Seizure-like activity and cellular damage in rat hippocampal neurons in cell culture.

Neurons dissociated from the hippocampal formations of neonatal rats were grown in medium containing kynurenic acid (a glutamate receptor antagonist) and elevated Mg2+. Such chronically blocked neurons, when first exposed to medium without blockers (after 0.5-5.0 months), generated intense seizure-like activity. This consisted of bursts of synchronous electrical responses that resembled paroxysmal depolarization shifts and sustained depolarizations that, in some neurons, nearly abolished the resting potential. Sustained depolarizations were usually reversed by timely application of kynurenate or 2-amino-5-phosphonovalerate, indicating that continuous activation of glutamate receptors was required for their maintenance. Prolonged periods of intense seizure-like activity usually killed most neurons in the culture. This system allows seizure-related cellular mechanisms to be studied in long-term cell culture.

Changes in event-related potentials in a three-stimulus auditory oddball task after mild head injury.

Previous research has demonstrated changes in event-related potentials in a variety of cognitive tasks after severe closed head injury. We sought to establish if similar changes were present in patients who had sustained only apparently mild head injury (MHI) by recording event-related potentials in a group of 24 mild head injured and 24 control participants during a three-stimulus auditory target detection task. For this "oddball" task participants were required to press a button every time they heard a rare target tone and to ignore rare novel sounds and frequent non-target tones. Neuropsychological test results indicated that the mild head injured group had mild memory and attention impairments. Analysis of behavioural performance on the three-stimulus "oddball" task showed no difference in reaction times or error rates between the two groups. Target condition N2 deflections appeared to be larger in the mild head injured but peak amplitude measures revealed that this effect was not significant. There were no significant differences in the amplitude or latency of the P3b evoked by target stimuli or the P3a evoked by novel stimuli. However, a putative "O-wave" or "reorienting negativity" following the P3a was more negative in the mild head injured group suggesting increased activation of components of the attention network. These findings lend support to the hypothesis that MHI can cause subtle cognitive impairments that are associated with abnormal allocation of attention resources in the context of normal behavioural performance.

Cognitive brain potentials in a three-stimulus auditory "oddball" task after closed head injury.

Event-related potentials (ERPs) were recorded in a three stimulus oddball task from 16 patients who had sustained a severe closed head injury at least 6 months before testing, and from 16 control subjects. The stimuli comprised a random sequence of frequent non-target tones (P = 0.70), rare target tones (P = 0.15), and rare novel sounds (P = 0.15). The task requirement was to respond promptly to each target tone. From a latency of 200 msec onwards, the ERPs evoked by frequent non-targets were substantially more negative-going in the head-injured than in the control group. When this difference in the ERPs to the frequent tones was taken into account, there was no evidence to suggest that either the latency or the amplitude of the target-evoked N2 and P3b components differed between the groups. The novel stimuli evoked a prominent P3a component. The amplitude and scalp distribution of this component differed little between the groups, but its peak latency was reliably longer in the head-injured subjects. The findings in respect of the N2 and P3b components suggest that impairments in early processing of task-relevant stimuli are not an invariant feature of closed head injury. The findings regarding P3a suggest that, in the majority of patients, head injury has only a limited effect on the neural systems underlying involuntary shifts of attention.

Specialized face processing and hemispheric asymmetry in man and monkey: evidence from single unit and reaction time studies.

Experimental and clinical studies have generally shown that the neural mechanisms for face processing in man are (1) designed to deal with the configuration of upright faces and (2) located predominantly in the right cerebral hemisphere. Monkeys would seem to process faces in a different manner to humans since they appear to show no hemispheric asymmetry and to treat upright and inverted faces equivalently. We re-examine these claims. Our reaction time studies reveal that monkeys do behave like human subjects since they process facial configuration faster when stimuli are presented upright as compared with horizontal or inverted. Single unit studies in the monkey reveal patches of neurones responsive to faces in the upper bank and fundus of the left superior temporal sulcus (STS). Recording from the right hemisphere also reveals cells responsive to faces but in this hemisphere such cells appear less numerous. These cells process upright faces faster than inverted faces. Face processing in monkeys and man appears to utilize qualitatively similar mechanisms, but the extent and/or direction of cerebral asymmetry in these mechanisms may not be similar.

Visual analysis of body movements by neurones in the temporal cortex of the macaque monkey: a preliminary report.

Movement provides biologically important information about the nature (and intent) of animate objects. We have studied cells in the superior temporal sulcus of the macaque monkey which seem to process such visual information. We found that the majority of cells in this brain region were selective for type of movement and for stimulus form, most cells responding only to particular movements of the body or some part of it. A variety of cell types emerged, including cells sensitive to: translation of bodies in view, movements into view (appearance) or out of view (disappearance) and the articulation and rotation of the body/head. Directional selectivity for cells sensitive to translation tended to lie along one of 3 orthogonal Cartesian axes centred on the monkey (towards/away, left/right and up/down). One type of rotation sensitive cell was tuned to rotation about one or more of these axes, a second type was sensitive to different head rotations which brought the face to confront the monkey or turned the face away. Reconstructions of cell positions indicated that cells of the same type were clumped anatomically both across the surface of the cortex and perpendicular to the surface.

Scopolamine impairs memory performance and reduces frontal but not parietal visual P3 amplitude.

It has been suggested that the P3 event-related potential (ERP) may mark the operation of certain working or long-term memory processes. It has also been reported that cholinergic blockade by scopolamine induces significant memory impairment and is associated with an increased latency, as well as amplitude reduction or abolition of the auditory P3, thus supporting hypothesised links between P3 and long-term memory function. An intriguing anomaly is that, while visual P3 latency is also increased by scopolamine, amplitude is not changed. The aim of this study was to make a more detailed assessment of the effects of scopolamine on the visual P3 at a drug dose known to induce memory impairment. After drug administration, memory performance was significantly impaired and visual P3 latency was significantly increased. There was little evidence of parietal P3 amplitude reduction, but frontal P3 amplitude was significantly reduced in both target and non-target conditions. These findings, when considered in the light of a more recent study of the effects of scopolamine on auditory P3, suggest that cholinergic blockade produces a common effect in both visual and auditory modalities of significant frontal P3 amplitude reduction, but no significant parietal P3 amplitude reduction. These results are consistent with the view that there are modality-independent generators of the parietal and frontal P3. The finding of drug-induced memory impairment and modulations of frontal ERP deflections is also consistent with recent evidence of a significant role for regions of the frontal lobe in encoding and retrieval of long-term memories.

Event related potentials, reaction time, and cognitive performance in idiopathic Parkinson's disease.

Sixteen non-demented patients with idiopathic Parkinson's disease (PD) with varying degrees of cognitive impairment and sixteen age-, sex- and education-matched normal controls were examined with (1) an auditory oddball paradigm requiring counting or a motor response in separate determinations, (2) a reaction time task with movement time component and (3) a detailed clinical and neuropsychological test battery. Patients were impaired on a number of neuropsychological tests. They also showed an increased P2 and N2 latency, but no significant increase in P3 latency. Their response initiation times and reaction times during the oddball experiment were not different from controls, whereas movement time was significantly increased. Increased peak latencies, particularly for N2, were moderately associated with Parkinsonian motor impairment in patients and with the Benton Multiple Choice Visual Retention Test in patients and controls. Movement time was associated with P3 latency only in controls and in both groups with the Benton Multiple Choice Visual Retention Test. The observed pattern of results suggests that in non-demented PD patients ERP peak latencies, visuo-spatial task performance and Parkinsonian motor impairment share a significant degree of variance. While impairments in neuropsychological tests and delay in the earlier peaks P2 and N2 do not appear to be sensitive to medication with L-DOPA, normal P3 latencies might indicate good pharmacological symptom control in the absence of dementia.

Co-culture of dissociated hippocampal and sympathetic cells from the neonatal rat.

When sympathetic neurones, obtained from superior cervical ganglia of postnatal rats, were grown in microcultures with cells of the postnatal hippocampal formation for 6-44 days, about 70% of the sympathetic neurones formed functional synapses on themselves or a neighbouring sympathetic neurone. In all forty-four cases in which hexamethonium (0.5-1 mM) was applied it strongly or completely blocked the synaptic interaction. This indicates that the synaptic interaction was cholinergic and raises the possibility that the denervated cells of the hippocampal formation induced the cholinergic function in the co-cultured sympathetic neurones.

Adrenergic-cholinergic dual function in cultured sympathetic neurons of the rat.

Sympathetic principal neurons, dissociated from the superior cervical ganglia of newborn rats and put into culture, exhibit plasticity with respect to the choice between noradrenaline (norepinephrine) and acetylcholine as transmitter. The neurons shift from an initial, immature adrenergic state to a cholinergic state in certain culture conditions, e.g in co-culture with a variety of non-neuronal cells or after exposure to a medium conditioned by such cells. To study the transition directly, we have grown single neurons in "microcultures" with cardiac myocytes, which provide a sensitive assay for the transmitters secreted by the neurons. We have shown previously that during the transition from adrenergic to cholinergic status such neurons secrete both transmitters and have terminals of mixed fine structure (dual function). We describe here experiments in which identified neurons were serially assayed over periods of 9-45 days. Partial transitions were observed, always in the direction adrenergic to cholinergic function, and one complete transition was observed from apparently purely adrenergic function to dual function and then to apparently purely cholinergic function. We also report observation of adrenergic-cholinergic dual function, in preliminary single and serial assays, in sympathetic principal neurons from the superior cervical ganglia of adult rats.

Transmitter status in cultured rat sympathetic neurons: plasticity and multiple function.

Considerable recent study of the development of transmitter status in sympathetic principal neurons, both in vivo and in culture, has produced several surprising findings. In this paper we review work on cultured immature and adult principal neurons dissociated from the superior cervical ganglia of rats. The main points are; 1) Immature principal neurons that display adrenergic properties during the first postnatal week in culture can be shifted to cholinergic status, including formation of functional cholinergic synapses, by coculture with nonneuronal cells (e.g., dissociated heart cells) or by medium conditioned by such cells. Through the use of microcultures that contain only a single neuron grown on heart cells, it has been possible to demonstrate the transition from adrenergic to cholinergic function directly by serial physiological assays of the same neuron at intervals of days or weeks. 2) During this transition, the cultured neurons display adrenergic/cholinergic dual function. This dual function has also been observed in principal neurons isolated from ganglia of adult rats. 3) Some cultured neurons secrete a third transmitter, probably adenosine or a phosphorylated derivative. This purinergic function is expressed with adrenergic or cholinergic function, or with both (triple function). In some cases, the main effect exerted by a neuron on cocultured cardiac myocytes is purinergic.

Dual function during development of rat sympathetic neurones in culture.

Many sympathetic principal neurones of the superior cervical ganglion of the newborn rat are known to be plastic with respect to the choice between norepinephrine (NE) and acetylcholine (ACh) as transmitter; when the neurones are dissociated and placed in culture, a majority of them can be shifted from an initial, immature, adrenergic state to a cholinergic state by co-culture with a variety of non-neuronal cells or by medium conditioned by such cells. To study this transition it has been helpful to grow single neurones, each in a microculture which also contains cardiac myocytes. The transmitter status of a neurone can be assayed by recording its effect on the myocytes (adrenergic excitation, cholinergic inhibition or dual function); then a fine structural assay of the neurone based on the appearance of the synaptic vesicles can be made and correlated directly with the physiology. In this paper we report the following findings on principal neurones developing in such microcultures. (i) During the transition period, a majority of the neurones were dual in function and in vesicular appearance. (ii) The physiological effects and vesicular appearance varied from mainly adrenergic to mainly cholinergic. (iii) In preliminary attempts to follow the transition by recording at least twice from the same microculture, partial transitions were observed, always in the direction adrenergic-to-cholinergic. (iv) The transitions were not synchronous or fixed in time course even in pairs of neurones grown side by side in the same microculture.

Synaptic functions in rat sympathetic neurons in microcultures. III. A Purinergic effect on cardiac myocytes.

In the first two of this series of papers, a sensitive microculture procedure was used to show that rat sympathetic neurons grown singly on small islands of heart cells release norepinephrine (NE) and/or acetylcholine (ACh). We report here the release of a third transmitter in response to stimulation of these neurons. This agent was recognized by its effect on the cocultured cardiac myocytes: an inhibition of beating or a hyperpolarization that, in contrast to cholinergic inhibition, was unaffected by atropine (up to 5 microM). Evidence described here indicates that this agent was primarily adenosine (or a closely related compound): the atropine-resistant myocyte inhibition was antagonized by adenosine-receptor blockers [8-phenyltheophylline, theophylline, 7-(2-chloroethyl) theophylline] and was attenuated by an enzyme (adenosine deaminase) that hydrolyzes adenosine to pharmacologically inactive inosine. Many of the neurons, whether initially dissociated from ganglia of newborn or adult rats, evoked this purinergic response, almost always in combination with adrenergic and cholinergic responses. In a few cases it was the only detectable response. The relative strength of the adrenergic, cholinergic, and purinergic responses varied widely from neuron to neuron, suggesting that the adrenergic and purinergic or the cholinergic and purinergic agents were not stored at constant stoichiometric ratios.

Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons.

Electrophysiological studies were made on microcultures (300-500 mum in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells. Some neurons inhibited,some excited, and others first inhibited and then excited the cardiac myocytes. Application of drugs provided evidence for secretion of acetylcholine by the first group, catecholamines by the second, and both acetylcholine and catecholamines by the third. Solitary neurons which inhibited themyocytes usually excited themselves at nicotinic synapses (autapses).

Evidence for cholinergic synapses between dissociated rat sympathetic neurons in cell culture.

Sympathetic principal neurons were dissociated from superior cervical ganglia of new-born rats, and grown in cell culture. In electrophysiological experiments two types of excitatory synapses were found. One, which was relatively rare, was shown to operate by electrical transmission. The other, the predominant type, had several characteristics of chemical transmission, and pharmacological evidence indicated it was cholinergic.

Synaptic functions in rat sympathetic neurons in microcultures. IV. Nonadrenergic excitation of cardiac myocytes and the variety of multiple-transmitter states.

In the first 3 papers of this series (Furshpan et al., 1986a, b; Potter et al., 1986), a sensitive microculture procedure was used to show that sympathetic principal neurons, dissociated from newborn or adult superior cervical ganglia and grown singly on cardiac myocytes, display adrenergic, cholinergic, and purinergic functions, sometimes in isolation but more often in combination. In this paper we describe additional effects on cardiac myocytes evoked by these neurons; the effects were excitatory and insensitive to adrenergic blocking agents (and to agents that block the inhibitory effects of acetylcholine and purines). In some of these microcultures, evidence consistent with secretion of serotonin was obtained; the nonadrenergic excitatory effect was diminished or abolished by serotonin blockers or reserpine. Further evidence for serotonergic transmission is presented in the accompanying paper by Sah and Matsumoto (1987). In other cases, an as-yet-unidentified agent "X" also produced a nonadrenergic excitation. The X effect characteristically required a prolonged train of neuronal impulses, had a time course of 50-200 sec, and was insensitive to agents that affected the other transmitters, including serotonin. In addition, we discuss 2 remarkable features of the transmitter repertoire of the microcultured sympathetic neurons: expression of the several transmitters in a variety of combinations, including at-least-quadruple function, and expression of the transmitters within a particular combination in varying relative strengths. The result is a diversity of transmitter release greater than that previously reported for vertebrate or invertebrate neurons.

Normal P300 following extensive damage to the left medial temporal lobe.

Event-related potentials (ERPs) were recorded during auditory and visual "oddball" tasks from a patient with a severe verbal memory deficit due to a low grade infiltrating glioma which involved the full extent of the left medial temporal lobe. In both sensory modalities, the patient's oddball-evoked P300s were symmetrical and of normal amplitude. These findings are difficult to reconcile with the hypothesis that the hippocampus, or any other medial temporal structure, makes a substantial contribution to the scalp P300.

The effect of different high-pass filter settings on peak latencies in the event-related potentials of schizophrenics, patients with Parkinson's disease and controls.

Eighteen schizophrenic patients, 16 patients with idiopathic Parkinson's disease, and the same numbers of age, sex and education matched controls were examined with oddball experiments for the generation of P3. Individual averages were high-pass filtered at different cut-off frequencies with single-pole digital filters with equivalent analogue Butterworth filter profiles. The purpose of this procedure was to simulate analogue high-pass filters used in clinical studies from different centres and to examine their potential effect on group differences. Increasing high-pass filters resulted in a phase lead for all peaks examined (N1, P2, N2, P3). The only group differences were found for P3, which showed a greater phase lead in controls than in the patient groups, usually resulting in a more pronounced group difference. Similar wave forms and filter properties could be modelled by synthetic wave forms consisting of sine waves of different frequencies.