The application of cross-point switch arrays as input selector switch devices for multi-channel electrophysiological experiments.

Integrated circuits (ICs) containing cross-point switch arrays were applied to create analog input selector switch devices for multi-channel electrophysiological experiments. The described analog input selector switch devices make it possible to connect to the main amplifier's inputs those microelectrode and preamplifier output wires that yield unit discharges of acceptable shape and amplitude, or yield other kind of acceptable electrophysiological signals (EEG, EP). This kind of selector allows to use higher number of preamplifier channels and to ignore the input channels without adequate signals or the channels with noisy inputs. No manual switching is required, as the work is done by computer controlled switches. The switch positions can be saved and reloaded at the next experimental session through an I/O port (e.g. the parallel port) of the computer.

Firing rates of hippocampal neurons are preserved during subsequent sleep episodes and modified by novel awake experience.

What determines the firing rate of cortical neurons in the absence of external sensory input or motor behavior, such as during sleep? Here we report that, in a familiar environment, the discharge frequency of simultaneously recorded individual CA1 pyramidal neurons and the coactivation of cell pairs remain highly correlated across sleep-wake-sleep sequences. However, both measures were affected when new sets of neurons were activated in a novel environment. Nevertheless, the grand mean firing rate of the whole pyramidal cell population remained constant across behavioral states and testing conditions. The findings suggest that long-term firing patterns of single cells can be modified by experience. We hypothesize that increased firing rates of recently used neurons are associated with a concomitant decrease in the discharge activity of the remaining population, leaving the mean excitability of the hippocampal network unaltered.

Behavior-dependent states of the hippocampal network affect functional clustering of neurons.

Local versus distant coherence of hippocampal CA1 pyramidal cells was investigated in the behaving rat. Temporal cross-correlation of pyramidal cells revealed a significantly stronger relationship among local (<140 microm) pyramidal neurons compared with distant (>300 microm) neurons during non-theta-associated immobility and sleep but not during theta-associated running and walking. In contrast, cross-correlation between local pyramidal cell-interneuron pairs was significantly stronger than between distant pairs during theta oscillations but were similar during non-theta-associated behaviors. We suggest that network state-dependent functional clustering of neuronal activity emerges because of the differential contribution of the main excitatory inputs, the perforant path, and Schaffer collaterals during theta and non-theta behaviors.

The application of printed circuit board technology for fabrication of multi-channel micro-drives.

A modular multichannel microdrive ('hyperdrive') is described. The microdrive uses printed circuit board technology and flexible fused silica capillaries. The modular design allows for the fabrication of 4-32 independently movable electrodes or 'tetrodes'. The drives are re-usable and re-loading the drive with electrodes is simple.

Firing rate and theta-phase coding by hippocampal pyramidal neurons during 'space clamping'.

In the hippocampus, spatial representation of the environment has been suggested to be coded by either the firing rate of pyramidal cell assemblies or the relative timing of the action potentials during the theta EEG cycle. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) to examine how 'spatial' and other inputs affect firing rate and timing of hippocampal CA1 pyramidal cells and interneurons. Nineteen per cent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel in a given direction ('wheel' cells). Spatial rotation of the apparatus showed that selective discharge of pyramidal cells in the wheel was under the combined influence of distal and apparatus cues. During steady running, both discharge rate and theta phase were constant. Rotation of the wheel apparatus resulted in a shift of both firing rate and preferred theta phase. The discharge frequency of 'wheel' cells increased threefold (on average) with increasing running velocity. In contrast, change in running speed had relatively little effect on the theta phase-related discharge of 'wheel' cells. Our findings indicate that mechanisms that regulate rate and phase of spikes are overlapping but not necessarily identical.

Replay and time compression of recurring spike sequences in the hippocampus.

Information in neuronal networks may be represented by the spatiotemporal patterns of spikes. Here we examined the temporal coordination of pyramidal cell spikes in the rat hippocampus during slow-wave sleep. In addition, rats were trained to run in a defined position in space (running wheel) to activate a selected group of pyramidal cells. A template-matching method and a joint probability map method were used for sequence search. Repeating spike sequences in excess of chance occurrence were examined by comparing the number of repeating sequences in the original spike trains and in surrogate trains after Monte Carlo shuffling of the spikes. Four different shuffling procedures were used to control for the population dynamics of hippocampal neurons. Repeating spike sequences in the recorded cell assemblies were present in both the awake and sleeping animal in excess of what might be predicted by random variations. Spike sequences observed during wheel running were "replayed" at a faster timescale during single sharp-wave bursts of slow-wave sleep. We hypothesize that the endogenously expressed spike sequences during sleep reflect reactivation of the circuitry modified by previous experience. Reactivation of acquired sequences may serve to consolidate information.

Fast network oscillations in the hippocampal CA1 region of the behaving rat.

This study examined intermittent, high-frequency (100-200 Hz) oscillatory patterns in the CA1 region of the hippocampus in the absence of theta activity, i.e., during and in between sharp wave (SPW) bursts. Pyramidal and interneuronal activity was phase-locked not only to large amplitude (>7 SD from baseline) oscillatory events, which are present mainly during SPWs, but to smaller amplitude (<4 SD) patterns, as well. Large-amplitude events were in the 140-200 Hz, "ripple" frequency range. Lower-amplitude events, however, contained slower, 100-130 Hz ("slow") oscillatory patterns. Fast ripple waves reversed just below the CA1 pyramidal layer, whereas slow oscillatory potentials reversed in the stratum radiatum and/or in the stratum oriens. Parallel CA1-CA3 recordings revealed correlated CA3 field and unit activity to the slow CA1 waves but not to fast ripple waves. These findings suggest that fast ripples emerge in the CA1 region, whereas slow (100-130 Hz) oscillatory patterns are generated in the CA3 region and transferred to the CA1 field.

Sustained activation of hippocampal pyramidal cells by 'space clamping' in a running wheel.

In contrast to sensory cortical areas of the brain, the relevant physiological inputs to the hippocampus, leading to selective activation of pyramidal cells, are largely unknown. Pyramidal cells are thought to be phasically activated by spatial cues and a variety of sensory and motor stimuli. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) and kept sensory inputs constant. Twelve percent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel. Cell firing was specific to the direction of running and disappeared after rotating the recording apparatus. The discharge frequency of pyramidal cells and interneurons was sustained as long as the rat ran continuously in the wheel. Furthermore, the discharge frequency of pyramidal cells and interneurons increased with increasing running velocity, even though the frequency of hippocampal theta waves remained constant. The discharge frequency of some 'wheel-related' pyramidal cells could increase more than 10-fold between 10 and 100 cm/s, whereas the firing rate of 'non-wheel' cells remained constantly low. We hypothesize that: (i) a necessary condition for place-specific discharge of hippocampal pyramidal cells is the presence of theta oscillation; and (ii) relevant stimuli can tonically and selectively activate hippocampal pyramidal cells as long as theta activity is present.

Oscillatory coupling of hippocampal pyramidal cells and interneurons in the behaving Rat.

We examined whether excitation and inhibition are balanced in hippocampal cortical networks. Extracellular field and single-unit activity were recorded by multiple tetrodes and multisite silicon probes to reveal the timing of the activity of hippocampal CA1 pyramidal cells and classes of interneurons during theta waves and sharp wave burst (SPW)-associated field ripples. The somatic and dendritic inhibition of pyramidal cells was deduced from the activity of interneurons in the pyramidal layer [int(p)] and in the alveus and st. oriens [int(a/o)], respectively. Int(p) and int(a/o) discharged an average of 60 and 20 degrees before the population discharge of pyramidal cells during the theta cycle, respectively. SPW ripples were associated with a 2.5-fold net increase of excitation. The discharge frequency of int(a/o) increased, decreased ("anti-SPW" cells), or did not change ("SPW-independent" cells) during SPW, suggesting that not all interneurons are innervated by pyramidal cells. Int(p) either fired together with (unimodal cells) or both before and after (bimodal cells) the pyramidal cell burst. During fast-ripple oscillation, the activity of interneurons in both the int(p) and int(a/o) groups lagged the maximum discharge probability of pyramidal neurons by 1-2 msec. Network state changes, as reflected by field activity, covaried with changes in the spike train dynamics of single cells and their interactions. Summed activity of parallel-recorded interneurons, but not of pyramidal cells, reliably predicted theta cycles, whereas the reverse was true for the ripple cycles of SPWs. We suggest that network-driven excitability changes provide temporal windows of opportunity for single pyramidal cells to suppress, enable, or facilitate selective synaptic inputs.

Reliability and state dependence of pyramidal cell-interneuron synapses in the hippocampus: an ensemble approach in the behaving rat.

Spike transmission probability between pyramidal cells and interneurons in the CA1 pyramidal layer was investigated in the behaving rat by the simultaneous recording of neuronal ensembles. Population synchrony was strongest during sharp wave (SPW) bursts. However, the increase was three times larger for pyramidal cells than for interneurons. The contribution of single pyramidal cells to the discharge of interneurons was often large (up to 0.6 probability), as assessed by the presence of significant (<3 ms) peaks in the cross-correlogram. Complex-spike bursts were more effective than single spikes. Single cell contribution was higher between SPW bursts than during SPWs or theta activity. Hence, single pyramidal cells can reliably discharge interneurons, and the probability of spike transmission is behavior dependent.

Severe spatial navigation deficit in the Morris water maze after single high dose of neonatal x-ray irradiation in the rat.

Ambiguous spatial behavior deficits induced in adult rats by different types of dentate gyrus lesions were examined by subjecting neonatal rats to x-ray irradiation, which reduces the granule cell population in fascia dentata without affecting the number of hilar neurons and pyramidal cells of Ammon's horn. Three- to six-month-old irradiated and intact male Long-Evans rats were tested in the Morris water maze. Four experiments were done. (i) Rats were trained to find an invisible escape platform, when started from any of four equidistant points at the circumference of the pool. (ii) The same rats then were trained to find a visible platform in the same pool. Poor performance of irradiated rats in both experiments suggested a visual deficit. (iii) Navigation in the absence of visual cues was studied in other rats trained in total darkness to find the escape platform under conditions of fixed start-fixed goal geometry. (iv) Contribution of nonvisual allocentric cues and egocentric path integration mechanisms to spatial performance of the above rats was tested in darkness after rotating both the start and goal positions by 90 degrees clockwise. Impairment of irradiated rats in Exp. 3 and 4 and histological examination of their brains support the conclusion that 60-70% reduction of granule cells in the dorsal hippocampus causes significant deterioration in both allocentric and egocentric orientation.

Distribution and time course of appearance of "dark" neurons and EEG activity after amygdaloid kainate lesion.

To determine the extent and time course of local and distant neuronal damage produced by microiontophoretic administration of kainic acid (KA) into the central amygdaloid nucleus, distribution of neuronal damage was compared in various brain areas after different survival times. For demonstration of damaged, so-called "dark" neurons, a newly developed silver stain was employed. In addition, silver staining method was used to visualize microglia cells. In a separate experiment, electroencephalographic (EEG) activity was recorded from the amygdaloid body, hippocampus, and the frontal cortex before and after microiontophoretic KA lesion of the central amygdaloid nucleus. It was observed that (1) even a minute amount of KA into this nucleus caused transient neuronal damage in distant brain areas; (2) the hippocampal formation, subiculum, entorhinal cortex, piriform cortex, and lateral septum were consistently affected; (3) the extent and time course of neuronal damage and appearance of microglia cells varied from area to area; (4) the KA neurotoxicity in distant brain areas appeared to depend on specific excitatory circuits, especially in the hippocampal formation; (5) the appearance and time course of pathologic EEG activity paralleled the appearance of dark neurons; and (6) the absence of pathologic EEG activity and the lack of massive neuronal loss or microglia proliferation in distant brain areas of rats surviving longer than 48 h suggested that these areas may have recovered both morphologically and functionally. Although details of cellular mechanism responsible for development of "dark" degeneration of neurons are not known, the silver method employed in the present study proved to be sensitive, useful tool for fine histological analyses of early and distant consequences of excitotoxic lesions.

Intracranial pressure waves generated by high-energy short laser pulses can cause morphological damage in remote areas: comparison of the effects of 2.1-micron Ho:YAG and 1.06-micron Nd:YAG laser irradiations in the rat brain.

BACKGROUND AND OBJECTIVE: Histological effects of 2.1-micron Ho:YAG and 1.06-micron Nd:YAG laser pulses were compared in the rat brain, with special regard to areas remote from the irradiated site. STUDY DESIGN/MATERIALS AND METHODS: Laser pulses were delivered through a 0.6-mm glass fiber, the tip of which was either introduced into the caudate nucleus (application mode I), or held at a 2-mm distance above the exposed intact dura. In the latter case, the space between the dura and the fiber tip was filled either with physiological saline (application mode II) or with air (application mode III). RESULTS: In application modes I and II, but not in application mode III, Ho:YAG laser pulses of 1.5 J and 200 microseconds, but not Nd:YAG laser pulses with the same parameters, immediately caused morphological damage to a considerable number of neurons and axons randomly distributed among apparently normal ones in certain areas remote from the irradiated site. A decrease in the energy and an increase in the length of the pulses lowered the incidence of the remote morphological damage. CONCLUSION: This novel finding may impose limits on the application of Ho:YAG lasers in human endoscopic neurosurgery.

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.

Appearance of deteriorated neurons on regionally different time tables in rat brain thin slices maintained in physiological condition.

Brain slices are widely used for electrophysiological experiments. However, the time table of the chronological cell deterioration and its regional difference in slices are unknown. The argyrophil III staining method can demonstrate deteriorated ('collapsed') neurons specifically. Therefore we studied the appearance of the 'collapsed' neurons with time in coronal brain 'thin' slices maintained in vitro to evaluate possible regional differences in vulnerability. In the hippocampus, CA1 pyramidal, dentate gyrus granule, and non-pyramidal cells in any subfield became argyrophilic most easily, as did layer V-VI pyramidal neurons in the neocortex. These results suggest that some subclasses of neurons in brain slices degrade earlier than others even when maintained in physiological condition.

Neuronal damage following transient cerebral ischemia and its restoration by neural transplant.

The middle cerebral artery (mca) was intraluminally occluded for one hour prior to reperfusion in the rat. Neuronal damage as well as motor imbalance were assessed in both acute and chronic stages with or without neural transplant in the striatum. In acute stage, argyrophil III staining demonstrated "collapsed" dark neurons in the ipsilateral striatum, cortex, reticular thalamus, amygdala and sometimes in the hippocampus. They had shrunken somata and corkscrew-like dendrites. In accordance with the appearance of dark neurons, the immunoreactivity for calpain of endogenous inactive form decreased or disappeared in ischemic areas. In chronic stage, ischemic core area (striatum and cortex) got into porencephaly, and animals made rotations following methamphetamine injection. Neural transplant (fetal striatal cells) was made during 2 to 4 weeks after the ischemia. Once the transplant survived and grew in the striatum, the methamphetamine rotations were attenuated. Using mca ischemic model rats we report here pathophysiological processes that lead to neuronal damage and infarct. Neural transplants into these animals brought partial restoration in motor disturbance, offering a valuable information concerning therapeutic possibility.

Pathophysiological process after transient ischemia of the middle cerebral artery in the rat.

For the understanding of pathophysiology of the cerebral ischemia, we made a transient intraluminal occlusion of the middle cerebral artery in the rat and investigated the appearance of collapsed dark neurons and the extravasation of serum proteins using argyrophil III method and immunohistochemistry. In the acute stage (minutes to 3 days), dark neurons appeared in the lateral half of the ipsilateral striatum and adjacent cortex which formed the ischemic core of this model. Dark neurons also appeared in the ipsilateral reticular thalamic nucleus, hippocampus, and amygdala. The extravasation of serum proteins, albumin, leucocyte common antigen, immunoglobulin G, complement factor C3, as well as heat shock protein 70, was observed not only in the ischemic but sometimes also in the contralateral hemisphere. Among these, the expression of IgG and C3 was most prominent in the ischemic core. In the chronic stage (1 to 3 months), the ischemic core changed into the porencephaly, and the ventrobasal nucleus of the thalamus got also involved in the necrosis. A strong microgliosis was observed in the substantia nigra pars reticulata. Data suggest, that among many mechanisms that contribute to ischemic neuronal death, the activation of immune response, due to the damage of blood-brain barrier and the extravasation of serum proteins could promote the ischemic cell death in the brain.

Appearance of immunoglobulin G and complement factor C3 in the striatum after transient focal ischemia in the rat.

The pathophysiological feature of brain ischemia-infarct was investigated using immunohistochemistry and Gallyas' silver staining after transient ischemia of the middle cerebral artery (MCA) in the rat. A very strong IgG infiltration with clear-cut borders was detected at one to 3 days in ischemic core areas (lateral striatum and adjacent cortex) that fell into porencephaly later. Complement factor C3 (C3) immunoreactivity (IR) appeared similarly while albumin IR more diffusely. Microglial activation could be observed at 1 day while rounded leukocyte-like elements at 3 days after reperfusion. Data suggest that the early appearance of the IgG/C3 IR bears particular importance after transient MCA ischemia as it could predict the ensuing porencephaly in the chronic stage.