To what extent can animal models be used to understand human brain developmental disorders?

Brain development in utero: Transplacental exposure to maternal drugs, and metabolic imbalances.
Perinatal insults and low-birth-weight infants: Developmental aspects of perinatal injury,
hypoxic/ischemia, pediatric epilepsy, congenital infections involving the CNS [excluding HIV].
Many of the genes for schizophrenia identified so far appear to impact biologically on some
of the most basic processes of brain development, involving neuronal differentiation ;
Porteous et al 2006), synapse
biology (eg. Harrison 2006) and various processes involved in neuronal plasticity

- decreases in PPI of startle, an operational measure of sensorimotor gating deficits that are evident
in patients with schizophrenia, have been demonstrated to result from socially isolating rats from
weaning until after puberty
- Recent studies have shown that 6 to 8 weeks of social isolation during development, but not
during adulthood, produces deficits in PPI that are at least partially reversible by the
administration of neuroleptic dopamine antagonists or by clinically effective atypical
antipsychotics having antagonist activity at multiple receptors (53,54).
- Furthermore, postweaning isolation rearing of rats also results in deficits in the gating of the N40
event-related potential, that are analogous to the deficits in P50 gating observed in schizophrenia
(55).
- Because schizophrenia commonly emerges in early adulthood, developmental factors have
provided the basis for Intrauterine viral encephalopathies. Also implicated in autism.
- Imaging of brain subsequently and behavioural paradigms. Social isolation. Potentially, in contrast
to the drug-induced models of gating deficits, these models might have etiologic validity and
might be sensitive to antipsychotic drugs having novel mechanisms of action

The British Union for the Abolition of Vivisection believes that whole-animal experiments are
“outmoded” and could, and should, be replaced by in vitro studies and computer models. Discuss both
the merits and the potential limitations of their position in the context of neuroscientific research.

Discuss the usefulness and limitations of the available animal models in the development of new
therapeutic interventions in the brain.

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3 (GOOD FOR REFERENCES- read it)

The best answers included an overview of the different types of animal models and critically discussed
the validity of each of them, using specific examples where appropriate
and a broader analysis of a wider spectrum of diseases would enhance the answer. And also some
future advances which are likely.

Financial/ethical/ mechanistic/ historical/ clinical/ scientific. Accepted view. Coounterviews. My view.

Can do genetic things......
 Nonhuman models enable the investigation of the neurobiology of the phenomena of interest
using invasive techniques that cannot be used in humans, and can also be implemented in the design of
new treatments. They can significantly reduce the cost of drug development by increasing the degree of
confidence in a particular pharmacologic approach before undertaking expensive and time-consuming
clinical trials. Animal models have led to tremendous advances in understanding brain
function, however, there is also suggestion that they are outmoded, and are the cause of a “bottle-
neck effect” in psychotropic drug discovery compared to some of the rapid in vitro studies for drug
development (tallmann 1999). Furthermore, several high profile failures of drugs which had proved
efficacy in mice, but not in humans (for example neurokinin receptor antagonists as analgesics), have
also fuelled the debate that animals have only minimal utililty for modelling human disease.

Most scientists would argue that the vertebrates, including rodent and primates, derived from a
common ancestor, suggesting that there would be certain degrees of homology between the two
species. Humans share over 99% of their genome with lower mammals including rats and mice and
most psychiatric diagnoses have a counterpart in animals. However many argue that no animal model
can fully reproduce the perceptual, cognitive and emotional features of the human disease. Whilst all
are in agreement that animal models have certain limitations, there is a divide over whether the relative
strengths outweigh the weaknesses.
An animal model may represent a disease on different levels: (1) it may reproduce aetiology
(2) it may mimic phenomena i.e. the clinical symptoms; or (3) it may predict responsiveness to
available treatments. Models that reconstruct the etiology and pathophysiological mechanisms of the
disease are of the highest order of fidelity and can they have so-called “construct validity.” For
example, recreating the effects of a loss of function mutation in Kv1.1 in episodic ataxia type I (herson
2003), where the causal gene KCNA1 can be knocked out, and effects of current treatment
acetazolamide on firing patterns can be studied. This is also the case with drugs of abuse where the
inducing factor is known to be excessive administration of a drug, and animal models have highlighted
the role of the mesolimbic pathway in reinforcement properties in the self-administration paradigm,
which measures how hard an animal will work to obtrain the drug of abuse (Koob 1992
However, caution must be observed with animal model even when the aetiology is identical,
as it is possible that the physiology or anatomy of the animal model does not identically replicate that
of humans. For example, in animal models of spinal cord injury, the aetiology is similar, i.e. a lesion of
the corticospinal tract, and researchers can record regeneration after administration of a drug, such as
rolipram, or chondroitinase ABC (Elizabeth Bradbury) by measuring locomotor function, but it has
been shown that in rodents it is actually the Rubro-spinal tract which serves primary motor function.

Because animal models involve an inducing condition (Geyer and Markou,

2002), the choice of an inducing condition by necessity involves a hypothesis
about the etiology of the disorder that may or may not be correct. This highlights a
fundamental point; that our knowledge, of psychiatric disorders in particular, is severely lacking. For
example, Experimental Allergic Encephalitis , is the model used for MS, motivated by observations
during convalescence from viral infections (Rivers and Berry 1933). However, this model may not
have good construct validity as the aetiology is as yet unproven, and accordingly, some characteristics
of MS such as epitope spread, are grossly under-represented in EAE (Yu et al 1996). Moreover there
are substantial immunologic differences between mice and humans, which has implications for
predictive validity. EAE models predicted the efficacy of anti-TNF-alpha treatment, as in Rheumatoid
arthritis, but the “Janus-like” properties of TNF-Alpha actually make relapse-remittance worse
(Feldmann 2005). Nevertheless, it has led directly to the development of 2 therapies in MS;
Glatiramer, an altered peptide ligand and Natalizumab, a monoclonal antibody to alpha4-Beta1
integrin.

Because of the complexity of understanding the aetiology (especially in psychiatry) and

discrepant nature of diagnostic categories in psychiatry, many animal models mimic only specific signs
or symptoms associated with the disease, rather than mimicking the entire syndrome. For example,
amphetamine models of schizophrenia model the positive mania-like symptoms in mice, yet fail to
recreate negative symptoms such as catatonic behaviour, hence the construct validity is questionable.
SZ involves uniquely human behaviours in the realms of perception, thinking and
the experience of emotions, however by modelling the specific symptoms a
fundamental problem plaguing animal models is minimized= face validity. These models have high
predictive validity as models for the efficacy of DA antagonist treatments for schizophrenia e.g
risperidone, however, there is also evidence suggesting abnormal neurodevelopment
(Beauregard), dysfunction of cortical glutamatergic neurons (e.g. ketamine an
NMDA antagonist induces SZ-like hyperactivity in rats), and genetic susceptibility
is important.

Many behavioural paradigms measure a variable that may or may not even be symptomatic of
the disorder in humans. For example defensive burying as a model of anxiety, a behaviour exhibited by
mice in response to fear-elicting stimuli, and tests of the approach-avoidance conflict that uses reduced
exploration of novel environments as a marker of anxiety. e.g. open-field and elevated plus mazes.
These models have good face validity for anxiety in rats, and are also predictive of anti-anxiolytic
effect, confirmed by the ability of clinically effective benzodiazepine anxiolytics to
increase exploration under these conditions (Crawley, 1985) . However, they are not
necessarily good at predicting efficacy in the clinic, for example, the clear efficacy
of antidepressants in the treatment of generalized anxiety disorders does not
back-translate to the elevated plus maze (Lister 1986) . Furthermore, we cannot
separate state from trait anxiety, so they may interact and confound the response
to pharmacological interventions (Steckler 2008). State anxiety is more likely to
reflect a natural biologically advantageous response to an unfamiliar
environment, whereas trait anxiety may mimic pathological anxiety states in the
absence of threatening stimuli. Moreover, pre-handling is also like to place the
animal in a pre-aroused state, and in chronic pain models was found to induce
hyperalgesias

Animals models are also used to test or model neurological phenomenon, as they are believed
to be objective measures. For example, stimulation by electrodes placed over the Peri-aqueductal gray
and Locus coeruleus can potentiate anxiety(Jenck 1995), or as an objective measures of functional
changes. For example, Prepulse inhibition and gating of P50 event related potentials are used as
measures of impaired sensorimotor gating, and is useful as human schizophrenics have impaired PPI of
startle (Parwani 2000).These models have helped to highlight deficits in our knowledge of SZ since the
disruptive effects of glutamate antagonists on PPI of startle in mice and humans are reversed by
atypical, but not by most typical, antipsychotics (Bakshi 1995).

Animal models have also been vital in investigating the function of single genes, and their
causality in disease. Techniques now allow very precise ‘‘lesions’’ (knockouts) or increased expression
(knockin) of specific proteins in selected brain sites in adult animals, and at selected times (using
inducible promoters). This technology has allowed us to model several diseases of unknown cause
which have genetic factors identified by linkage, for example Oddo and colleagues developed triple
transgenic mice for PS1, APP and MAPT overexpression, and they displayed both neurofibrillary
tangles and amyloid plaques, and also displayed effects in hippocampus on synaptic plasticity.
Moreover, transgenic mice expressing the wild-type human gene for -synuclein develop several of the
clinicopathological features of PD, including accumulation of Lewy bodies in hippocampus and basal
ganglia, and loss of dopaminergic terminals in basal ganglia. ALSO, ALS-like pathology occurs in
mice bearing the human mutated SOD1 gene; hyaline inclusion bodies, muscle atrophy, astrocytic
damage and the extensive loss of large myelinated axons of motor neurons (Gurney 1994).

However, not only is their likely to be considerable redundancy in gene expression which
would make interpretation of results difficult, but there may be post-tanslational modifications and
epigenetic mechanisms which add to complexity and transgenic anbimals may fail to reproduce. In the
context of Alzheimers, Amyloid Beta can exist in several forms due to post-translational processes, and
it has been suggested that the oligomeric form might actually underlie the toxic property (Cheng et al
Nat. Neurosci; Walsh et al 2002). Whereas in SZ, alternatively spliced isoforms and post-tranlational
modification of COMT, a risk-associated gene for schizophrenia in its val/met variant. In addition
genes interact with the environment and with other genes and COMT interacts with NRG1(Nicodemus,
2000). The complexity implied by these discoveries would suggest pathways and their interactions
impact most on human perception, cognition and behaviour so modelling disease by individual gene
perturbation would be completely futile.

Because several diseases such as Schizophrenia and autism are believed to be developmental,
an inducing condition, whether it be environmental manipulations, drug manipulations, lesions or
genetic manipulations can also be implemented at different stages to investigate a neurodevelopmental
component. However, progression of disease is rapid in the animal model compared with the clinical
condition, so the narrow time window for intervention decreases the probability of selecting the
optimum time for treatment. Moreover, when attempting to model a developmental or age-related
disorder, assumptions have to be made about the corresponding age in the animal model that most
accurately represents that in the humans, for example when activating an inducible knockout. In fact,
many studies in AD-an age-related disease- are conducted in young mice simply because the cost of
maintenance of the animals is too high, and when using knockout mice, their viability is diminished
after a certain amount of time.

This raises an interesting point about the animals themselves. How much can we extrapolate
from animals which are most often inbred and reared in controlled conditions. Furthermore, the choice
of animal is usually based on cost or ease of manipulation (for example mice where embryonic stem
cell lines are easily propagated), rather than for approximation to humans. For example Sprague
Dawley rats are chosen for study into neuropathic and chronic pain because they tend to be more
responsive to thermal nociception. This highly subjective choice of subject seems wholly unscientific,
but also, how much can we learn about chronic pain from an acute stimulus in one particular modality
of pain (Mogil 2000). In fact, consistent phenotypes are rarely obtained by modification
of the same gene even in mice. The disruption of a gene in one strain of mice may
be lethal, whereas disruption of exactly the same gene in another strain of mice
may have no detectable phenotypic effect. If this is true of the impact on one
gene of the rest of the mouse genome, how much more is it likely to be true of
the impact of the rest of the genes in the human genome (Pearson 2002)

COMPARED TO IN VITRO;

There are calls that we should abandon animal models in favour of in vitro tests and computer
modelling. However, increase in cost of drug development, and emphasis on translational medicine
means animal models have become more important again.Techniques using high throughput
screening and a barrage of in vitro tests can be used to predict and measure
various biochemical parameters; the likely metabolism of drug compounds;
assessing gross safety concerns and cellular toxicity. However whilst in vitro
testing can address certain practical concerns, there is no substitute for study of
an integrated organism in vivo, where we can test for efficacy in the whole
animal, and ascertain in vivo EC50 for example to get some idea of safe dosage in
humans. Moreover, study of the whole brain in vivo allows us to study brain
circuitry in disease and the complex interactions that take place between
networks and make predictions about the potential effects of psychotropic drugs in patients.

CONCLUSION

Animal models have led to the development of treatments in a number of disease settings, and
can be useful as long as we can interpret them correctly. The identification of surrogate markers of
diseases and the use of multifactorial approaches to assess both behavioural and biochemical
parameters should increase their utility and validity. It is unrealistic to attempt to go from the ‘‘test
tube’’ to the clinic when attempting to treat complex mental, cognitive, and emotional disturbances that
do not yet have clearly defined neurobiological substrates, or even correlates. Clinical studies need to
be informed by results from animal studies as much as the reverse is true. But caution must be
exercised, and it is accepted that our methods need to be refined (National centr for 3R’s)

- Multifactorial; lend support to a Proof of concept for a hypothesis for

neurological function or in drug development. For example the behavioural
assay for pain of paw licking which is sensitive to error, can be supported by
measuring neuronal firing rates or using functional imaging.
DEPRESSION;

TALK ABOUT ASSUMPTIONs- life events assumptions in depression models. Also

Example where drug would was developed without an animal model;

Indeed, if the forced swim test (Porsolt et al, 1978) had been the most
frequently used preclinical assay for the detection of antidepressant-like
compounds when fluoxetine was developed (the Investigative New Drug
application for permission to examine the drug in man was filed in 1977; Wong e
al, 2005), it is interesting to consider fluoxetine’s potential fate given its lack of
activity in the original rat forced swim test before the test was modified to detect
the efficacy of compounds such as fluoxetine (Cryan et al, 2002). If activity in the
original forced swim test had been taken as a necessary requirement before
deciding to develop the compound fully, depressed patients would not have
benefited from the availability of fluoxetine.

AN example of a confound;
For example, in an animal model of anhedonia with relevance to depression, the
combination of a selective serotonin reuptake inhibitor (eg fluoxetine or
paroxetine) with a serotonin-1A receptor antagonist permanently reversed the
anhedonia associated with psychostimulant withdrawal in rats, while inducing a
transient anhedonic state in control rats (Harrison et al, 2001

learned helplessness;
It has been argued that the model has good face validity because there is similarity between the
behavioral characteristics of learned-helpless animals and signs of depression in humans (for critical
reviews, see 87, 88). For example, learned-helpless animals exhibit loss of appetite and weight,
decreased locomotor activity, and poor performance in both appetitively and aversively motivated
tasks. These behavioral characteristics of learned-helpless animals are considered equivalent to loss of
appetite and weight, psychomotor retardation, and anhedonia, respectively, demonstrated by depressed
humans (DSM-IV). As discussed above, however, symptom similarity (i.e. face validity) does not
necessarily imply homology. Thus, these claims for the model's face validity do not address the
question of whether the model has any utility. The predictive validity in terms of pharmacological
isomorphism of the model is indicated by the fact that pharmacological treatments clinically effective
in depression, such as tricyclics, MAO inhibitors, atypical antidepressants, and electroconvulsive shock
therapy are effective in reducing the behavioral and "physical" abnormalities seen in animals exposed
to uncontrollable stress.
BUT, the assumption of the model is that learned helplessness is exhibited by depressed individuals.
Very little evidence supports this assumption, however (8). Clinical observations describing depressed
patients as exhibiting behavioral despair or negative cognitive sets do not constitute experimental
evidence for the existence of the specific psychological process of learned helplessness, on which the
model is based.

Some depression animal models- chronic mild stresses e.g. tilted cage/ food
deprivation
Developmental; Maternal separation.
5-HT; tryptophan free diet.

Escapable shock (learned helplessness); Forced swim test ; Intracranial self

stimulation (anhedonia)
GENETIC= 5-HT transporter knock in OR CRF knock in.

CHRONIC PAIN
We use acute pain assays to model chronic pain!

Capsaicin to activate TRPV1 (Caterina et al 1997). Again, Problems with modality.

OR intradermal injection of endogenous nociceptor-sensitizing molecules such as

bradykinin, pro-inflammatory cytokines, prostaglandins, serotonin and substance P70.

Epigenetic mechanisms of pain. In animal models;

It is now appreciated, for example, that dietary factors (such as soy40 and omega-3
fatty-acid41 content) can interact with genotype and affect neuropathic pain-related
behaviours in rats (Shir et al 2001). Also handling and novelty stress can also produce
hyperalgesia.

The behaviours typically measured are either spinal reflexes (such as withdrawal from experimenter-
applied stimuli)132, spino-bulbospinal reflexes (such as jumping or abdominal stretching)133 or simple
innate behaviours (such as vocalization, scratching, biting, licking and guarding) that can be performed
even by decerebrate animals134, 135. Reflexive withdrawals may be affected by surgical damage to axons
of motor neurons136, 137, for example, and are not obviously relevant to clinical pain, which involves
cognitive and emotional appraisal and learning, and extensive supraspinal processing

PARKINSON’S

PD is characterised by intraneuronal cytoplasmic deposits (Lewy bodies) of the PD-associated gene

product α-synuclein. Transgenic expression of α-synuclein recreated hallmark features of PD in mice
and fruit flies, establishing α-synuclein as PD-causing drug target. Moreover, environmental risk
factors such as the pesticide rotenone have been used successfully to generate rodent models of PD.
Lesion models of PD are being exploited for the development of experimental gene therapy and
transplantation approaches.

GENE ENVIRONMENT INTERACTIONS----MULTI-FACTORIAL DISEASE WHICH HAVE

DEGREE OF SUMMATION. E.G. GENETIC INFLUENCE IN SZ AND IN PD

Neurotoxins such as 6-hydroxydopamine or 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP),are widely used
as animal models of PD.... Neurotoxins such as 6-hydroxydopamine or 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP),are widely used
as animal models of PD....

.... However, the animal model is

an acute model and non-progressive.,,,,very important for progressive diseases.

PARK1,4 (SNCA) has been identified as a causative gene for autosomal

dominant PD, its gene product, SNCA has
been considered as one of major components of Lewy
bodies.
Although a number of transgenic mice
models overexpressing SNCA have been developed, and some show increased
vulnerability to MPTP
(Table 1), none of the transgenic SNCA mice accurately
model PD. In contrast to mice models, Drosophila overexpressing
human wild or mutant types SNCA revealed a remarkably
selective age-dependent loss of subsets of dopaminergic
neurons. Moreover, the neuronal loss was associated with
the presence of LB-like, filamentous intracytoplasmic
inclusions

Fruit flies and Nematode

Drosophilia (fruit fly) and nematode (C. Elegans). useful.... identifying the biologically
relevant targets and effectors of a given neuroactive substance. Detailed maps of the genomes of
both these organisms are available, and they both have short generation times and are amenable to
germline transformation, and are easy practically.
Several drugs mechanisms of action has been determined in these models.
;Lithium salts are widely used for treatment of bipolar affective disorder. Effective treatment for acute
mania, and is effective mood stabilizaing agent. Klein and Melton (15) investigated whether lithium
might affect GSK-3 signaling, because effects are mimicked by mutations in GSK-3. They
subsequently demonstrated in vertebrates that vertebrate GSK-3 is directly inhibited by lithium in
XenopusOocytes. Thus showed some effects are phospho-inositide independent, may be the side
effects including potentiation of glycogen release.. (nb/ Lithium is also shown to be non-
competitive inhibitor of inositol-monophosphatase, which converts IMPs to inositol, required for
generation of IP3 a critical signalling molecule (Berridge et al)). Gives good example of the
power of genetic neuropsychopharmacology in simple model systems. Studies
in Dictyostelium were instrumental in identifying the GSK-3 pathway as a
possible mediator of lithium’s deleterious side effects. . Work in Drosophila
has provided an important lead into discovering how lithium’s effects on
phosphoinositide signaling affect neuronal function. (although obvious
limitation is that there is no guarantee it will be conserved across the
evolutiobary gulf between these disparate animals)But at molecular level,
mice and humans have remarkable similarities.

In recent years, the DA hypothesis of schizophrenia has evolved into the narrower hypothesis that the
mesolimbic DA system, as distinct from the nigrostriatal DA system, is most relevant to schizophrenia.
The nigrostriatal DA system is now seen as most relevant to the dyskinetic side effects of antipsychotic
treatments. The mesolimbic system appears to mediate the locomotor activating effects of low doses of
amphetamine, while the nigrostriatal system mediates the stereotypies that predominate at higher doses,

......Inducible knockouts and tissue-specific promoters.

Mice can now be engineered to carry genes driven by tissue-specific promoters to ensure specific
neural expression. Yet this method, for all its advantages, is not ideal. It is constrained by the promoters
available, and it is both slow and resource intensive, requiring a new strain of mice for each new gene.

..... Recently, a new approach using designer zinc-finger

nucleases (ZFNs) has succeeded in introducing targeted
mutations to the zebrafish genome. 20 Woods IG, Schier AF. Targeted mutagenesis in zebrafish.
Nat Biotechnol 2008; 26: 650–651

Furthermore,
the measures used both preclinically and clinically should
have construct validity, defined as measuring accurately the
theoretical behavioral and neurobiological variables that are
considered core to the disorder of interest (eg Cronbach and
Meehl, 1955; Geyer and Markou, 1995). The reliance on
construct validity is particularly relevant in cases in which
no clinically effective medications exist for a particular
indication and insufficient information about etiology exists
to support the demonstration of etiological validity

TREVOR SHARPE NOTES

MODELS
Look up, marmoset, non-human primates, zebrafish, chick (embryos), leech
(nervous system has demonstrated capacity for repair.