Which techniques do you consider to have the greatest potential for understanding disorders

of the brain?

Consider how different experimental methodologies have been used in the study of a
neurological brain disorder.

ALSO; SYSTEMS VS MOLECULAR NEUROSCIENCE essay- write an intro. (systems

poor understanding- need to know this before we can figure out cognitive)
Since the time when Ramon Cajal made his famous drawings of individual neurons, huge advances in
molecular biology, electrophysiology, and computational neuroscience in the last century have revolutionized
our understanding of the complex processes occurring within the brain. Molecular techniques have provided us
with the molecular determinants of building the nervous system, and how signalling cascades transduce signals
originating outside of the cell into the regulation of gene expression, whilst at the cellular level our
understanding of neurotransmission has progressed significantly. Nonetheless, our understanding on the systems
level is still relatively poor; how these neural circuits function and the mechanisms through which behaviours
are generated. Neuroscience is still in its infancy, and in the future we may be able to unravel brain function and
how it is altered pathphysiologically by taking an integrative approach using the various tools that we have.

Electrophysiology has been key to neuroscience. Hodgkin and Huxley (1952) used the voltage clamp to
explain the propertied of the action potential in the giant squid axon, and this technique has continued to be
important in the study of membrane potential changes, and capacitance measurements can also be used to
indirectly inform us about vesicular release at synapses. The patch clamp which was developed by Neher and
Sakmann has been hugely influential, as it has allowed the investigation of unitary conductances. Investigating
the properties of single ion channels has allowed us to understand how mutations in the gene encoding a
particular type of channel can lead to disease. For example KCNA mutations which cause episodic ataxia.

These intracellular recording techniques however, are invasive and require disruption of the plasma
membrane. Hence extracellular recording techniques such as the EEG, where electrodes are placed over the
scalp to measure event-related potentials, or local field potential recrdings can be used in an awake animal to
investigate populations of neurons. The EEG has been important in understanding and diagnosing the aberrant
activity seen in epilepsy. The advent of juxtacellular single cell recording is increasingly useful as it allows
recording or stimulation of a single cell and can be used in conjunction with the EEG or local field potential
recordings in order to study the characteristics of populations of neurons, but then can be used to label the
neuron with biocytin to study its anatomy. Electrical stimulation techniques are also important in showing
causality of neural activity in behavioural activity.

Ramon Y Cajal made his first observations that supported the neuron doctrine through a microscope,
and the principle of building up an image from scattered radiation still has an important part to play today for
visualization of intact tissue at high resolution. Most optical microscopy makes use of aromatic dyes or
fluorescent molecules which provide a high signal to noise ratio, and we use immunofluorescence and proteins
tagged with GFP to identify a particular molecule or to report its expression. Microscopy is so important in
investigating the histological markers of disease, and not only behavioural signs, and so is vital in the diagnosis
of diseases. For example we can readily identify the toxic protein species in Alzheimers (A-beta), Parkinson’s
(lewy bodies) and ALS (inclusion bodies).

Microscopy can also been used quantitatively, using signal changes to report concentration changes or
receptor binding for example, usind radiolabelled or fluorescent tagged markers. For example dyes such as
FURA-2 have been important in understanding the intracellular calcium signalling pathways, and also in
demonstrating the role of elevated Ca2+ levels in excitotoxic damage and cell death in the region of penumbra
of following acute damage to the CNS. Moreover, fluorescence has been important in the study of
neurotransmission and cellular neuroscience, for example styryly dyes such as FM-1-43 to investigate
neurotransmitter release, which has begun to replace quantal analysis, and also characterizing protein
trafficking, for example of Glutamatergic receptors, to the post-synaptic membrane. Due recognition of the
importance of these techniques was given, when Roger Tsien, a giant in this field was awarded the Nobel Prize
in 2007. Today microscopy can be used at ever higher resolution, and electron micrographs can provide high
magnification of the CNS, whilst techniques such as 2-photon imaging and TIRF acn be used for subcellular
visualization. Thes techniques and Forster resonance enerhy transfer for understanding molecular dynamics will
be of increasing importance.
Techniques such as x-ray crystallography are becoming increasingly useful in understanding
determining the arrangement of atoms within a crystal, and how structure relates to function. This is used in
determining how drugs interact with their protein target, and will be important in understanding how a mutation
in the primary protein sequence can alter the conformation and properties of a protein pathologically, for
example in certain channelopathies, something which is not readily apparent just from isolating the
polymorphism. XXXX

The impetus of much research in neurobiology has turned towards the molecular, and we now can
identify the mediators and modulators that are responsible or are involved in neurological disorders. Molecular
biology has also allowed the purification of unknown proteins and quantification of gene expression. This is
exemplified by Martin Schwab’s chacterization of the NoGo protein as the proteinaceous species which was
responsible for some inhibitory property of the adult CNS to regeneration after SCI or MS. He used ultra-
centrifugation to separate the protein and generated a monoclonal antibody to the protein, and found that its
blockade allowed some recovery of locomotor function in mouse models. Gel electrophoresis and affinity
electrophoresis are other techniques for separating proteins or nucleic acids, and identification of DNA species
is now facilitated by DNA microarrays and gene expression can be measured using RT-PCR. These techniques
will be important in the identification of regulatory proteins, and for clonal expression. Monitoring changes in
gene expression has been important for studying a number of brain disorders, (e.g. c=fos expression as a marker
of resilience to depression) and also in allowing us to understand the effects that pharmacological agents have
on gene expression. For example it has been postulated that the therapeutic effects of anti-depressants are
mediated by increases in BDNF levels XXHowever, as the field of LTP demonstrates, in the future the impetus
will be on making a link between molecules and how they affect cellular processes.

The use of linkage analysis has helped identify SNP’s and genetic correlates in a number of
neurological diseases whose cause is unknown, and we can now clone and express the protein in a suitable
system. This may be in an expression system, for example the oocytes of xenopus laevis, which has been
extensively used for characterizing the properties of membrane proteins, and is a powerful method for testing
pharmacological agents, XXXX, Alternatively it may involve the transduction of a DNA-containing vector into
a host in order to investigate a particular genetic manipulation. We have several animal models including mice
and several invertebrate models such as drosophilia and nematode, which are used for their ease of
manipulation. Mouse embryonic stem cell lines can be readily propagated, and detailed maps of the
Drosophila (fruitfly) and nematode genomes are available and they have short
generation times and are amenable to germline manipulation. These models have been
important in developmental neurobiology,and have also helped us identyify the target of
neuroactive substances, such as Lithium and its effects on phosphor-inositide signalling
(Berridge).

Technical advances now allow us to insert particular mutations in a protein in order to investigate its
function, with temporal and spatial specificity XXXX. We now have the ability to target almost any component
of the genome, and the tools that may allow us to explore the effects of single genes on global assemblies of
neurons, and how this can affect behaviour in psychiatric diseases for example. There are 3 ways by which we
target the genome. Insertion of exogenous gene sequences are into the genome of a mouse; gene targeting
technologies, in which mutations are targeted to inactivate or modify an endogenous gene of interest; and
conditional manipulations, in which mutations are restricted to particular stages of development or to particular
regions of the CNS.

Recent progress in this rapidly advancing field includes the use of post-transcriptional gene silencing/
RNA interference to knockdown a particular gene. Long lasting RNAi-mediated gene knockdown can be
achieved using lentiviral vectors that express the siRNAs. These techniques are used extensively in neurosceice
and could be possible therapeutiv avenues in the future. For exmple, studies by Raoul et al. and Ralph et al.,
showed that the injection of the lentiviral vectors led to substantial delays in the onset and progression of the
ALS by SOD-1 knockdown. RNAi reduced the expression of mutant SOD1 in culture and in vivo, prevented the
death of spinal and brainstem motor neurons and improved the motor performance of the mice in different
behavioural tasks. Raoul et al. also measured electromyographic responses in their animals, and found them to
be preserved in the mice treated with the lentiviral vectors. In fact, superoxide dismutase has not yet been
demonstrated as causal in ALS.
Gene targeting has been vital for identifyting the causality of certain genes, and has highlighted numerous novel
findings too numerous to explain, for example, knockout mice have demonstrated the key role of the
endogenous opioid system in addiction and the fefcts of alcohol, as µ-opioid receptor K.O’s do not self-
administer alcohol. These techniques may also be important therapeutically in the future, if we could target
insertions, and mouse models have shown therapeutic benefits, for example alcohol self-administration is
reduced alcohol in rats after D2 receptor expression is increased by viral vector transduction, in line with
findings that striatal D2 receptor availability is reduced in patients with addictive illness.

However, there may be significant redundancy in gene expression, and several genes may contribute to
a behavioural phenotype, which confuses the interpretation of results of KO studies. Genetic manipulations in
animal models also do not account for post-translational modifications that take place in humans, and there may
be considerable differences between the two. This is relevant in human diseases where it is believed that protein
misfolding is causal, for example in ALS, PD and Alzheimers it has been postulated that this is the case, and
certainly in AD, there is increasing eveidence that oligomerization underlies the toxic property of the protein.

There is now an acceptance that many brain disorders are influenced, at least in part, by epigenetic
modifications, demonstrated by the benefits of XXXXX histone deacetylases. ����
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Nevertheless, animal models have been essential to our understanding of disease. An inducing
protocol is needed, which can be designed to reproduce the aetiology e.g. drugs of abuse to model drug
addiction; to mimick symptoms e.g amphetamines to model acute psychoses in schizophrenia; or for modelling
the efficacy of drugs, and then we are able to study various elements of neurological disorders. In vivo models
are useful because we can study the brain in an interacting brain, within the context of the physiology of a whole
organism, whilst in vitro techniques are often confined to investigation at the single cell level, which cannot
accurately reproduce the hormonal,/////////////// in vivo, nor can it reproduce the oscillating, interacting networks
of neurons that determine brain function.

There is now a commitment to reduce the use of animal models (the National centre for 3R’s), and it is
accepted that animal models have their limitations for translation to humans. Hence there is far greater emphasis
now on improving in vitro methods, and computer models of cortical circuitry, but most importantly to
developing objective techniques for studying the brain in vivo. This has centred around imaging techniques.
New techniques aim to improve our understanding of the anatomy of the brain, but also of the function. Initially
MRI allowed us to investigate the anatomy of the brain, by providing contrast between the different soft tissues
in the body, using a powerful magnetic field to align the nuclear magnetization of hydrogen atoms in water in
the body, and this has helped identify structural differences in disease states, for example enlarged ventricles in
schizophrenia xxxxxx. But now improved techniques also focus on individual neurons. For example neuron-
specific dyes for tagging a whole neuron. In fact, recent work by Josh Sanes has led to a novel method for
studying all neurons together, and it is hoped that we may be able to generate a cortical map or “connectome,”
which will enable us to understand the faulty wiring in a diseasesuch as SZ. In brainbow mice transgenes,
Cre/lox recombination is used to create a stochastic choice of expression betwee 3 or more fluorescent proteins
colours .

In vivo functional imaging is also increasingly important. Functional MRI (fMRI) measures stimulus-
related changes in blood flow, and is a valuable method to study task-related brain activation in
patients with neurological or neuropsychiatric illness. With neuronal activation, relatively
increased oxyhaemoglobin concentration results in enhanced signal changes in proportion to blood flow and
blood volume (blood-oxygen level dependent (BOLD)). This technology gives high temporal and
spatial resolution, and can be used to investigate the neuroanatomy of particular brain states, for example Irene
Tracey’s work at fMRIb has led to the characterization of a “pain signature” . It has also been useful in the study
of functional cortical re-arrangement after brain injuries, for example showing enlarged hand representation in
the ventral premotor cortex after an M1 lesion in squirrel monkeys. fMRI has also been useful in characterizing
the default network- brain activity when an individual is inactive, and this default network has been shown to be
disrupted in autism, schizophrenia (Harrison 2007) and Alzheimer’s disease (Lustig 2003), and it has been
suggested that some drugs might mediate their efficacy by effects on the default network, for example
GABApentin, the most clinically useful chronic pain treatment (Raichle). Furthermore pharmacologic fMRI is
increasingly used to study the effects of neurochemical perturbations on brain function at
a regionally-specific or systems level. phMRI has been applied to investigations of
cocaine dependence(Breiter 1997),77 depression (Kalin 1997), schizophrenia (Honey
1999), and Parkinson’s disease. A recently emerging, exciting application of phMRI is in
the prediction of clinical response to pharmacological treatment. For example, patients
with depression who are treated with antidepressant medications demonstrate changes
in activation of affective circuitry that are associated with clinical response to
pharmacological treatment. The addition of genetic factors to these investigations holds
promise for illuminating pharmacogenetic mechanisms of CNS drug effects.
Positron Emission tomography is another functional imaging tool, and uses positron-emitting isotopes
of carbon, nitrogen, oxygen or fluorine which are incorporated into H2O15, deoxyglucose (2-flouro-2-
deoxyglucose), or any neurotransmitter, and so act as tracers of metabolic process or drug activity, and can be
used for assessment of neuroreceptor number and affinity. PET technology has been particularly well employed
in identifying functional deficits or excesses in neurotransmitter levels, for example in measuring deficits in
serotonergic transmission in mood disorders, and drug induced changes. These techniques are widely lauded,
and certainly fMRI has been highly publicized for use as a lie detector. However they provide only indirect
correlates of brain activity and no understanding of function, and they may also be sensitive to transient brain
states such as arousal, attention or sleep deprivation. Hence caution must be exerc ised when reading articles
titled “fMRI can reveal your secrets.”

New study- Nicola Fillipini- shows APOE-e4 causes increased greater brain activity in earlier life....

Imaging has been useful because it allows in vivo study that is non-invasive. However, in order to
study several aspects of brain disorders, an intervention is required, for example in studying brain circuitry, the
simplest way to achieve this is to lesion it and see how it affects neurological function and behaviour. Now
however, transcranial magnetic stimulation is a technique which is available to us that can selectively lesion, or
upregulate function in a specific brain area, in a way that does not necessitate destructive lesions. TMS
introduces electrical noise into a specific cortical region, and will therefore disrupt any useful signals passing
through that region, and so can investigate causality of a particular brain region associated with tasks. (Coil
sitting over cortex. Sudden high magnetic field. Good spatial rsolution.) Most study with TMS has been carried
out in the primary motor cortex, but TMS may have therapeutic effects in a range of disorders including SZ,
depression and ALS. It has been shown that temporary interference of motor function brought about with TMS
can lead to temporary reorganization of the pattern of activity associated with voluntary hand/arm movements
that closely resembles the pattern seen after stroke. So there is a strong rationale for applying TMS in stroke to
promote or speed restoration of function through natural processes of neural reorganization (Stefan et al).

Our advances in neurosciecn has been phenomenal in recent years. Future techniques should aim to
integrate our knowledge in order to develop a picture of the nervous system which resolves network activity
with the subcellular mechanisms which have been identified. As Pascal said, “it is as impossible to know the
parts without knowing the whole, as to know the whole without knowing the individual parts.”

Pharm- understanding how drugs actually work?�� �����������

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�Genetics- addiction- systems--- mesolimbic. NT levels. Detecting antibodies and radioreceptor assays....
Nowadays use fluorescence
Depression- behavioural techniques. Animal models. But also DBT has proven useful possibly.
Animal models for testing drugs. Imaging too.....

Computer models too in drug development

MS/ etc......Also characterization of the inhibitory proteins. Labelling etc.

SZ- pharmacology- neuro pharmacogenomics

Alzheimers- anial models, genetics definitely. Linkage analysis. Susceptibility genes. Dementia; memory and
learning. Moecular in definng oligomers are important....RNAinterference techniques

HUGE....pharmacology?
Ion channels- molecular cloning, genetics. Electrophysiology.

Chronic pain- also fMRI as important. AND electrophys- maladaptive pain and phantom limb pain...

BUT ethics of imaging. Correlation based

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Despite new perspectives in neuroscience, drug discovery seems to have

Molecular approach and systems

Pharmacology, computer, knockouts- mention siRNA and new methods- knockins etc, lesioning, imaging

Small molecule inhibitors

Examples; ion channels, where pharmacological blockade is not yet possible....

Modelling in animals

....talk about cis mutation substitutions and point mutations and what this has contributed to
ion channel field....can be used in conjunction with field potential/patch clamps to determine
function...(i.e. can do electrophys in vitro, as well as doing observation of behavioural
phenotype....e,g, seizures

An advantage of lesion manipulations over chronic

drug manipulations is that lesions may lead to deficits and/
or neuroadaptations in a variety of brain systems rather than
just the one or few affected by a drug

For example, Churchland and Segjnowski [1994] and

Amit [1989] use artificial neural networks that incorporate
biological parameters, to explore interactions
among neurones and how they learn and remember

in vivo imaging.... http://www.nature.com/nrn/journal/v7/n6/full/nrn1905.html

Roger Tsien, design and biological application of novel fluorescent probes.

Genetics
Genomics and Proteomics allows the sequencing, processing and identifying individual gene products.
This will provide a template for exploring this vast array of proteins. Neuropharmacology in the post-genomic
era will be involved in the development of drugs aimed at a new set of proteins and a more profound
understanding of the mechanisms by which these drugs regulate behaviour and function.

Pharmacogenetics represents the ultimate application of the molecular era to neuroscience and medicine. Holds
the promise of personalized medicine, where an individual’s genetic make-up can be used to predict their
responsiveness to drugs, and may be able to identify distinct aetiologic subtypes of complex disorders such as
autism, schizophrenia and epilepsy.
e.g. use of fruit flies (drosophilia) and nematode (C.elegans) as models have been useful.... identifying the
biologically relevant targets and effectors of a given neuroactive substance

Knockout knock in
EEG

PET and SPECT

Remain the only viable techniques for studying logand binding in the brain, and there are new radiotracers
which allow the stidy of post-receptor signal transduction, and even gene expression.

Also, Magnetic resonance spectroscopy; allows mappig of distribution and concentration of molecules in the
brain.

fMRI advances; Some of the biggest advances in functional MRI (fMRI) methodology for the indirect
measurement of neural activity have been in the time domain. Bandettini describes new
methods for improving the temporal resolution of fMRI, in particular event-related designs. In more
traditional blocked-trial designs, the BOLD signal is averaged for many seconds, typically for
several trials of a behavioral task. However, with event-related designs, one can measure BOLD
changes for events lasting less than 2 seconds, which allows one to distinguish activity changes in
one part of a trial from another, e.g., from the encoding to the retrieval phase of a trial in a
memory task. As described by Hillyard and Kutas,
new analytic techniques have improved the spatial resolution
of ERPs, and there has been considerable progress in
combining the spatial resolution of fMRI with the temporal
resolution of ERPs.

Transcranial magnetic stimulation

RATher than elsioning..... ethically it is also better. Will allow them to recover
function afterwards?

BRAINBOWS

In the future, in vivo imaging will be key to understanding the minute biochemical
changes