Patch-clamp recording in brain slices with improved slicer technology.

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality. In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis. We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leaf-spring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency ( approximately 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation. A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a light-emitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 microm, significantly less than many commercial tissue slicers. Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

Rapid signaling at inhibitory synapses in a dentate gyrus interneuron network.

Mutual synaptic interactions between GABAergic interneurons are thought to be of critical importance for the generation of network oscillations and for temporal encoding of information in the hippocampus. However, the functional properties of synaptic transmission between hippocampal interneurons are largely unknown. We have made paired recordings from basket cells (BCs) in the dentate gyrus of rat hippocampal slices, followed by correlated light and electron microscopical analysis. Unitary GABA(A) receptor-mediated IPSCs at BC-BC synapses recorded at the soma showed a fast rise and decay, with a mean decay time constant of 2.5 +/- 0.2 msec (32 degrees C). Synaptic transmission at BC-BC synapses showed paired-pulse depression (PPD) (32 +/- 5% for 10 msec interpulse intervals) and multiple-pulse depression during repetitive stimulation. Detailed passive cable model simulations based on somatodendritic morphology and localization of synaptic contacts further indicated that the conductance change at the postsynaptic site was even faster, decaying with a mean time constant of 1.8 +/- 0.6 msec. Sequential triple recordings revealed that the decay time course of IPSCs at BC-BC synapses was approximately twofold faster than that at BC-granule cell synapses, whereas the extent of PPD was comparable. To examine the consequences of the fast postsynaptic conductance change for the generation of oscillatory activity, we developed a computational model of an interneuron network. The model showed robust oscillations at frequencies >60 Hz if the excitatory drive was sufficiently large. Thus the fast conductance change at interneuron-interneuron synapses may promote the generation of high-frequency oscillations observed in the dentate gyrus in vivo.

A hippocampal interneuron associated with the mossy fiber system.

Network properties of the hippocampus emerge from the interaction of principal cells and a heterogeneous population of interneurons expressing gamma-aminobutyric acid (GABA). To understand these interactions, the synaptic connections of different types of interneurons need to be elucidated. Here we describe a type of inhibitory interneuron of the hippocampal CA3 region that has an axon coaligned with the mossy fibers. Whole-cell patch-clamp recordings, in combination with intracellular biocytin filling, were made from nonpyramidal cells of the stratum lucidum under visual control. Mossy fiber-associated (MFA) interneurons generated brief action potentials followed by a prominent after-hyperpolarization. Subsequent visualization revealed an extensive axonal arbor which was preferentially located in the stratum lucidum of CA3 and often invaded the hilus. The dendrites of MFA interneurons were mainly located in the strata radiatum and oriens, suggesting that these cells are primarily activated by associational and commissural fibers. Electron microscopic analysis showed that axon terminals of MFA interneurons established symmetric synaptic contacts predominantly on proximal apical dendritic shafts, and to a lesser degree, on somata of pyramidal cells. Synaptic contacts were also found on GABAergic interneurons of the CA3 region and putative mossy cells of the hilus. Inhibitory postsynaptic currents (IPSCs) elicited by MFA interneurons in simultaneously recorded pyramidal cells had fast kinetics (half duration, 3.6 ms) and were blocked by the GABA(A) receptor antagonist bicuculline. Thus, MFA interneurons are GABAergic cells in a position to selectively suppress the mossy fiber input, an important requirement for the recall of memory traces from the CA3 network.

Evidence for the existence of non-GABAergic, cholinergic interneurons in the rodent hippocampus.

Previous studies have revealed a small number of hippocampal interneurons immunoreactive for choline acetyltransferase, the acetylcholine-synthesizing enzyme. It remained an open question, however, whether these neurons represented a subgroup of inhibitory GABAergic neurons co-localizing acetylcholine. In this study, we have combined immunocytochemistry for choline acetyltransferase and in situ hybridization for glutamate decarboxylase messenger RNA, the GABA-synthesizing enzyme. None of the choline acetyltransferase-immunoreactive neurons in the various layers of the hippocampus proper and fascia dentata were found to co-localize glutamate decarboxylase messenger RNA. The lack of an in situ hybridization signal in these neurons is unlikely to result from the combination of the two labeling techniques. When combining in situ hybridization for glutamate decarboxylase messenger RNA with immunostaining for parvalbumin, a calcium-binding protein expressed by many GABAergic hippocampal interneurons, numerous double-labeled cells were observed. These data provide neurochemical evidence for the existence of non-GABAergic, supposedly cholinergic non-principal cells in the hippocampus.

Distal initiation and active propagation of action potentials in interneuron dendrites.

Fast and reliable activation of inhibitory interneurons is critical for the stability of cortical neuronal networks. Active conductances in dendrites may facilitate interneuron activation, but direct experimental evidence was unavailable. Patch-clamp recordings from dendrites of hippocampal oriens-alveus interneurons revealed high densities of voltage-gated sodium and potassium ion channels. Simultaneous recordings from dendrites and somata suggested that action potential initiation occurs preferentially in the axon with long threshold stimuli, but can be shifted to somatodendritic sites when brief stimuli are applied. After initiation, action potentials propagate over the somatodendritic domain with constant amplitude, high velocity, and reliability, even during high-frequency trains.

Unitary IPSPs evoked by interneurons at the stratum radiatum-stratum lacunosum-moleculare border in the CA1 area of the rat hippocampus in vitro.

1. Hippocampal non-principal neurons at the stratum radiatum-stratum lacunosum-moleculare border (R-LM interneurons) of the CA1 area may constitute several cell classes and have been implicated in the generation of GABAergic unitary IPSPs. Using biocytin-filled electrodes we recorded R-LM interneurons intracellularly in vitro and determined their postsynaptic effects in concomitantly recorded pyramidal cells. 2. Light microscopic analysis revealed four populations of R-LM interneurons with distinct axons: (1) basket cells (n = 4) with axons predominantly ramifying in the pyramidal cell layer; (2) Schaffer collateral/commissural pathway-associated interneurons (n = 10) stratifying in stratum radiatum and, to a lesser extent, stratum oriens; (3) perforant pathway-associated interneurons (n = 6) innervating the perforant path termination zone in stratum lacunosum-moleculare of the CA1 area as well as equivalent portions of the dentate gyrus and subiculum; and (4) neurogliaform interneurons (n = 2) characterized by their dense, compact axonal and dendritic arbour. 3. Random electron microscopic sampling of synaptic targets revealed a preponderance of pyramidal neurons as postsynaptic elements. Basket cells had a synaptic target preference for somata and proximal dendrites, whereas the remainder of R-LM interneurons innervated dendritic shafts and spines. The axon of dendrite-targeting cells formed up to six putative contacts with individual postsynatpic pyramidal cells. 4. Anatomically recovered R-LM interneurons (n = 22) had a mean resting membrane potential of -56.7 +/- 3.6 mV, a membrane time constant of 12.9 +/- 7.7 ms and an input resistance of 86.4 +/- 29.2 M omega. Depolarizing current pulses generally elicited overshooting action potentials (70.8 +/- 6.9 mV) which had a mean duration, when measured at half-amplitude, of 0.7 +/- 0.1 ms. In response to prolonged (> 200 ms) depolarizing current pulses all R-LM interneurons displayed (a varying degree of) spike frequency adaptation. 5. Basket cells, Schaffer-associated and neurogliaform interneurons elicited small-amplitude (< 2 mV), short-latency IPSPs in postsynaptic pyramids (n = 5, 13 and 1, respectively). Those interactions in which an effect was elicited with the repetitive activation of the presynaptic neuron (n = 13) showed a substantial degree of postsynaptic response summation. Unitary IPSPs had fast kinetics and, whenever tested (n = 5; 1 basket cell and 4 Schaffer-associated interneurons), were abolished by the GABAA receptor antagonist bicuculline. 6. Thus, R-LM interneurons comprise several distinct populations which evoke fast GABAA receptor mediated IPSPs. The domain-specific innervation of postsynaptic pyramidal cells suggests functionally diverse effects on the integration of afferent information in functionally non-equivalent compartments of pyramidal cells.

Synaptic effects of identified interneurons innervating both interneurons and pyramidal cells in the rat hippocampus.

GABAergic interneurons sculpt the activity of principal cells and are themselves governed by GABAergic inputs. To determine directly some of the sources and mechanisms of this GABAergic innervation, we have used dual intracellular recordings with biocytin-filled microelectrodes and investigated synaptic interactions between pairs of interneurons in area CA1 of the adult rat hippocampus. Of four synaptically-coupled interneuron-to-interneuron cell pairs, three presynaptic cells were identified as basket cells, preferentially innervating somata and proximal dendrites of pyramidal cells, but one differing from the other two in the laminar distribution of its dendritic and axonal fields. The fourth presynaptic interneuron was located at the border between strata lacunosum moleculare and radiatum, with axon ramifying within stratum radiatum. Action potentials evoked in all four presynaptic interneurons were found to elicit fast hyperpolarizing inhibitory postsynaptic potentials (mean amplitude 0.35 +/- 0.10 mV at a membrane potential of -59 +/- 2.8 mV) in other simultaneously recorded interneurons (n=4). In addition, three of the presynaptic interneurons were also shown to produce similar postsynaptic responses in subsequently recorded pyramidal cells (n=4). Electron microscopic evaluation revealed one of the presynaptic basket cells to form 12 synaptic junctions with the perisomatic domain (seven somatic synapses and five synapses onto proximal dendritic shafts) of the postsynaptic interneuron in addition to innervating the same compartments of randomly-selected local pyramidal cells (50% somatic and 50% proximal dendritic synapses, n=12). In addition, light microscopic analysis also indicated autaptic self-innervation in basket (12 of 12) and bistratified cells (six of six). Electron microscopic investigation of one basket cell confirmed six autaptic junctions made by five of its boutons. Together, these data demonstrate that several distinct types of interneuron have divergent output to both principal cells and local interneurons of the same (basket cells) or different type. The fast synaptic effects, probably mediated by GABA in both postsynaptic interneurons and principal cells are similar. These additional sources of GABA identified here in the input to GABAergic cells could contribute to the differential temporal patterning of distinct GABAergic synaptic networks.

A current-source density analysis of the long-term potentiation in the hippocampus.

Tetanic stimulation of presynaptic fibres induces long-term potentiation (LTP) which means enhancement of synaptic efficacy in the stimulated pathway for hours or days. In addition to that component, a permanent change occurs in the postsynaptic cells promoting their discharges. This latter effect called 'EPSP-to-spike (E-S) potentiation' is thought to be mediated by voltage-sensitive channels in the dendrites. Current-source density (CSD) analysis was made in the CA1 area of hippocampal slice preparations to find if LTP causes changes of the transmembrane currents in the stratum radiatum which can be detected with this technique. Some increase of currents associated with synaptic transmission itself at distant dendritic areas was accompanied by a disproportional enhancement of other currents attributed to activation of dendritic membranes at approximately 150 microns away from the pyramidal layer. When this current grew sufficiently large, it propagated towards the cell body layer. In slices where LTP had less E-S potentiation component, the increase in CSD at distant and more proximal portions of the dendrites remained proportional. Paired pulse facilitation induced in the same slices did not produce disproportional enhancement of proximal dendritic currents either. Our findings support the assumptions that during LTP associated with E-S potentiation the probability of activation of voltage-sensitive channel is enhanced on the dendrites of CA1 pyramidal cells.

Responses of forebrain neurons to the MAO-B blocker L-deprenyl.

Despite the large amount of neuropharmacological data concerning catecholamine (CA) mechanisms of the mammalian brain, little is known yet about the effects of MAO-inhibitors on single neurons. The present series of experiments aim to elucidate these specific neurochemical attributes of forebrain cells. Single neuron activity was recorded by means of multi-barreled microelectrodes in the caudate nucleus, globus pallidus, and amygdala of both anesthetized rats and anesthetized or alert monkeys during microelectrophoretic application of the MAO-B blocker L-deprenyl (DEPR). CAs (dopamine and noradrenaline), glutamate, GABA, and acetylcholine were also applied. Nearly the half (46%) of all forebrain neurons tested responded, exclusively with inhibition, to DEPR, and the CA-sensitive cells were especially responsive to the MAO-B inhibitor. The time course of DEPR-induced neuronal suppression was short. In some cases, amphetamine (AMPH) and clorgyline (CLOR) were also applied microelectrophoretically. AMPH elicited similar activity changes to those seen after DEPR administrations, whereas CLOR applications were less effective. Our results provide evidence that DEPR can effectively modulate the activity of CA-sensitive neurons in the three different forebrain regions of two different species. On the basis of this data, the possible neurochemical mechanisms of DEPR action are discussed.

Glucose-sensitive neurons of the globus pallidus: I. Neurochemical characteristics.

The lateral hypothalamic area (LHA) and globus pallidus (GP) are basically involved in the regulation of feeding and metabolic processes. In the LHA, glucose-sensitive (GS) neurons were described: their activity was found to be specifically suppressed by electrophoretic application of glucose, and these neurons appeared to be also influenced by various feeding-associated neurochemical signals. The main goal of the present experiments was to examine whether similar GS neurons exist in the GP. In addition, neurochemical attributes of the cells were also tested. In anesthetized rats and anesthetized or awake monkeys, single-neuron activity of the GP was recorded by means of carbon fiber multibarreled microelectrodes and the effects of glucose, glutamate (Gt), GABA, dopamine (DA), noradrenaline (NA) and acetylcholine (Ach) were studied. In both the rat and monkey GP, approximately 12% of the neurons examined responded, with inhibition, to glucose. GP neurons, in a high proportion, were also inhibited by GABA and NA. After application of Gt, DA, or Ach, activity increase or decrease occurred. GS neurons exhibited remarkable sensitivity to these neurochemicals previously identified as neurotransmitters of the complex pallidal, extrapyramidal-limbic neuron loops. The results, along with previous data, indicate that GS cells of the GP, while possessing complex neurochemical characteristics, may belong to a hierarchically organized central glucose-monitoring system essential in the regulation of feeding.

Glucose-sensitive neurons of the globus pallidus: II. Complex functional attributes.

The globus pallidus (GP) is intimately involved in regulation of various aspects of hunger- and thirst-motivated behaviors. Our parallel neurochemical studies demonstrated the existence of GP neurons whose discharge rates are suppressed by glucose applied microelectrophoretically. In the present series of experiments, we aimed to provide complex, feeding-associated functional characterization--similar to that previously accomplished in the case of lateral hypothalamic and amygdaloid chemosensitive neurons--of these glucose-sensitive (GS) and the glucose-insensitive (GIS) pallidal cells. To do so, extracellular single neuron activity of the GP was recorded in anesthetized rats and anesthetized or awake rhesus monkeys by means of carbon fiber, multibarreled glass microelectrodes during: a) microelectrophoretic administration of chemicals, b) gustatory, and c) olfactory stimulations. In alert primates, activity changes were also recorded during presentation of food and nonfood objects as well as during the performance of a conditioned, high fixed-ratio bar-press feeding task. The half of pallidal cells examined showed firing rate changes during phases of the conditioned alimentary task. In both species, about 1/7 of all neurons tested proved to be GS, while the proportion of cells responding to gustatory and olfactory stimulations was 19% and 16%, respectively. Task-related and taste- and smell-responsive units were mainly found among the GS neurons of the pallidum. These data, along with previous findings, indicate that chemosensitive cells of the GP, in an apparent overlap with units of the central gustatory representation, are involved in a hierarchically organized glucose-monitoring neural network, through which pallidal neurons exert their integrative functions in the central feeding control.

Computer analysis of single neuron activity during conditioned feeding task.

A computer controlled complex electrophysiological set-up employing the multibarrel micro-electrophoretic technique is reported in this paper. The laboratory equipped for this technique is used for recording single neuron activity from various sites of the central nervous system of rhesus monkeys during: 1) performing conditioned behavioral tasks, 2) intracerebral microelectrophoretic administration of chemicals, and 3) oral application of gustatory stimuli.

Feeding disturbances and EEG activity changes after amygdaloid kainate lesions in the rat.

Kainic acid (KA), in various concentrations, was applied iontophoretically into the central nucleus of the amygdala. Microlesions with this cell specific neurotoxin caused body weight loss, hypo- or aphagia and hypo- or adipsia in a dose-dependent manner. EEG-examinations proved that even low doses of KA produced seizure activity; however, these epileptiform symptoms disappeared within the first 48 h after the operations. Thus, the lasting feeding disturbances produced by iontophoretic KA applications to the central nucleus of the amygdala (i.e., even these fine microlesions) were not related causally to the pathological EEG activity changes. Our findings, along with previous data, indicated that the body weight loss and feeding deficits were due to the KA-induced impairment of complex regulatory mechanisms.

[Aneurysm of the internal carotid artery with fatal epistaxis]. / Anevrism al arterei carotide interne, cu epistaxis letal.

The paper reports on a case of aneurysm of the internal carotid artery that broke out by the sphenoidal sinus and manifested itself by a violent, lethal nasal hemorrhage. The necropsy established the cause of the hemorrhage. The elements of diagnosis and therapeutical behaviour required by these extremely severe cases are discussed.