In vitro and in vivo techniques in CNS drug discovery

In spite of an increased understanding of brain mechanisms in recent years, there has been a lack of major new drugs being registered for psychiatric and neurological conditions1,2. To prioritise drug discovery resources and provide early proof-of-concept studies for novel compounds and mechanisms, the pharmaceutical industry is increasingly focusing on trying to identify and develop early biological markers (biomarkers).

A biomarker has been defined as a characteristic that can be objectively measured as an indicator of a normal biological process; a pathogenic process; or a pharmacological process caused by a therapeutic intervention3,4.

Electrophysiological recording from single cells can contribute a great deal towards discovering pharmacological mechanisms and measuring drug activity at the receptor level, which has already been the focus of several articles in European Pharmaceutical Review5,6. However, for CNS indications, pharmacological activity in vivo is often more difficult to predict, as neurons do not exist in isolation but form complex networks, generating different modes of activity based on their integration within that circuitry. In this article, we describe how both in vitro and in vivo electrophysiological techniques employing ensemble recordings of neuronal network activity are being utilised to tackle this approach to CNS drug discovery. We have focussed on the discussion of three such techniques: in vitro brain slice recordings to study the modulation of synaptic transmission and plasticity; multi-electrode array recordings to identify the action of psychoactive drugs on simple neuronal networks; and the use of electroencephalogram recordings to identify biomarkers of drug efficacy.

Studying synaptic plasticity in vitro

Deficits in cognition underlie a large number of CNS disorders whose treatment remains a major challenge in healthcare. These deficits range from mild cognitive impairment (MCI) without overt dementia7; age-related memory impairment (AAMI)8, to Alzheimer’s disease (AD), estimated to affect 25 million people worldwide in 20009, in addition to other neurodegenerative conditions such as Parkinson’s10. Cognitive dysfunction has also been recognised as a primary and encoring core deficit in schizophrenia, affecting approximately 1 per cent of the population11.

A central tenet in neurobiology is that long-lasting changes in the efficacy of synaptic transmission in the mammalian brain are considered to be of fundamental importance for the storage of information. The leading experimental model for such changes has been long-term potentiation (LTP) – the finding that brief, high frequency activation of excitatory pathways can evoke an increase in synaptic efficiency12 – as well as the converse phenomena, long term depression (LTD). LTP has a long duration, often lasting days, but is not permanent. The formation of LTP also requires a number of convergent synapses to be activated and shows input specificity as well as associativity13. These ‘Hebbian’ qualities render this form of plasticity ideally suited to underlie, at least in some instances, the neural basis of memory formation and certainly some evidence to support this hypothesis has emerged (reviewed in14,15).

The hippocampus itself is a crucial element of the neurobiological bases of higher cognitive function and can serve as a simple model for cortical processing in general because of its regular and relatively simple cytoarchitecture. In an acute hippocampal brain slice preparation, stimulation of either the perforant path emanating from the entorhinal cortex or the schaeffer commissural fibres emanating from CA3, using bipolar stimulating electrodes, can be used to elicit glutamatergic excitatory post-synaptic potentials, recorded as extracellular field potentials from either CA3 or CA1 respectively, and a pattern of activation employed which will allow the experimenter to determine the action of drugs on basal synaptic transmission, as well as both short–term (such as paired pulse facilitation) and long-term plasticity (such as LTP / LTD). Therefore, the possibility of the discovery and development of compounds through the enhancement or modulation of hippocampal excitatory transmission and synaptic plasticity has become an increasingly attractive ‘proof of concept’ for potential drug efficacy in the management of dementia and cognitive impairment. Both academia and the pharmaceutical industry have been utilising this approach to develop potential nootropic drugs to enhance learning and memory. These include the AMPAkines16,17, GABAα5 inverse agonists18-20, selective inhibition of the glycine transporter Glyt1 to enhance NMDA receptor function21-23, HDAC inhibitors to improve cognitive dysfunction in Rubinstein-Taybi syndrome24 and their use in schizophrenia25, and the development of selective PDE4 inhibitors as cognition enhancers26-28. Further targets that have been identified and tested in this way are more extensively reviewed elsewhere29.

Previously, a major drawback of these types of experiments was that throughput was severely limited with conventional brain slice recording apparatus. Significantly higher throughput can now be achieved with commercially available multi-chamber brain slice recording systems, such as the SliceMaster (Scientifica), which allows recording from eight slices simultaneously yet independently. This not only optimises tissue use from animals, but also improves the quality of data since control and treatment groups can be clustered within the same experimental design30.

Multiple electrode array technology

Multi-electrode arrays provide a means to measure ensemble neuronal network activity in a simple in vitro system and thus determine how drugs might influence the behaviour of neuronal networks. Two instruments are commercially available: the Multichannel Systems and the MED64 instruments, both providing 64 channel recording facilities.

There are a number of different applications for multi-electrode array (MEA) recording systems. Dissociated neuronal tissue can be cultured onto MEA chips and maintained in vitro during the course of several weeks. During several days the network of neurons will become spontaneously active and this firing activity can be recorded from all channels simultaneously, offering a measure of spatial and temporal dynamics over a period of time. Often cultures will develop regular bursting activity which will persist over days or weeks. This offers the opportunity to investigate the effects of psychoactive drugs on neuronal network activity, giving insights into neuronal synchronisation and temporal organisation. Extracellular activity can also be measured in acute brain slices laid directly onto the electrode array. The ability to record from 64 electrodes simultaneously offers a lot more flexibility and information on the spatial distribution of field potentials across the slice. This technique has been applied to the study of synaptic plasticity and the investigation of oscillatory activity in areas such as the hippocampus31. Under certain conditions brain slices may be cultured directly onto MEA chips and longer term experiments carried out to study development of connectivity or regeneration of connections between closely associated areas32. In addition to the ability to record extracellular activity, each electrode can be stimulated to evoke activation of neurons. This ability enables the study of synaptic plasticity mechanisms such as long term potentiation (LTP) and long term depression (LTD) as described in the previous section.

The culture of retinal ganglion cells has proved extremely useful on MEA. Experiments using whole retina and retinal cultures have led to a clearer understanding of retinal function and the effects of drugs on the electroretinogram33. Other applications for MEA technology also have merit in improving throughput within the pharmaceutical industry. The ability to culture cardiac myocytes onto MEA chips and measure directly electrical activity from contracting cells has been utilised to quickly assess cardiovascular risk for hit to lead compounds. The potential for a drug to produce QT prolongation leading to adverse cardiovascular events is a major problem throughout the industry. By applying drugs to cultures of cardiac myocytes, a very rapid measure of action potential structure across the whole array can give a quick and easy measure of any effect the drug may have on the ventricular field potential34. Recently it has been possible to culture embryonic stem cells onto MEAs in order to study the development and identification of differentiated cell types such as cardiac pacemaker, atrial and ventricular subtypes35. This tool should prove extremely useful in furthering our understanding of the development of activity in neuronal stem cells.

Multielectrode arrays offer many advantages against conventional single cell or single electrode brain slice electrophysiology. Since recordings are made simultaneously from 60 electrodes and many cells are intrinsically connected, the system is very sensitive to perturbation or pharmacological intervention. The sensitive nature of this system has led to the development of portable biosensors for the detection of neurotoxins using cultured neurons36. Since neuronal networks can be cultured over several weeks and recordings can be made at intermittent time intervals, the development of connectivity and the influence of drugs on synaptogenesis can be measured37. In addition, the effects of chronic drug exposure can be measured on spontaneous neuronal activity during the course of weeks.

As well as using multielectrode recordings in vitro, it is now possible to extend this technology to 3-dimensional arrays for use in vivo. These are now available in a number of versions for either recording from the surface of the brain or implanting into various regions to measure extracellular electrical activity from particular parts of the brain and develop our understanding of network behaviour at the in vivo level38.

Electroencephalogram (EEG) in drug discovery

The electroencephalogram (EEG) has been shown to be a powerful method for classifying psychotropic agents and evaluating their pharmacodynamics39. The EEG quantitatively assess brain activity with a better sensitivity and temporal resolution than many other current imaging methods40. The electroencephalogram (EEG) is recorded from the scalp in humans and is generated by inhibitory and excitatory postsynaptic potentials of cortical nerve cells. The rhythmic cortical EEG activity arises from the interaction of thalamic, thalamocortical and cortical neurons with intrinsic oscillatory properties and involves large neuronal populations that include all major neurotransmitter systems. Therefore any deficiencies or over-activities in any of the neurotransmitter systems caused by a drug or underlying pathophysiology will cause an alteration of the ‘normal‘ EEG profile. The normal EEG profile has been well characterised and has also been shown to be very reproducible, which makes any consistent changes of the EEG profile a potential biomarker41,42. Subjects suffering from different psychiatric disorders (e.g. schizophrenia, Alzheimer’s disease, depression, general anxiety disorder) show different EEG profiles compared to normal control subjects (for review see39,43). Drugs from a wide range of psychopharmacological classes cause specific changes in brain activity, usually opposite to the EEG profiles seen in subjects with the psychiatric disorder. This would suggest that new drugs and potentially novel treatments could be identified based on the specific EEG profile that the drug causes i.e. the drug normalises the EEG changes recorded in psychiatric patients – the Key-Lock principle43. EEG profiling could potentially play a role in individualised drug treatment. However, there is another dimension to the Key-Lock principle that involves the temporal structure of brain activity. Recent evidence suggests that changes in the normal dynamics of brain function could be an underlying cause of psychiatric disease and it has therefore been suggested that psychotropic drugs should focus on restoring the normal temporal structure of brain activity44-46. However, conventional EEG analysis is limited and newer, more advanced analysis of EEG micro-structures is being introduced45.

Extensive literature shows that the EEG pattern changes dramatically with aging and cognitive decline. It has also been shown that specific features of the qEEG can distinguish patients with Alzheimer’s disease, vascular dementia and normal control subjects and that qEEG can also be used to predict, with a 90 per cent accuracy, future cognitive decline47. Interestingly, changes in the qEEG profile after a single dose of a cognitive enhancer (i.e. acetylcholine esterase inhibitor) was consistently found to be a good predictor of clinical cognitive response48,49.

EEG has also proven useful in the study of schizophrenia. In spite of the heterogeneity of the disease, a number of specific abnormalities in distinct frequency bands compared to a ‘normal’ EEG profile have been reported and these changes can be reversed with antipsychotic treatment (see 40,50). John and colleagues51 also showed that cluster analysis could be used to separate schizophrenics into a number of subgroups based on their qEEG profile. Furthermore, some of the subgroups have been shown to respond differently to antipsychotics52,53. Specific changes in EEG profile has also been reported for a wide range of disorders including anxiety and depression, attention deficit disorder, eating disorders, epilepsy and alcohol abuse (for references see 40). Numerous studies have reported changes in the EEG profile in depressed patients and also shown that antidepressants normalise the EEG profile, consistent with the Key-Lock principle discussed above54,55. It was also recently shown that one of the most consistent biomarkers of a selective serotonin reuptake inhibitor (SSRI) was a change in the EEG profile56.

This article has focused primarily on the developments that have been reported with regard to the changes in the EEG profile in humans. Another important marker of drug effect, which potentially shows greater species translatability than EEG measurements, is alterations in sleep and sleep architecture. A detailed discussion of sleep changes caused by sleep enhancers, antidepressants, antipsychotics and nootropics in rats and humans is a separate subject for review.

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