Biomedical subjects

J O Willoughby

Publications and source records attributed to J O Willoughby.

At least 19 recordsLinked to original sources

PubMed2006

Cell swelling, seizures and spreading depression: an impedance study.

The cellular processes that take place during the transition from pre-seizure state to seizure remain to be defined. In this study in awake, paralyzed rats, we used an electrical impedance measure of changes in extra-cellular intracranial volume to estimate changes in cell size in acute models of epilepsy. Animals were prepared with extradural electroencephalographic (EEG)/impedance electrodes and a venous catheter. On a subsequent day, animals were paralyzed, ventilated and treated with picrotoxin, kainic acid or fluorocitrate in doses that usually induce epileptiform discharges. We now report that increases in baseline impedance were induced by kainic acid and smaller increases by picrotoxin. We also demonstrated that epileptiform discharges were preceded by small, accelerated increases in impedance. Increases in baseline impedance were highly correlated with increases in power of non-ictal high frequency EEG activity. Seizures were accompanied by increases in impedance and all treatments induced transient, relatively large, increases in impedance often associated with unilateral reductions in low frequency EEG, likely periods of spreading depression. We conclude: cerebral cells swell in convulsant models of epilepsy, that there are pre-ictal accelerations in cell swelling, and that spreading depression-like events are frequently associated with seizures.

Action Potentials↗

PubMed2004

Gamma rhythms are not integral to EEG spindle phenomena.

OBJECTIVE: Gamma rhythms (30-100 Hz) have been shown to be associated with spindling activity induced by picrotoxin. To determine if gamma power is unique to picrotoxin spindles or is an integral part of physiological and pathological spindling activity we analysed and compared the strength and brain distribution of gamma EEG power during 4 spindling activities in the rat. METHODS: The electroencephalogram (EEG) was recorded from rats with chronically implanted electrodes during natural sleep, barbiturate anaesthesia, during naturally occurring absence epilepsy spike and wave discharges and following the systemic application of picrotoxin. Spectral analysis was applied off-line to compare the strength and brain distribution of gamma EEG power during the 4 spindling activities. RESULTS: Each spindle type contained significantly different levels of gamma power. Gamma power was significantly increased over background levels during picrotoxin spindles, slightly increased during absence spindles, slightly decreased during sleep spindles and significantly suppressed during barbiturate spindles CONCLUSIONS: Changes in the power of gamma frequencies during spindle types suggest that gamma frequencies are neither the cause of nor an integral part of a spindle. They appear to be correlated with levels of consciousness and may contribute to the process of epileptogenesis. SIGNIFICANCE: The findings are consistent with high frequency EEG activity being related to seizure-tendency.

Animals↗

PubMed2004

Localised astroglial dysfunction disrupts high-frequency EEG rhythms.

We used cerebral cortex injections of fluorocitrate to determine if selective astrocytic disturbances affect the electroencephalogram (EEG). Rats were halothane-anaesthetized and 0.8 nmol of sodium fluorocitrate was injected into hindlimb (motor-sensory) cortex. Extra-dural EEG electrodes were implanted after which the anaesthesia was ceased. EEG was recorded at 1, 3, 5, 7, 24 and 48 hours. There was a broad-band reduction in frequencies in the EEG between 20 and 100 Hz commencing within 1 hour of injection and largely restricted to the side of injection and to frontal cortex, and maximal at 3 hours. Halothane had a suppressive effect on gamma power after citrate injection, but also prevented EEG suppression caused by fluorocitrate, consistent with the hypothesis that some of the action of fluorocitrate depended on gap-junctions. The findings are consistent with the hypothesis that primary astroglial dysfunction leads to reduced neuronal transmission and further supports gap-junctions as mediating fluorocitrate-induced astroglial effects.

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PubMed2004

Cognitive tasks augment gamma EEG power.

OBJECTIVE: Gamma EEG oscillations are low amplitude rhythms in the 30-100 Hz range that correlate with cognitive task execution. They are usually reported using time-locked averaging of EEG during repetitive tasks. We tested the hypothesis that continuous gamma EEG would be measurable during mental tasks. METHODS: We investigated sustained human gamma EEG oscillations induced by 8 cognitive tasks (Visual Checkerboard, Expectancy, Reading, Subtraction, Music, Expectancy, Word learning, Word recall, and a Video Segment) in 20 subjects using standard digital EEG recording and power spectral analysis. RESULTS: All of the cognitive tasks augmented gamma power relative to a control condition (eyes open watching a blank computer screen). This enhancement was statistically significant at more than one scalp site for all tasks except checkerboard. The Expectancy, Learning, Reading and Subtraction tasks expressed the most impressive gamma response, up to 5 fold above the control condition and there was some task-related specificity of the distribution of increased gamma power, especially in posterior cortex with visual tasks. CONCLUSIONS: Widespread gamma activation of cortical EEG can easily be demonstrated during mental activity. SIGNIFICANCE: These results establish the feasibility of measuring high frequency EEG rhythms with trans-cranial recordings, demonstrate that sustained gamma EEG activity correlates with mentation, and provides evidence consistent with the temporal binding model.

Adult↗

PubMed2003

Persistent abnormality detected in the non-ictal electroencephalogram in primary generalised epilepsy.

OBJECTIVES: Gamma oscillations (30-100 Hz gamma electroencephalographic (EEG) activity) correlate with high frequency synchronous rhythmic bursting in assemblies of cerebral neurons participating in aspects of consciousness. Previous studies in a kainic acid animal model of epilepsy revealed increased intensity of gamma rhythms in background EEG preceding epileptiform discharges, leading the authors to test for intensified gamma EEG in humans with epilepsy. METHODS: 64 channel cortical EEG were recorded from 10 people with primary generalised epilepsy, 11 with partial epilepsy, and 20 controls during a quiescent mental state. Using standard methods of EEG analysis the strength of EEG rhythms (fast Fourier transformation) was quantified and the strengths of rhythms in the patient groups compared with with controls by unpaired t test at 1 Hz intervals from 1 Hz to 100 Hz. RESULTS: In patients with generalised epilepsy, there was a threefold to sevenfold increase in power of gamma EEG between 30 Hz and 100 Hz (p<0.01). Analysis of three unmedicated patients with primary generalised epilepsies revealed an additional 10-fold narrow band increase of power around 35 Hz-40 Hz (p<0.0001). There were no corresponding changes in patients with partial epilepsy. CONCLUSIONS: Increased gamma EEG is probably a marker of the underlying ion channel or neurotransmitter receptor dysfunction in primary generalised epilepsies and may also be a pathophysiological prerequisite for the development of seizures. The finding provides a new diagnostic approach and also links the pathophysiology of generalised epilepsies to emerging concepts of neuronal correlates of consciousness.

Adult↗

PubMed2002

Picrotoxin-induced generalised convulsive seizure in rat: changes in regional distribution and frequency of the power of electroencephalogram rhythms.

OBJECTIVES: It is unknown how generalised discharges in primary generalised epilepsy (PGE) develop from background brain electrical activity or how widespread these discharged are throughout the brain. Here we address this by determining which neural structures and rhythms lead to and participate in generalised discharges in the picrotoxin rat model of PGE. METHODS: Rats with chronically implanted electrodes were infused with picrotoxin until a seizure occurred. This process we refer to as acute epileptogenesis. The electroencephalogram (EEG) was recorded and spectral analysis applied off-line to determine changes in the spectral power of contributing frequencies in 13 brain regions. RESULTS: Two types of generalised discharge occurred, spindles and seizure, which were present in all brain regions studied. None of the frequencies (1-100 Hz) were significantly increased in background EEG before either spindles or seizure. Within the generalised discharges, power changes revealed significant increases in 6-8 Hz, most powerful in ventrolateral thalamus and neocortex. Gamma frequencies were increased significantly in neocortical structures during spindles with further increases in most structures at seizure onset. 1 Hz was significantly increased in parietal cortex during spindles with differential increases at seizure onset. CONCLUSIONS: We conclude that gamma, 1 and 6-8 Hz frequencies do not appear to contribute to picrotoxin epileptogenesis but do play a role in generalised seizures. The distribution of these frequencies during discharges suggests that the spindles are thalamocortical events and that the seizure is a cortical event with downstream effects on other brain regions.

Animals↗

PubMed2001

Kainic acid and seizure-induced Fos in subtypes of cerebrocortical neurons.

Kainic acid injected in vivo into adult rats evokes the expression of the immediate early gene c-fos in the dentate gyrus and associated structures before a seizure occurs and in these and additional regions after a single motor seizure. The aim of this study was to identify cortical cell classes expressing Fos that correlate with these phenomena. Fos expression occurred before a seizure in the middle layers of entorhinal cortex in excitatory neurons and predominantly in calbindin D28-K-containing inhibitory neurons. Given the early Fos-labeling of these cells, we suggest they are associated with the hippocampal EEG events also seen at this stage of the effects of kainic acid. After a motor seizure Fos induction occurred in primary motor, sensory, piriform and entorhinal cortices, mainly in excitatory neurons, but also in a proportion of calcium binding protein-containing neurons proportionate to the degree of activation of the region as determined by Fos. Nearly 100% of neurons were Fos+ in entorhinal cortex, whereas 80% of excitatory and 50% of calcium binding protein-containing neurons were Fos+ in piriform cortex with lower proportions in neocortex. Of the calcium binding protein-containing neocortical neurons, calbindin D28-K cells exhibited the highest proportion of double labeling with Fos. This pattern of neocortical activation by kainic acid, a glutamate agonist, is only slightly different to that seen after seizures caused by blockade of gamma aminobutyric acid receptors suggesting that seizures caused by different mechanisms utilize similar neo-cortical circuitry.

Animals↗

PubMed2000

Kainic acid induces distinct types of epileptiform discharge with differential involvement of hippocampus and neocortex.

Systemic administration of kainic acid (KA), an excitatory amino acid agonist, provides a model of epilepsy due to increased neural excitation. We examined discharges using multi-channel EEG recording and spectral analysis in rats implanted with neocortical and hippocampal electrodes after intravenous infusion of KA (10 mg/kg), until and including the first convulsive seizure. Gamma activity (30-80 Hz) increased in hippocampus from 3-9 min after KA administration. Two types of preconvulsive bilateral rhythmic discharges were observed, both consisting of generalised high voltage sharp waves at low frequencies (<10 Hz) mixed with fast oscillations (<20 Hz): (1) generalised non-convulsive discharges (GNCD) occurred in all animals and (2) spike-wave discharges (SW), predominantly localised in neocortex, occurred in 45% of animals. Convulsive seizure evolved out of a GNCD. Spectral profiles of epileptiform discharges were characterised by an increase in power of low (<10 Hz) and high (beta and gamma range, 20-80 Hz) frequencies which were differently expressed in neocortex and hippocampus. Thus, in this model of convulsive epilepsy caused by increased excitation, there is an early increase in gamma activity, a process that might contribute to synchronisation, and two distinct types of bilateral discharges, hippocampal-neocortical (GNCD) and preferentially neocortical (SW). Neocortical, not hippocampal, changes in EEG power correlated with development of convulsive behaviours.

Animals↗

PubMed2000

Confocal microscopic estimation of GABAergic nerve terminals in the central nervous system.

We describe a method for estimating the average proportion of GABAergic terminal area relative to total nerve terminal area with confocal microscopy. Nerve terminal regions were identified with dual colour immunofluorescence on Vibratome sections with an antibody to synaptophysin (SYN), and GABAergic processes, including axon terminals, were identified with an antibody to glutamic acid decarboxylase (GAD). Sections were viewed in an Olympus AX70 microscope attached to a Biorad 1024 MRC scanning confocal system. Images were collected with a 100 x objective from the same tissue locations and imported into the NIH-Image program, where black and white binary images were obtained for co-localisation and quantitation. Measurements were made separately of areas of SYN/GAD (GABAergic terminals) and SYN labelling (all terminals). The relative proportion of GABAergic terminal areas in visual cortex (6.1+/-1%; mean +/- SE), CA1 hippocampus (2.6+/-0.5%) and deep cerebellar nuclei (46.6+/-3%) are consistent with what is known of the relative levels of inhibitory input to these structures. The assumptions that SYN labelling is restricted to axon terminals, and that SYN labels all axon terminals was tested by ultrastructural localisation of SYN in the three brain regions examined. Only 7.4+/-0.4% of SYN-labelled profiles could not be positively identified as synaptic vesicle-containing axon terminals, and between 93.4 and 99.2% of vesiculated axon profiles were SYN-positive. These results suggest that SYN is a very reliable marker for axon terminals, and validates the confocal analytical approach. The confocal method allows rapid sampling of many brain regions and would be suitable for examining terminals containing any neurotransmitter that can be detected immunocytochemically.

Animals↗

PubMed2000

Mechanisms underlying partial (focal, or lesional) epilepsy.

Neuronal mechanisms underlying focal convulsions and secondary generalised convulsions have been extensively investigated at many different levels, from the biochemical, through the cellular to the intact brain. Numerous pathogenic processes relevant to epilepsy are now known. Experimental models suggest that alterations to the shape, connectivity and receptor-chemistry of individual neurons increase their excitability and the resultant increases in activity lead to changes of the local ionic environment that further causes enhanced excitability. As a consequence of the latter, neuronal firing rates increase and there is a shift from single-firing- to burst-firing-behaviours in populations of neurons and, possibly, also several changes in the way populations of neurons communicate, namely, from 'synaptic' to 'electric' and from orthodromic to antidromic. As a consequence, massive neuronal synchronisation occurs, the correlate of the focal or secondarily generalised attack. Because of experimental difficulties in studying the widespread and evolving neuronal activities in freely behaving animals, a precise correlation between states of neuronal activation and convulsive behaviour is still unclear.

Animals↗

PubMed1999

Picrotoxin-, kainic acid- and seizure-induced Fos in brainstem, with special reference to catecholamine cell groups.

One of the original views about motor convulsions with bilateral synchronous EEG discharges was that ascending projections from deep midline brainstem neurons subserve discharge synchronisation. We examined this in kainic acid- and picrotoxin-induced seizures using the distribution of Fos protein expression in the brainstem as a possible marker of brainstem neuronal activation. Before seizure, Fos was present in parabrachial nucleus, central grey and A1/C1 cell groups and, to a lesser extent, in A2/C2 cell groups and A6 neurons. Seizure correlated with further induction of Fos in these cell groups as well as in A6 neurons. There was no Fos in other cell groups with bilateral cerebro-cortical projections nor in cell groups likely to participate in the expression of seizures, reflecting a limitation of the Fos method. This study provides evidence of locus coeruleus involvement in these two models of seizure.

Animals↗

PubMed1999

Autoregressive modeling of the EEG in systemic kainic acid-induced epileptogenesis.

Background activity as well as three kinds of bilateral epileptiform discharges, recorded from the cerebral cortex and hippocampus of freely behaving rats treated with intravenous kainic acid (KA), were analysed by the directed transfer function (DTF) method within multivariate autoregressive modeling of the EEG. This method reveals statistical influence (flow of activity) between brain regions at different frequencies. There was no significant influence between rhythms in different brain regions in the background EEG. Early after KA administration, low frequency rhythms (< 10Hz) in the frontal cortex began to lead slow rhythms in other areas and high frequency rhythms (20-60 Hz), possibly gamma oscillations, intensified in the hippocampus. In spike-wave discharges, frontal cortex led both low and high frequency rhythms. Initially during generalised non-convulsive discharges, slow rhythms originated from frontal cortex and high frequency rhythms from hippocampus while later, slow rhythms as well, often arose from hippocampus. During the convulsive discharge, the flow of activity of dominant slow rhythms repeatedly changed between hippocampus and neocortex, with more frequent dominance of the hippocampus, while hippocampus continued to lead high frequency rhythms. We conclude that KA-induced epileptiform discharges are cortical and hippocampal events, specifically that the frontal cortex is early to express low frequency rhythms and the hippocampus, high frequency rhythms. More generally, the findings suggest that epileptiform discharges result from interacting rhythms of different frequencies that arise from different structures, and that gamma oscillations possibly contribute to widespread synchronisation during some forms of epileptogenesis.

Animals↗

PubMed1998

Laminar distribution of Fos/calcium-binding protein and Fos/neurofilament protein-labeled neurons in rat motor and sensory cortex after picrotoxin-induced seizures.

Cerebrocortical Fos induction after picrotoxin-induced seizure occurs in spiny neurons and, to a lesser extent, in neurons defined by calcium-binding protein immunoreactivity. In motor and sensory cortex of rats we have defined the laminar distribution of Fos expression in these neurons. Initially we defined the laminar distributions of parvalbumin-, calbindin-D 28K-, and calretinin-immunoreactive aspiny neurons; these were unique for each class and similar across cortical regions. Spiny cells defined by SMI32 immunoreactivity were distributed with two peaks and there were differences between cortical regions. Parvalbumin-immunoreactive neurons exhibited peak numbers where numbers of SMI32-immunoreactive neurons were low. The distribution of Fos induction across laminae matched that of its class for calbindin-D 28K and calretinin neurons; however, Fos induction was less in infragranular compared with supragranular for parvalbumin in motor cortex and SMI32 containing neurons in both cortices. In both these latter cell classes Fos induction was inversely correlated with neuronal size. It is suggested that cell size within some cell classes is one factor that determines the extent of Fos induction within that class following seizures.

Animals↗

PubMed1997

Fos induction following systemic kainic acid: early expression in hippocampus and later widespread expression correlated with seizure.

We determined the distribution of Fos protein expression in a model of generalised epilepsy caused by excessive neuronal excitation. Fos immunoreactivity was mapped in forebrain in unrestrained rats, previously prepared with an indwelling venous catheter, after the intravenous administration of kainic acid (10 mg/kg). We determined cerebral activation following various periods of exposure to kainic acid by using intravenous administration of pentobarbitone to prevent further activation. Within a few minutes, kainic acid caused episodes of staring, sniffing, wet dog shakes, nodding and chewing. Fos induction occurred initially and simultaneously in hippocampus, subiculum, septum and entorhinal cortex as early as 9.5 min after kainate injection. After up to 40 min of staring, sniffing, wet dog shakes, nodding and chewing, Fos induction was not further increased above levels present within the first 9.5 min. After 56 +/- 6 min a motor convulsion occurred, initially affecting the jaw, head and tail and variably extending to the forelimbs, trunk or hindlimbs. Following the convulsive event, additional Fos was expressed in hippocampus, thalamus, caudate-putamen and other subcortical structures and in the cerebral cortex. Fos induction was sometimes asymmetric in entorhinal, visual, piriform, cingulum, parietal and frontal cortices and in amygdala and dorsal endopiriform area. Electroencephalographic recordings after a few minutes exposure to kainic acid revealed an increased amplitude of fast frequencies in hippocampus which appeared to correlate with Fos induction in this structure. The findings are generally consistent with the reported distribution and slow development of kainic acid-induced seizure activity using electrophysiological and deoxyglucose methods. However, the Fos distribution suggests that (i) hippocampal, possibly dentate, activation precedes significant activation elsewhere, (ii) extensive involvement of other cerebral structures and cerebral cortex occurs simultaneously and correlates with motor seizures and (iii) brain structures can be recruited asymmetrically.

Animals↗

PubMed1996

Fos induction in subtypes of cerebrocortical neurons following single picrotoxin-induced seizures.

In adult rats single seizures of varying behavioural severities were caused by slow, systemic infusion of picrotoxin, an antagonist of the C1- channel at the GABAA receptor. We used a double labelling immunohistochemical method to define the subclasses of neurons that contained Fos protein following seizures. In four cortical regions (piriform, entorhinal, motor and sensory) neuronal subclasses were defined with antibodies against the calcium-binding proteins D-28K, parvalbumin and calretinin (aspiny neurons), and neurofilament protein (spiny neurons). The remaining spiny neuron population was estimated by subtraction of defined subclasses from total neuronal numbers determined from Nissl stain. After seizures, most of the calbindin D-28K immunoreactive interneurons (> 80%) and many of the unlabelled spiny neurons (60-80%) were FOs positive. Co-localisation of Fos was found in about 30% of parvalbumin, calretinin and neurofilament protein immunoreactive neurons. Paradoxically, mild seizures were associated with induction of Fos in up to 80% of cortical cells and more severe seizures with 60%, the difference being due to different levels of Fos induction in spiny neurons. These results also demonstrate that seizures induce Fos predominantly in excitatory cortical neurons.

Animals↗

PubMed1996

Pentobarbitone induces Fos in astrocytes: increased expression following picrotoxin and seizures.

Fos immunoreactivity was observed in astrocytes identified by immunoreactivity to glial fibrillary acidic protein, 1.5 h following intravenous pentobarbitone anesthesia (20-30 mg/kg). Fos-positive astrocytes were seen only in the hilus of the hippocampus. Pentobarbitone administered after picrotoxin but without seizures resulted in an increase in both the intensity of Fos labeling and areal distribution of Fos-positive hilar astrocytes. Pentobarbitone administered after single picrotoxin-induced seizures resulted in an increased number of Fos immunoreactive astrocytes in the hilus and other hippocampal regions and their presence in the upper layers of Fr1, Fr2, cingulate, visual, and perirhinal cortex. Our observations that Fos is expressed in astrocytes following pentobarbitone, provides support for the current view that glial cell processes can be activated by pentobarbitone stimulation of GABAa receptor-chloride channels.

Animals↗

PubMed1996

Frontal cortex leads other brain structures in generalised spike-and-wave spindles and seizure spikes induced by picrotoxin.

Generaliszed spike-and-wave (SW) spindles (5-7 Hz) associated with myoclonic jerks precede the occurrence of regular spikes (2-3 Hz) associated with convulsive seizure induced by picrotoxin. SW spindles occur spontaneously in rodent and cat under some experimental conditions and are considered to be models of human generalised epilepsy. These spindles have been proposed as being led by a thalamic pacemaker. To examine this possibility in picrotoxin-induced SW spindles and seizure spikes, we recorded EEG using chronically implant unipolar electrodes during intravenous picrotoxin infusion in freely behaving rat. The 6 EEG signals were digitally sampled at 1000 Hz. Linear correlation, spectral, coherence and phase analyses were undertaken to determine time differences (TDs) between EEG channels and the brain structure leading seizure activity. One frontal cortex led all other structures during SW spindles. TD between SW spindles in the leading frontal cortex (Fr1) and the contralateral Fr1 was 3.6 + / - 0.5 msec. All ipsilateral structures (hippocampus, thalamus, amygdala, caudate nucleus and occipital cortex) were delayed by more than 3 msec from Fr1 (intralaminar thalamic nuclei - by 6.3 + / - 0.9 msec). TDs of SW spindles between subcortical regions were less than 1.5 msec. Similar relationships with slightly smaller TDs were found with spikes during convulsive seizure except TDs between frontal cortices did not significantly differ from zero. We suggest that seizure activity induced by picrotoxin is led by one Fr1 during SW spindles and by both frontal cortices working as one system during convulsive seizure.

Animals↗

PubMed1996

Opioid receptor activation resets the hypothalamic clock generating growth hormone secretory bursts in the rat.

Plasma growth hormone (GH) concentrations were measured serially every 20 min for 6 h in unrestrained chronically-catheterised male rats to define physiological GH pulsatile secretory patterns. Bursts of GH secretion lasted 69 +/- 5 min and occurred every 177 +/- 4 min. Intravenous administration of the opioid receptor agonist morphine (200 micrograms/kg) caused an immediate GH burst of normal duration (63 +/- 3 min) in all animals. This burst of secretion occurred whatever the phase of the background GH cycle and was followed by normal trough GH levels; a second GH burst occurred 177 +/- 6 min later, an inter-burst period not different from controls. Opioid receptor blockade with naloxone (5 mg/kg) administered i.v. every 20 min during spontaneous GH bursts significantly lengthened the interburst interval from 177 +/- 4 to 200 +/- 9 min (P = 0.015). Naloxone did not affect synchronisation of the GH rhythm induced by morphine but lengthened the duration of GH secretory bursts from 69 +/- 5 to 94 +/- 9 min (P = 0.017). The findings indicate that opioid receptor activation resets the hypothalamic mechanism generating pulsatile GH secretion and that both the period of the GH rhythm and duration of the GH burst is normally shortened by opioid mechanisms.

Animals↗