytin showed that NRT cells that project to Pf arborize extensively only on the ipsilateral side. The same approach, however, has revealed NRT cells projecting to both ipsi- and contralateral ventromedial thalamic nuclei. The axon of these NRT neurons arborizes more profusely ipsilaterally than contralaterally. These results reveal that the NRT projection to Pf in rodents is strictly unilateral. These findings are at variance with the emerging concept that NRT exerts a prominent bilateral influence upon most thalamic nuclei.

The axonal arborization of single thalamic reticular neurons in the somatosensory thalamus of the rat.

This study describes the axonal projections of single neurons of the thalamic reticular complex within the somatosensory thalamic nuclei in rats. Experiments were performed under urethane anaesthesia and reticular cells were labelled by extracellular microiontophoretic applications of biocytin. The axonal arborization of 25 thalamic reticular cells projecting to the ventrobasal (VB) nucleus and/or to the posterior thalamic (Po) complex were reconstructed from serial horizontal sections. Reticular cells labelled with biocytin display somatodendritic features similar to those reported previously. Their cell body is fusiform and their dendrites bear few spines and show a high degree of streaming along the horizontal curved axis of the nucleus. In most cells, axon-like beaded processes stem out from dendrites but, contrary to previous descriptions, no intrareticular axonal collateral was observed. The axonal arborization of most thalamic reticular cells is confined within the limits of a single thalamic nucleus; only two neurons were seen projecting to both the VB and the Po nuclei. In VB, termination fields form short rods (diameter approximately 150 microns, length approximately 200-300 microns) densely packed with grape-like boutons and varicosities; termination fields in Pro are larger, much less dense, and they are contained within a horizontal slab of tissue (thickness approximately 200 microns, mediolateral width approximately 400 microns, rostrocaudal length approximately 1 mm. By charting the position of all labelled cells within the thickness of the thalamic reticular complex, a strip-like arrangement was revealed. Cells projecting to Po occupy the innermost portion of the nucleus whereas those projecting to the ventral-posteromedial and ventral-posterolateral nuclei are located respectively in the middle and in the outer tiers of the nucleus. This strip-like reciprocity was confirmed by separate biocytin injections performed in VB and in Po. These results show that inhibition of reticular origin is distributed within the rat dorsal thalamus in a highly specific manner, most likely according to a principle of reciprocity within the somatotopic representation of the body.

Corticothalamic projections from the cortical barrel field to the somatosensory thalamus in rats: a single-fibre study using biocytin as an anterograde tracer.

This study investigated the pattern of axonal projections of single corticothalamic neurons from the cortical barrel field representing the vibrissae in the rat. Microiontophoretic injections of biocytin were performed in cortical layers V and VI to label small pools of corticothalamic cells and their intrathalamic axonal projections. After a survival period of 48 h, the animals were perfused and the tissue was processed for biocytin histochemistry. On the basis of the intrathalamic distribution of axonal fields and of the types of terminations found in the thalamus, four types of corticothalamic projections were identified. (i) Cells of the upper part of layer VI projected exclusively to the ventral posteromedial (VPm) nucleus, where they arborized in long rostrocaudally oriented bands or 'rods'. (ii) All cells of the lower part of layer VI projected to the medial part of the thalamic posterior group (Pom) but the vast majority of them also collateralized in VPm where they participated in the formation of rods. (iii) A minority of corticothalamic cells in the lower portion of layer VI, possibly located under the interbarrel spaces (septae), arborized exclusively in Pom. (iv) The corticothalamic projection of layer V cells originated from collaterals of corticofugal cells whose main axons ran caudally towards the brainstem. These collaterals arborized exclusively in Pom or in the central lateral nucleus. All corticothalamic cells from layer VI displayed the same type of axonal network, made of long branches decorated by terminal buttons emitted en passant at the tip of fine stalks. Corticothalamic fibres arising from layer V pyramids, however, remained smooth as they ran across the lateral thalamus and they generated in Pom one or two clusters of large boutons. All corticothalamic axons derived from layer VI cells, but not those derived from layer V cells, gave off collaterals as they traversed the thalamic reticular complex. These observations are discussed in the light of previous studies bearing on the topological organization and function of corticothalamic projections to VPm and Pom in rats. The possibility that a similar cellular specificity and a similar organizational plan may characterize corticothalamic relationships in other sensory systems is also considered.

Thalamic reticular input to the rat visual thalamus: a single fiber study using biocytin as an anterograde tracer.

This study describes the axonal projections of single thalamic reticular (TR) neurons within the visual thalamus in rats. Experiments were performed under urethane anesthesia and reticular cells were labeled by extracellular or juxtacellular microiontophoretic applications of biocytin. The axonal arborizations of 19 TR cells projecting to the dorsal lateral geniculate nucleus (DLG) or to the lateral dorsal/lateral posterior complex (LD/LP) were reconstructed from serial horizontal sections. It was found that single TR cells projected within the limits of a single thalamic nucleus, either the DLG or the LD/LP complex, where their terminal fields formed rostrocaudally oriented rods (length: approximately 800 microns; diameter: approximately 100 microns) densely packed with grape-like boutons and varicosities. In addition, none of the labeled TR cells possessed recurrent axonal collaterals that ramified within the reticular complex itself. The functional implications of these morphological data for the synchronization of thalamic oscillations are discussed.

Corticothalamic projections from layer V cells in rat are collaterals of long-range corticofugal axons.

The vast majority of corticothalamic (CT) axons projecting to sensory-specific thalamic nuclei arise from layer VI cells but intralaminar and associative thalamic nuclei also receive, to various degrees, a cortical input from layer V pyramidal cells. It is also well established that all long-range corticofugal projections reaching the brainstem and spinal cord arise exclusively from layer V neurons. These observations raise the possibility that the CT input from layer V cells may be collaterals of those long-range axons projecting below thalamic level. The thalamic projections of layer V cells were mapped at a single cell level following small microiontophoretic injections of biocytin performed in the motor, somatosensory and visual cortices in rats. Camera lucida reconstruction of these CT axons revealed that they are all collaterals of long-range corticofugal axons. These collaterals do not give off axonal branches within the thalamic reticular nucleus and they arborize exclusively within intralaminar and associative thalamic nuclei where they from small clusters of varicose endings. As layer V cells are involved in motor commands everywhere in the neocortex, these CT projections and their thalamic targets should be directly involved in the central organization of motor programs.

Golgi-like labeling of a single neuron recorded extracellularly.

We describe a novel and powerful extracellular method for the staining of a single neuron identified by electrophysiological criteria. This single-unit technique involves the use of glass micro-electrodes (tip diameter: 1.5-2.5 microns) filled with a saline solution (NaCl; 0.5 M) containing 1.5% of biocytin or Neurobiotin. Once a neuron is recorded, isolated and identified, the tracer is delivered by anodal current pulses of a few nA. The cell must remain well isolated and alive during the ejection procedure to ensure optimal staining. Evidence is provided that the labeled neuron is actually the one that was recorded. Our simple method is reliable with a success rate exceeding 85%. The advantages and pitfalls are discussed. This single-unit labeling technique could further be combined with any other procedures ranging from biological to behavioral studies.

Muscarinic inhibition of reticular thalamic cells by basal forebrain neurones.

Pressure injections of a Ringer's solution containing glutamate (10 or 20 mM) into the nucleus basalis of Meynert inhibited the tonic firing of reticular thalamic (RT) neurones which were recorded in rats under deep urethane anaesthesia. When the inhibition was strong enough to stop the discharges, a burst firing mode was induced in RT cells before the recovery of tonic discharges. This glutamate-induced inhibition of RT cells was completely abolished following the systemic administration of the muscarinic antagonist, scopolamine (150 micrograms kg-1, i.v.). These results indicate that the cholinergic cells of the nucleus basalis of Meynert can control the mode of discharges of RT neurones through the activation of muscarinic receptors.

Control of 40-Hz firing of reticular thalamic cells by neurotransmitters.

This study bears on the control exerted by neurotransmitters on the expression of a 40-Hz pacemaker activity observed in reticular thalamic cells. Experiments were conducted in urethane-anaesthetized rats using extracellular recordings and local applications of antagonists against the neurotransmitters involved in the modulation of reticular thalamic cells. All drugs were dissolved in a Ringer's solution (pH 7.4) and were applied in small quantities (25-150 nl) by pressure through one barrel of a micropipette assembly. Forty-Hertz firing was abolished by local application of the alpha 1 antagonist prazosin and by bilateral lesion of the locus coeruleus. Local applications of glutamate antagonists reduced the rate of discharges by 30-50% as did cortical cooling or complete transection of the internal capsule. Conversely, scopolamine exerted a permissive action on the expression of 40-Hz activities; many spontaneously bursting units started firing at 40 Hz under the influence of this muscarinic antagonist. Since reticular thalamic cells are GABAergic and synaptically coupled via axonal collaterals, we investigated how GABAergic drugs affected the regular firing of these cells. Local applications of bicuculline produced a transient increase of the firing rates while the application of GABA induced intermittent pauses on a background of regular discharges. The application of piperidine-4-sulphonic acid, a GABAA receptor agonist, produced a similar effect. The length of pauses generated by piperidine was statistically analysed. It was found that the duration of short pauses was a multiple integer of the mean interspike interval of surrounding discharges. The preservation of the period and phase of the rhythm across the pauses implies that a subthreshold oscillation was presented into the cells during the arrests of discharges. Given the mode of action of noradrenaline and acetylcholine on reticular thalamic neurons, and considering a possible metabotropic action of glutamate, the above results suggest that deactivation of a leaky K conductance is critically involved in the regular firing of these cells in urethane-anaesthetized rats. Alternatively, because reticular cells are coupled via inhibitory synapses, it is proposed that the 40-Hz firing frequency reflects, in the frequency domain, a point of equilibrium in the reticular thalamic network when the leaky K conductance is fully deactivated by the metabotropic effects of monoamines and/or excitatory amino acids.

The origin of rhythmic fast subthreshold depolarizations in thalamic relay cells of rats under urethane anaesthesia.

Intracellular recordings were performed in relay neurons of the dorsal thalamus in rats under urethane anaesthesia. In 77 out of 127 neurons of the ventro-posterolateral and ventral lateral nuclei, but not in neurons of the ventro-posteromedial and posterior nuclei, a highly rhythmic pattern of subthreshold depolarizations was present at rest. The average frequency of these rhythmic depolarizations in ventro-posterolateral cells was 23.36 +/- 11.48 Hz (range: 6-60 Hz); in ventral lateral relay cells higher frequencies were observed (65.86 +/- 17.42 Hz; range: 17-95 Hz). The rhythmic subthreshold events were identified as excitatory postsynaptic potentials generated by the regular firing of prethalamic afferents located in dorsal column and deep cerebellar nuclei. Indeed, in cells of the ventro-posterolateral nucleus these spontaneous potentials had a waveform similar to that of synaptic potentials triggered by somatosensory stimulation. They increased in amplitude with membrane hyperpolarization and their rhythmic occurrence was not affected by the injection of large inward currents. Moreover, they persisted after capsular transection, but they could no more be recorded in ventro-posterolateral cells after lesion of dorsal column nuclei. Finally, it was found that prethalamic afferents within the deep cerebellar nuclei discharged spontaneously in a rhythmic manner within the same frequency band as that of the rhythmic synaptic potentials recorded in ventral lateral cells. On the basis of these results, it is concluded that the rhythmic subthreshold depolarizations observed in thalamic neurons of animals under urethane anaesthesia are not generated intrinsically but that they represent excitatory postsynaptic potentials of prethalamic origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-dependent 40-Hz oscillations in rat reticular thalamic neurons in vivo.

Extra- and intracellular recordings of thalamic reticular and relay neurons were performed in rats under urethane anaesthesia. Under this type of anaesthesia it was found that, throughout the whole reticular thalamic nucleus, a large proportion of cells (approximately 34%) discharged like clocks within a 25-60 Hz frequency band width (i.e. 40 Hz). Simultaneous recordings of pairs of reticular cells showed that the regular discharges of nearby units were not synchronous. Thus, the asynchronous 40-Hz firing of reticular thalamic cells was not correlated with any 40-Hz extracellular activity as revealed by the spectral analysis of the electroencephalogram and by recordings performed in various thalamic nuclei. In relay cells of the ventrobasal, ventral lateral and posterior thalamic nuclei, the regular firing of reticular thalamic neurons induced a rhythmic inhibitory modulation that was detected by the time-series analysis of the inhibitory postsynaptic potentials. In many relay cells, however, the disclosure of this inhibitory modulation required cellular depolarization since the resting potential in these cells was maintained at the reversal potential of the inhibitory events. Intracellular recordings of reticular thalamic cells showed that their regular firing was not driven in an all-or-nothing manner by 40-Hz synaptic inputs but rather that it depended upon the activation of a voltage-dependent pacemaker mechanism; this pacemaker activity was manifested by the presence of subthreshold oscillations that drove spike discharges and whose frequency was voltage dependent. In the context of data already published on the genesis of 40-Hz oscillations in the brain, and given the key position of reticular thalamic neurons in thalamocortical networks, the present results indicate that the reticular thalamic nucleus might play a pacemaker function in the genesis of 40-Hz oscillations in the thalamus and cortex during states of focused arousal.

Ectopic axonal firing in an epileptic cortical focus is not triggered by thalamocortical volleys during the interictal stage.

The triggering of ectopic action potentials (APs) at axon terminals in a chronic epileptic cobalt focus was investigated in thalamocortical (TC) neurons of rats under urethane anesthesia. TC cells which were in register with an active epileptic aggregate discharged bursts of 2-11 APs. According to the rules of the collision test, we ascertained that bursts contained APs of ectopic and/or somatic origin. During a transient blockage of TC orthodromic discharges produced by raising the extracellular concentration of Mg2+, ectopic bursts which were in close time relationship with the focal interictal electrocorticographic spikes persisted. These results demonstrate (i) the antidromic nature of identified ectopic APs and (ii) that, during the interictal stage, such axonal APs were not a consequence of TC discharges. The possible mechanisms for the triggering of ectopic axonal APs in the chronic cobalt focus are discussed.