Memories of John Eccles.

This article contains an account of the career of John Eccles that can be divided into two parts. The first extends from 1925, when he won a Rhodes scholarship to Magdalen College Oxford, to 1975, when he took voluntary retirement from the State University of New York at Buffalo. During this period, he set up six different laboratories in which he carried out research on synaptic mechanisms that provide the basis of neuroscience. The second period is the 20 years between his retirement and death in Switzerland, which he spent on the problem of the relationship between mind and brain.

Close temporal coupling of neuronal activity and tissue oxygen responses in rodent whisker barrel cortex.

Neuronal activity elicits metabolic and vascular responses, during which oxygen is first consumed and then supplied to the tissue via an increase in cerebral blood flow. Understanding the spatial and temporal dynamics of blood and tissue oxygen (To2) responses following neuronal activity is crucial for understanding the physiological basis of functional neuroimaging signals. However, our knowledge is limited because previous To2 measurements have been made at low temporal resolution (>100 ms). Here we recorded To2 at high temporal resolution (1 ms), simultaneously with co-localized field potentials, at several cortical depths from the whisker region of the somatosensory cortex in anaesthetized rats and mice. Stimulation of the whiskers produced rapid, laminar-specific changes in To2. Positive To2 responses (i.e. increases) were observed in the superficial layers within 50 ms of stimulus onset, faster than previously reported. Negative To2 responses (i.e. decreases) were observed in the deeper layers, with maximal amplitude in layer IV, within 40 ms of stimulus onset. The amplitude of the negative, but not the positive, To2 response correlated with local field potential amplitude. Disruption of neurovascular coupling, via nitric oxide synthase inhibition, abolished positive To2 responses to whisker stimulation in the superficial layers and increased negative To2 responses in all layers. Our data show that To2 responses occur rapidly following neuronal activity and are laminar dependent.

Characterisation of carbon paste electrodes for real-time amperometric monitoring of brain tissue oxygen.

Tissue O2 can be monitored using a variety of electrochemical techniques and electrodes. In vitro and in vivo characterisation studies for O2 reduction at carbon paste electrodes (CPEs) using constant potential amperometry (CPA) are presented. Cyclic voltammetry indicated that an applied potential of -650 mV is required for O2 reduction at CPEs. High sensitivity (-1.49 ± 0.01 nA/µM), low detection limit (ca. 0.1 µM) and good linear response characteristics (R² > 0.99) were observed in calibration experiments performed at this potential. There was also no effect of pH, temperature, and ion changes, and no dependence upon flow/fluid convection (stirring). Several compounds (e.g. dopamine and its metabolites) present in brain extracellular fluid were tested at physiological concentrations and shown not to interfere with the CPA O2 signal. In vivo experiments confirmed a sub-second response time observed in vitro and demonstrated long-term stability extending over twelve weeks, with minimal O2 consumption (ca. 1 nmol/h). These results indicate that CPEs operating amperometrically at a constant potential of -650 mV (vs. SCE) can be used reliably to continuously monitor brain extracellular tissue O2.

Brain tissue oxygen amperometry in behaving rats demonstrates functional dissociation of dorsal and ventral hippocampus during spatial processing and anxiety.

Traditionally, the function of the hippocampus (HPC) has been viewed in unitary terms, but there is growing evidence that the HPC is functionally differentiated along its septotemporal axis. Lesion studies in rodents and functional brain imaging in humans suggest a preferential role for the septal HPC in spatial learning and a preferential role for the temporal HPC in anxiety. To better enable cross-species comparison, we present an in vivo amperometric technique that measures changes in brain tissue oxygen at high temporal resolution in freely-moving rats. We recorded simultaneously from the dorsal (septal; dHPC) and ventral (temporal; vHPC) HPC during two anxiety tasks and two spatial tasks on the radial maze. We found a double-dissociation of function in the HPC, with increased vHPC signals during anxiety and increased dHPC signals during spatial processing. In addition, dHPC signals were modulated by spatial memory demands. These results add a new dimension to the growing consensus for a differentiation of HPC function, and highlight tissue oxygen amperometry as a valuable tool to aid translation between animal and human research.

The role of lactate in brain metabolism.

According to the astrocyte-neurone-lactate shuttle (ANLS) hypothesis, activated neurones use lactate released by astrocytes as their energy substrate. The hypothesis, based largely on in vitro experiments, postulates that lactate is derived from the uptake by astrocytes of synaptically released glutamate. The time course of changes in lactate, derived from in vivo experiments, is incompatible with the ANLS model. Neuronal activation leads to a delayed rise in lactate followed by a slow decay, which greatly outlasts the period of neuronal activation. The present review proposes that the uptake of stimulated glutamate release from astrocytes, rather than synaptically released glutamate, is the source of lactate released following neuronal activation. This rise in lactate occurs too late to provide energy for neuronal activity. Furthermore, there is no evidence that lactate undergoes local oxidative phosphorylation. In conclusion, under physiological conditions, there is no evidence that lactate is a significant source of energy for activated neurones.

In vivo neurochemical monitoring and the study of behaviour.

In vivo neurochemical monitoring techniques measure changes in the extracellular compartment of selected brain regions. These changes reflect the release of chemical messengers and intermediates of brain energy metabolism resulting from the activity of neuronal assemblies. The two principal techniques used in neurochemical monitoring are microdialysis and voltammetry. The presence of glutamate in the extracellular compartment and its pharmacological characteristics suggest that it is released from astrocytes and acts as neuromodulator rather than a neurotransmitter. The changes in extracellular noradrenaline and dopamine reflect their role in the control of behaviour. Changes in glucose and oxygen, the latter a measure of local cerebral blood flow, reflect synaptic processing in the underlying neuronal networks rather than a measure of efferent output from the brain region. In vivo neurochemical monitoring provides information about the intermediate processing that intervenes between the application of the stimulus and the resulting behaviour but does not reflect the final efferent output that leads to behaviour.

Glutamate infusion coupled with hypoxia has a neuroprotective effect in the rat.

Traumatic brain injury leads to a rise in glutamate, interference with oxygen supply and secondary neuronal death in the region surrounding the primary lesion. In the present experiments we have examined the effect of combining glutamate infusion with hypoxia on both brain metabolism and neuronal death. We have used microdialysis in unanaesthetised rats with a novel dual assay for glucose and lactate to monitor the temporal relation of changes in these metabolites resulting from infusion of 100 mM glutamate alone or combined with a reduction of inspired oxygen to 8%. In a parallel series of experiments we have compared the size of neuronal lesions under the same experimental conditions. We have used MAP2 antibody staining to measure the size of the neuronal lesion. Our results demonstrate that a 30 min glutamate infusion causes an immediate increase in neuronal glucose utilisation and a rise in lactate production. When hypoxia is added during the last 15 min of glutamate infusion there is a small rise in glucose and a large additional increase in lactate. The size of the neuronal lesions produced by infusion of 100 mM glutamate is reduced by the addition of hypoxia.

Striatal Ascorbate and its Relationship to Dopamine Receptor Stimulation and Motor Activity.

We have used the techniques of microdialysis and in vivo voltammetry to monitor striatal dopamine and ascorbate, as well as motor activity in unanaesthetized, freely-moving rats. Systemic administration of the non-selective dopamine receptor agonist apomorphine (0.5 mg/kg, s.c.) caused a decrease in dopamine, an increase in ascorbate, stereotyped behaviour and a generalized increase in motor activity. Separate systemic applications of the D1 receptor agonist SKF 38393 (10 mg/kg, s.c.) and the D2 receptor agonist Quinpirole (0.1 mg/kg s.c.) caused a decrease in dopamine but had no effect on ascorbate or motor activity. After coadministration of these drugs, there was an increase in both ascorbate and motor activity. Local application of apomorphine (0.01 mM) caused a reduction in dopamine similar to that seen following systemic application but had no effect on ascorbate or motor activity. The present results demonstrate that dopamine, via D1 and D2 receptors outside the striatum, plays an important role in the control of ascorbate release. These results lend further support to the hypothesis that changes in ascorbate levels are an index of glutamatergic neurotransmission.