Defensive Probe Burying :

Introductions The probe burying test has been used to study both anxiolytic and antidepressant drugs. It is based on the effects that a mild aversive stimulus in a particular context has on behavior. The stimulus is a mild shock given when the animal touches a probe. Typically, after exploration and contact with the probe, animals bury the probe using the bedding available in the experimental apparatus.
Several processes may affect performance in this test: locomotor activity, sensitivity to shock, and memory for the context and the contingencies used. As some of the behavior reflects stress and anxiety responses to anxiolytic and anxiogenic drugs can be studied. It can also been used to study memory as animals bury the probe upon re-exposure even after several days after first exposure.
Method Mice are placed in a chamber with a probe for 10 minutes. The probe gives a .5 mA shock upon contact. Enough bedding is provided to allow for digging and burying behavior. Percent of the probe buried, number of contacts, approaches, latency to bury, directed and undirected burying and other behaviors can be scored.
References:
* Martinez-Mota, L., Estrada-Camarena, E., Lopez-Rubalcava, C., Contreras, C. M., Fernandez-Guasti, A. (2000). Interaction of desipramine with steroid hormones on experimental anxiety. Psychoneuroendocrinology, 25, 109-20.

Elevated Plus Maze:

Introductions   The elevated plus maze is widely used as an anxiety paradigm and represents a test based on unconditioned responses to a potentially dangerous environment(1). The plus (or X-) maze comprises two (opposite) closed and two open arms, each arm located on a central pole. The combination of height, luminosity and open space is assumed to induce fear or anxiety in the mouse. The degree of anxiety is assessed by measuring the time spent on the open and closed arms and the number of entries made into each arm(2). When mice are placed in the center of the maze, they spend most of their time in the closed arms, avoiding the open arms(3). The concomitant behavioral, physiological and endocrinological phenomena occurring in the open arms lend strong support for face and construct validity of this test for anxiety(4). Moreover, the paradigm has relatively good predictive validity, although many false positives are found(5).
Benzodiazepines, barbiturates, and (sometimes) 5-HT1A receptor agonists are found to be anxiolytic, but antidepressants generally do not have anxiolytic activity in the test. The test is also sensitive to anxiogenic drugs, which lends strong support for its predictive validity. A comprehensive analysis of 16 inbred strains has shown that genetic differences have a large influence on plus-maze anxiety measures(6). Age, gender, and a substantial number of procedural variables (height of walls, level of light, pre-handling experience) also contribute to the basal level of anxiety in the plus-maze(6).
This paradigm is very promising, not only to identify drug effects, but also to identify putative effects of mutations. Several transgenic or knockout mutants (e.g. 5-HT1A- knockout, histamine H1-knockout, DA-D3-knockout, CRF-transgenic) have shown anxious-like phenotypes in this test.
Method   The elevated plus maze consists of two closed arms (15 x 6 x 30 cm) and two open arms (1 x 6 x 30 cm) forming a cross, with a quadrangular center (6 x 6cm). The maze is placed 50 cm above the floor and is made of black plastic. A test lasts 5 minutes and starts by placing a mouse on the center facing an open arm. Using a video camera the behavior of the animal is recorded and scored by an observer blind to genotype and/or drug treatment. Open and closed arm entries and time, open arm end exploration, head-dips, scanning, stretched attend postures, center pauses, defecation and urination are scored. In addition abnormal behavior and freezing can be scored.
References:
* Montgomery KC (1955) The relation between fear induced by novelty stimulation and exploratory behaviour. Journal of Comparative and Physiological Psychology 48:254-260.
* Handley SL, Mithani S (1984) Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of 'fear'-motivated behaviour. Naunyn-Schmiedeberg's Archives of Pharmacology 327:1-5.
* Lister RG (1987) The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 92:180-185.
* Pellow S, Chopin P, File SE, Briley M (1985) Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 14: 149-167.
* Rodgers RJ, Cole JC (1994) The elevated plus-maze: pharmacology, methodology and ethology. In: Ethology and Psychopharmacology. SJ Cooper and CA Hendrie (eds) John Wiley & Sons Ltd, pp9-43. 6. Trullas R, Skolnick P (1993) Differences in fear motivated behaviors among inbred mouse strains. Psychopharmacology 111:323-331.
* Trullas R, Skolnick P (1993) Differences in fear motivated behaviors among inbred mouse strains. Psychopharmacology 111:323-331.

Forced Swim Test:

Introductions   When mice are forced to swim in a cylinder from which no escape is possible they readily adopt a characteristic immobile posture and make no further attempts to escape except for small movements needed to keep floating. The immobility is considered by some to reflect a 'depressive mood'(1) in which animals cease to struggle to escape the aversive situation. The immobility induced by the procedure is influenced by a wide variety of antidepressants(2) and has a good predictive validity in that it detects antidepressants with different mechanisms of action (TCAs, SSRIs, MAOIs, and other atypical ones), but its construct validity is weak. There are false positives (psychostimulants, anticholinergics and antihistaminergics) but relatively few false negatives (?adrenergic agonists). The test is sensitive to muscle-relaxant (benzodiazepines) and sedative (neuroleptics) effects, leading to enhanced immobility(2).
Method    Mice are placed singly into a cylinder (46 x 30 cm) containing fresh water at 23 oC for 6 minutes. The activity (or immobility) of the animal is measured by an observer minute by minute.
References:
* Porsolt RD, LePichon M, Jalfre M (1977). Depression: a new animal model sensitive to antidepressant treatment. Nature 266:730-732.
* Porsolt RD, Lenegre A, McArthur RA (1991). Pharmacological models of depression. In: Animal models in Psychopharmacology.B. Olivier, J. Mos, J.L. Slangen (eds) Birkhauser Verlag, Basel, pp. 137-159.

Functional Neurobiology & Physiology (non-behavioral) :

• Radiotelemetry
  
• In vivo microdialysis
  
• In situ hybridization (radioactive, chromogenic and fluorescent)
  
• Neurohistology
  
• Immunohistochemistry
  
• Microdissection
  
• Microinjection technology
  
• Standard histological staining procedure
  
• Golgi staining
  
• Neuroplasticity studies
  
• Genotyping
  
• Microarray analysis
  

Holeboard :

Introductions    The holeboard is a simple test that measures exploration and can be used for the study of spatial learning and memory with an appetitive reward. Lighting conditions can be varied to provide more or less anxiogenic conditions. Drugs that affect memory can be studied in this test that allows training and testing to be separated by a duration of any desired length. As with other test such as the Morris water maze, this test allows for the study of the role of the hippocampus in spatial information processing.
Method    In the exploration version of this test mice are simply allowed to roam freely the squared arena and to poke their noses into the non-baited holes. The computer records each entry time and location.
In the learning and memory version of the test, mice are food deprived to obtain high motivation levels. Training consists of two to three days of visits to the holeboard arena in which 4 of the 16 holes have been baited. Each day there are two training and one testing session. In testing sessions mice explore the arena with no holes baited. Differential exploration of the previously baited holes (i.e. performance above chance levels) reflects memory of the reinforcement and its place.
References:
* Galey, D. & Jaffard, R. (1992). Post-training medial septal stimulation improves spatial information processing in BALB/c mice. Neuroscience Letters, 143, 87-90.
* Micheau, J., Destrades, C. and Jaffard, R. (1985). Physostogmine reverses memory deficits producedd by pretraining electrical stimulation of the dorsal hippocampus in mice. Behavioral Brain Research, 15, 75.

Home Cage Observations:

Introductions    Group housed male mice (5/cage) are observed (videotaped) for two 2-hour periods in one 24 hour period, 2 hours in the light and 2 hours in the dark. In this 24 hour period ongoing individual and social behavior is recorded during an active (dark phase) and an inactive (light phase) period of the normal circadian cycle. All behaviors are scored from the videotapes using event recorders. Information is thus gathered about activity, sleeping, social interactions, self-grooming, and feeding. Spontaneous abnormal behavior or exaggeration of normal behavior (stereotypy) can be assessed. This test provides quick information on possible abnormalities in certain (mutant) phenotypes, which will be of help in determining and designing appropriate consecutive tests. This test can also be used to determine drug effects, although this will less typically be the case. The test preferably is performed on homogeneous groups of mice (all same genotype) although heterogeneous groups may also be assessed by marking individual mice.
Method    Group housed mice (2 to 5 per cage) are held in the colony room for at least a week on a standard 12h light-12h dark light schedule. During a subsequent 24 hour period, two 2-hour periods, one in the light phase and one in the dark phase, are videotaped for home cage behavioral observations. The behavior is scored by a trained observer using an event recorder and noting the occurrence of many behaviors, like grooming, social grooming, huddling, sleeping, eating, digging, scratching, hanging from the wire top, stereotyped movements, gnawing, social activities (aggression, homosexual activities) and any other behavior or activity of importance.

In-Vivo Microdialysis:

Introductions   In-vivo microdialysis provides a means whereby the extracellular concentration of neurotransmitters can be measured and the effects of pharmacological, behavioral, or physiological challenges can be determined in wild type (WT) and transgenic animals. Thus, the basal extracellular levels of a neurotransmitter in response to a pharmacological challenge or resulting from gene overexpression or knockout (KO) can be assessed.
PsychoGenics has established the microdialysis technique for use in awake, freely moving mice and rats. This technique is optimally suited to get insight into the effects of certain drugs on neurotransmission, or the effects of a mutation on brain functioning, specifically if small numbers of animals are available and time-course of a drug effect are of interest. The example shown in the figure demonstrates the effects of a selective serotonin reuptake inhibitor (SSRI) antidepressant (paroxetine) on the extracellular serotonin levels in male wildtype and serotonin-1B receptor knockout mice. It can be clearly seen that the reuptake inhibitor paroxetine induces a higher extracellular concentration of serotonin in the hippocampus of 5-HT1B-KO mice versus WT mice.
Method    Mice (or rats) are equipped with a microdialysis probe in a certain area of the brain (e.g. hippocampus or striatum) via precise placement using stereotaxic surgery. After recovery from the operation, the animals can be used for two to three sequential days to measure the extracellular levels of certain chemicals, including neuro-transmitters and their metabolites. The sampling is performed in freely moving animals and pharmacological, behavioral or physiological challenges or tests can be combined with direct measurement of the chemical of interest (dopamine, serotonin, etc.).
References:
* Chen, L., He, M., Sibille, E., Thompson, A., Sarnyai, Z., et al. (1999). Adaptive changes in postsynaptic dopamine receptors despite unaltered dynamics in mice lacking monoamine oxidase B. J Neurochem, 73, 647-55.
* Gainetdinov, R.R., Jones, S.R., Caron, M.G. (1999). Functional hyperdopa-minergia in dopamine transporter knock-out mice. Biol Psychiatry, 46, 303-11.

Locomotor Activity (Open Field):

Introductions    The Locomotor Activity (Open Field) test is used to study anxiety-like and exploratory responses in animals. Mice are placed in a novel and relatively well lit arena. Because rodents are neophobic and find bright light aversive, the animal avoids brightly lit open spaces, preferring to stay close to the walls (thigmotaxis). The test measures a range of behaviors induced by anxiety and exploratory processes and thus it is very suitable to detect a broad variety of (subtle) effects, either induced by drugs or by a mutation that affects CNS function.
Drug or genetic effects are assessed on exploratory or locomotor behavior by measuring distance covered and autonomic activity such as urination and fecal bolus counts. Benzodiazepine anxiolytics reduce the behavioral and autonomic parameters in this test(1). However, the procedure yields false-positives with psychostimulants and anticholinergics(2), and it does not reliably detect 5-HT1A receptor agonists and SSRIs(3). The behavior evoked in the open field is remarkably sensitive to a variety of internal and external factors and can yield information about several central processes involved in anxiety, depression, mania, schizophrenia, ADHD, and addiction.
Method    A mouse is placed for a 40 minute session in a simple squared test box (27 x 27 x 20 cm) without a cover or with a transparent lid. By using an infrared beam array system locomotion, rearing and time spent in certain predefined areas of the Open Field are measured.
References:
* Anisman H, Kokkinides L, Glazier S, Remington G (1976) Differentiation of response biases elicited by scopolamine and d-amphetamine: effects on habituation. Behavioral Biology 18:401-417.
* Simon P, Soubrie P (1979) Behavioral studies to differentiate anxiolytic and sedative activity of the tranquillizing drugs. In: Modern problems of pharmacopsychiatry, Vol. 14: Differential Psychopharmacology of Anxiolytics and Sedatives, J.R. Boissier (ed), pp. 99-143; Basel, S. Karger.
* Olivier B, Miczek KA (1998) Fear and anxiety: mechanisms, models and molecules. In: N Dodman, I Shuster (eds) Psychopharmacology of animal behavior disorders. Blackwell Sciences, pp 105-121.

Metabolism/Micturition :

Introductions    In the development of a behavioral profile, it can be important to assess the general metabolic activity of the animal as it may reflect behavioral effects produced by a drug or genetic manipulation. For example, a drug that produces an increase in locomotor activity (hyperactivity) would be expected to produce an increase in metabolic activity and a concomitant increase in food and water consumption. Another example would be the alteration in circadian rhythms that could result from changes in mood regulation such as those seen in models of depression. Other examples would be changes in metabolic activity and consummatory behavior resulting from eating disorders/obesity, neuro-muscular disorders, and sleep disorders.
PsychoGenics has developed a paradigm that assesses several activity and metabolic parameters simultaneously through the use of a metabolic chamber equipped with activity monitors, sensors that measure food and water consumption, and a system to measure the time of occurrence and the amount of individual micturition events (Wood et al. 2001). PsychoGenics has used the system to asses strain differences in metabolic activity (see Moody et al. 1997).
Method
The metabolic chamber consists of a circular plastic enclosure with a metal grid floor and cutouts allowing the mouse access to food and water containers. The metal grid floor allows both urine and feces to pass through. The urine ultimately falls onto paper mounted on the pan of a balance that measures the weight of urine in each micturition event. Both the food and water containers are attached to special balances that continuously monitor the weight of each container. Additionally, activity within the chamber is monitored by two infrared beam arrays that record movements in both the horizontal (locomotor activity) and vertical planes (rearing).
The mice are housed in the metabolic chambers and therefore can be monitored for a number of days. The mice are placed into the chamber for a period of time to acclimate them to the enclosure and to the change in flooring and food and water containers. No measurements are taken during this period. On succeeding days, activity, consummatory behavior and micturition can be recorded .
References:
* Moody, D. E., Pomp, D., Nielsen, M. K. (1997). Variability in metabolic rate, feed intake and fatness among selection and inbred lines of mice. Genet Res, 70, 225-35.
* Wood, R., Eichel, L., Messing, E.M. & Schwarz, E. (2001). Automated noninvasive measurement of cyclophosphamide-induced changes in murine voiding frequency and volume. J Urology, 165, 653-59.

Morris Water Maze :

Introductions    The Morris Water Maze is the favored test for the study of hippocampal-dependent learning and memory. It consists of a water pool with a hidden escape platform where the subject must learn the location of the platform using either contextual or local cues.
Performance in the Morris Water Maze depends on several mechanisms, from attention, learning and memory, to vision and motor coordination. The cognitive processes that underlie performance in this test are dependent on many biochemical pathways, the most notable and well studied, the cholinergic system.
It has been consistently shown that lesions of the hippocampus or its cholinergic input impair performance in this test. As an example we show the detrimental effect that scopolamine, a muscarinic antagonist, has on performance (latency to reach the platform location) when injected before each training session over four days in the hidden platform version. Whereas physostigmine, a cholinesterase inhibitor, did not block the scopolamine-induced performance deficit, it partially rescued performance on a testing day free of drugs, showing that scopolamine has dual effects (on performance and on memory processes).
Method    There are several versions of the protocol. One popular protocol consists of training sessions with 3 blocks of 4 trials (although once single session with four trials leads to apparent learning as shown by the graph), with a 2 min interval between trials and a 1 h interval between blocks. During training a mouse is placed in one quadrant of the pool and allowed to swim until the platform is found. Mice are left on the platform for few seconds. If the platform is not found within a minute, mice are placed on the platform for a few seconds, and the trial is terminated. The starting point is randomly rotated for every mouse every trial. In the hidden platform version the platform is located in a fixed position with respect to the surrounding area. In the visible platform version, the platform position is rotated with respect to the surrounding area, but its position is visible above the water surface. Sessions must be repeated over several days before good memory and recall becomes apparent. Test sessions consist of placing the mice in the pool without the platform and recording the swimming path during the session. Behavior is tracked by a video camera and analyzed by video track software.
References:
* Morris, R. G. M., Garrud, P., Rawlins, J. N. P., O'Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature, 297, 681-683.

Neurological Evaluation (Irwin test, rotarod, visual cliff, and grip strength):

Introductions    A systematic series of observations is made, from which a broad range of information about the functional status of a mouse can be obtained. The procedure assesses behavioral, neurological, and autonomic functions of a mouse. The advantage of this approach is that multiple measurements are gathered from the same animal. These types of procedures have been applied in pre-clinical drug evaluation(1) and have been used in slightly modified forms more recently to evaluate the putative abnormalities of newly created transgenic and knockout mice(2,3).
This battery of tests sequentially measures various capabilities of mice including sensory (visual, auditory and touch), motor (gait, balance, ataxia), and autonomic (salivation, defecation, urination) responses. The evaluation gives extensive information about basal functions of the (mutant or drug-treated) mouse under study or the interaction of a drug with a certain genotype (either strain or mutant). Information from this series of tests may point to otherwise undetected effects, may suggest further testing in other behavioral paradigms, or may help in interpreting results obtained in the more complex follow-up tests evaluating higher brain functions(2,4).
Test Neurological Function

• VISUAL CLIFF/PLACING - vision and depth perception
• GRIP STRENGTH - muscle strength
• ROTAROD - motor coordination
• EYE OBSERVATION - exophthalmia, enophthalmia, lacrimation
• STARTLE - hearing, stimulus reactivity
• ARENA LOCOMOTION - walking, equilibrium and gait
• HANDLING - muscle tone, reflexes, somatosensoric and motoric integration
• GEOTAXIS - motor coordination, integrative response to tilting
• TAIL, TOE, EYE and PINNA RESPONSE - reflexive response to pain, pressure or touch stimuli
• GENERAL - abnormal reaction, behavior, posture, or seizures
References:
* Irwin S (1968) Comprehensive observational assessment:1a. A systematic, quantitative procedure for assessing the behavioral and physiological state of the mouse. Psychopharmacologia (Berl.) 13:222-257.
* Crawley J, Paylor R (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Hormones and Behavior 31:197-211.
* Rogers DC, Fisher EMC, Brown SDM, Peters J, Hunter AJ, Martin JE (1997) Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mammalian Genome 8:711-713.
* Picciotto MR, Wickman K (1998) Using knockout and transgenic mice to study neurophysiology and behavior. Physiological Reviews 78:1131-1163.

Novel Object Recognition :

Introductions    The novel object recognition task is based on the natural tendency of mice to investigate a novel object rather than a familiar object when both are simultaneously present in an open field where the animals have been habituated. On the first test day mice, habituated to the open field, are exposed to two novel objects, which differ from each other in shape, texture and color. After a pre-determined time delay (minutes to hours) mice are re-tested in the same open field, but now the animals are exposed to one familiar and one novel object. Mice explore the novel object more than the familiar object, reflecting the involvement of learning and (recognition) memory processes. Scopolamine blocks learning about the objects as measured by a reduction in the amount of time spent exploring the novel object2 during testing.
Method    The object recognition task is performed in the open field apparatus (see Locomotor Activity test). After the 40-minute exploration test, two novel objects are placed in the open field for 10 minutes and the amount of exploration is measured using the automated video track system. Twenty-four hours later (or less) the mouse is replaced into the same open field, but it is now exposed to one familiar object and one completely novel object. Over a ten-minute period the amount of time spent exploring the familiar and the novel objects is measured.
References:
* Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of memory in rats. Behav. Brain Res. 31:47-59. 2. Dodart JC, Mathis C, Ungerer A (1997) Scopolamine-induced deficits in a two-trial object recognition task in mice. Neuroreport 8:1173-1178.

Time Perception (Peak Procedure) :

Introductions    The Peak procedure is a psychophysical task that requires the reproduction of temporal information. Several processes underlie steady state performance in a temporal task: attention, motivation, short and long term memory, motor coordination and instrumental learning. The psychophysics of temporal information processing has been studied in both humans and animals, such as pigeons, monkeys, rats, mice and starlings (Brunner, Gibbon et al. 1994) and is affected in schizophrenia, depression and other psychiatric and neurodegenerative disorders.
Timing is very sensitive to dopaminergic effects. In Parkinson's patients, for example, dopaminergic dysfunction in the striatum results in a temporal information processing deficit, which disappears when patients are on L-dopa, and reappears when patients go off the drug (Malapani, Rakitin et al. 1998).
Evidence that the cholinergic system is involved in temporal information processing comes from studies showing that cholinergic supplementation in the neonates improves and choline deficit impairs temporal processing (Meck and Williams 1997). The cholinergic input to the hippocampus is probably involved in the consolidation of temporal information (Meck 1988).
Method    Isolated mice are maintained at 85% of their free-feeding body weight and trained to expect reward at a fixed delay. Mice are trained to collect the reinforcement (a liquid or solid food reinforcer) with one leverpress or nosepoke response. The timing precision can be assessed by withdrawing reinforcement in some trials, and measuring the point at which response rate is maximal.
References:
* Brunner, D., J. Gibbon, et al. (1994). "Choice between fixed and variable delays with different reward amounts." J Exp Psychol Anim Behav Process 20(4): 331-46.
* Malapani, C., B. Rakitin, et al. (1998). "Coupled temporal memories in Parkinson's disease. A dopamine-related dysfunction." J Cogn Neurosci 10(4): 316-31.
* Meck, W. H. (1988). "Hippocampal function is required for feedback control of an internal clock's criterion." Behav Neurosci 102(1): 54-60.
* Meck, W. H. and C. L. Williams (1997). "Simultaneous temporal processing is sensitive to prenatal choline availability in mature and aged rats." Neuroreport 8(14): 3045-51.
* Hinton, S. H., & Meck, W. H. (1996). Increasing the speed of an internal clock: The effects of nicotine on interval timing. Drug Development Research, 38, 204-211.

Pentylenetetrazol-Induced Seizures :

Introductions    The excitability level of the CNS is under the influence of many different neural mechanisms, including glutamatergic and GABA-ergic neurotransmission. Many anti-epileptic drugs accordingly influence the brain via these mechanisms(1). One means to measure the excitability of the CNS is to challenge it chemically, e.g. with pentylenetetrazol (PTZ), which acts as a convulsant via the GABAA benzodiazepine-receptor complex. By determining the minimal dose to induce convulsions in mice, a good estimate of the general excitability of the CNS can be obtained. A large variation in this parameter has been found among several strains and mutants(2,3,4,5).
Method   Group housed mice are brought into the experimental room and allowed to acclimate for a minimum of one hour prior to testing. Each animal receives a bolus IP injection of PTZ at different doses (20, 40, 60, or 80 mg/kg; 5-10 ml/kg dosing volume). Animals are then placed singly into a new cage for observation of seizure profile and recorded seizure latency times. Seizures are scored over a 30 minute observation period, according to the latency of the first twitch, latency of the first clonic/tonic convulsion (>5 second duration with loss of the righting reflex), latency time to full tonic convulsion, and latency time to death6. A seizure profile is described for three consecutive 10 minute observation periods post PTZ injection. Animals are observed for another 30 minutes post seizure profile (1 hour post PTZ dosing) for event of death. Anti-convulsant effects of drugs are tested against a fixed dose of PTZ (40 to 60 mg/kg) that induces seizures in all mice. A dose-response study determines the anticonvulsive properties (ED50 or lowest effective dose). Mice are euthanized after completion of the test.
References:
* Macdonald RL, Greenfield LJ (1997) Mechanisms of action of new antiepileptic drugs. Current Opinion in Neurology 10:121-128.
* Kosobud AE, Crabbe JC (1990) Genetic correlations among inbred strain sensitivities to convulsions induced by 9 convulsant drugs. Brain Research 526:8-16.
* Kosobud AE, Cross SJ, Crabbe JC (1992) Neural sensitivity to pentylenetetrazol convulsions in inbred and selectively bred mice. Brain Research 592:122-128.
* McIntyre TD, Alpern HP (1989) Patterns of convulsive susceptibility in the long-sleep and short-sleep selected mouse lines. Brain Research Bulletin 22:859-865.
* Sugaya E, Ishige A, Sekiguchi K, Iizuka S, Ito K, Sugimoto A, Aburada M, Hosoya E (1986) Pentylenetetrazol-induced convulsion and effect of anticonvulsants in mutant inbred EI mice. Epilepsia 27:354-358.
* Corda MC, Orlandi M, Lecca D, Giorgi O, (1992) Decrease in GABAergic function induced by pentylentetrazol kindling in rats. Antagonism by MK-801. Journal of Pharmacology and Experimental Therapeutics 262:797-800.

Phencyclidine (PCP) Model for Schizophrenia :

Introductions
PCP is a powerful psychotogen that in healthy humans elicits a wide range of symptoms that resemble both the positive and the negative symptomatology observed in schizophrenia, and acutely exacerbates existing behavioral and cognitive impairments in schizophrenia patients. Neurochemically, PCP alters excitatory neurotransmission in the brain that has been implicated in the pathogenesis of schizophrenia. In rodents, subchronic administration of PCP leads to hyperactivity, stereotyped behavior, disruption of social behavior, and pre-pulse inhibition of the startle response. This behavioral phenotype closely resembles some of the positive and negative symptoms observed in schizophrenia. Furthermore, PCP administration in primates results in a disruption of working memory, a key neuropsychological finding in schizophrenia. Clinically effective antipsychotic drugs can alleviate some of the schizophrenia-like behavioral pathology induced by PCP. The involvement of excitatory neurotransmission in schizophrenia, the resemblance of the PCP-induced behavioral pathology to the human disease and its reversibility by antipsychotics lend construct, face and predicitive validity to this model.
Method    Effects of different doses of PCP on pre-pulse inhibition of startle in C57 male mice. Increasing doses of PCP lead to disrupted inhibition compared to vehicle treatment (0 mg/kg).
References:
* Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR. (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacol., 156:117-154.

Prepulse Inhibition (PPI) :

Introductions    The acoustic startle reflex is a very basic response to strong exteroceptive stimuli. It is widely used to assess sensorimotor reactivity in animals and humans(1) yields important information about attentional and informational processes in the brain. Typically, whole-body startle in reaction to a loud acoustic stimulus is measured by placing a mouse into an animal holder that is placed onto a transducer platform in an acoustic chamber and measuring the amount of the animal's 'reaction' to the stimulus. The maximum startle response force and timing for each stimulus is measured. A weak sound preceding the loud acoustic stimulus inhibits the startle reflex; this is called pre-pulse inhibition(2). Pre-pulse inhibition (PPI) is impaired in schizophrenic patients, and is considered a test with good predictive, face and construct validity for schizophrenia(3).
Several psychotomimetic drugs (e.g. DA receptor agonists, PCP, ketamine) disrupt PPI(4) and simulate a psychotic-like state in animals, which can be antagonized by anti-psychotics(5). All strains of mice display startle responses and pre-pulse inhibition, but there are large strain differences(6). The acoustic startle reflex and PPI are sensitive to a broad variety of drugs, indicating that the parameters measured are very useful to discover subtle effects, either of drugs or of some mutation in the CNS. Effects of such manipulations on the basal startle reaction may point to interference with basic sensory and motor processes in the brain, which may influence other (higher) neural and mental processes. Effects on PPI may be indicative of specific effects on sensorimotor gating and point to putative processes involved in psychosis.
Method    ice are individually placed in the startle chamber with a background white noise of 70 dB, and left undisturbed for 10 minutes. Then there is a 15 minute session consisting of 56 trials. The startle stimulus is a 40 ms-120dB white noise sound, pre-pulses are 20 ms white noise sound of 72, 74 and 78 dB preceding the startle by 100 ms. Eight types of trials are given: prepulse plus startle (7 trials per prepulse intensity), prepulse alone (7 trials per prepulse intensity), startle alone (7 trials), and no stimulation (7 trials). The variable intertrial interval averages 15 seconds (between 10 and 20 seconds). In the no-stimulation trials, baseline measurements are taken. In the startle alone trials, the basic auditory startle is measured and in the prepulse plus startle trials, the amount of inhibition of normal Prepulse Inhibition (PPI)startle is measured and expressed as percentage of the basic startle. In the prepulse alone trials, the normal response to a weak noise is measured as a control.
During drug tests, various agents that disrupt PPI can be used (apomorphine, d- amphetamine, PCP, ketamine, DOI) and the effects of putative anti-psychotics can be assessed on the disrupted PPI. However, the effects of putative antipsychotics can also be measured directly, without disrupting the PPI by administration of psychotomimetics. Alternatively, mutant mice with or without drugs can be screened using the startle and PPI procedure.
References:
* Davis M (1980) Neurochemical modulation of sensory-motor reactivity: acoustic and tactile startle reflexes. Neuroscience and Biobehavioral Reviews 4:241-263.
* Geyer MA, Braff DL (1982) Habituation of the blink reflex in normals and schizophrenic patients Psychophysiology 19:1-6.
* Braff DL, Geyer MA (1990) Sensorimotor gating and schizophrenia. Human and animal studies. Archives of General Psychiatry 47: 181-188.
* Dulawa SC, Geyer MA (1996) Psychopharmacology of prepulse inhibition in mice. Chinese Journal of Physiology 39:139-146.
* Swerdlow NR, Geyer MA (1998) Using an animal model of deficient sensorimotor gating to study the pathophysiology and new treatments of schizophrenia. Schizophrenia Bulletin 24: 285-301
* Paylor R, Crawley JN (1997) Inbred strain differences in prepulse inhibition of the mouse startle response. Psychopharmacology 132:169-180.

Radiotelemetry:

Introductions    Radiotelemetry is a powerful technology for long-term monitoring of physiological functions, including heart rate, blood pressure, core body temperature, activity, electrocardiograms (ECG) and electroencephalograms (EEG) in a stress-free setting of the home cage. Mice are equipped with small, rechargeable telemetric implants in the abdominal cavity that send signals to a receiver placed underneath the home cage. The signals then are analyzed by a computer program to provide continuous recording of several different parameters for an extended period of time (up to one year). Mice equipped with a telemetric device can be used for practically all experimental conditions and tests, including mutant phenotyping, pharmacology, physiology, behavior and toxicology.
Stress and drug effects on heart rate and body temperature in mutant mice.
References:
* Bouwknecht JA, Hijzen TH, van der Gugten J, Maes RA, Olivier B. (2000) Stress-induced hyperthermia in mice: effects of flesinoxan on heart rate and body temperature. Eur J Pharmacol 400:59-66

Serotonin receptor 1A (5-HT1A) knockout mouse model for affective disorders :

Introductions    5-HT1A receptor deficiency has been implicated in mood disorders, such as depression and post-traumatic stress disorder (PTSD) and panic disorder (Mann, 1999). Decreased 5-HT1A binding was found in the brain of depressed suicide victims, and recent brain imaging studies have revealed decreased 5-HT1A receptor densities in the medial temporal lobe and other limbic brain regions of patients with major depression. Also, chronic stress, which is well known to be a major factor in the development of mood disorders, has been shown to lead to a specific down-regulation of 5-HT1A receptors in the hippocampus of experimental animals. These results strongly suggest that down-regulation of 5-HT1A receptors, caused by either genetic or stress-related processes, may significantly contribute to the development of mood disorders in humans. 5-HT1A receptor knockout (KO) mice were used to demonstrate a link between the receptor and anxiety. The recent production of three independent groups of 5-HT1A receptor KO in three different genetic backgrounds (C57Bl/6J, 129/Sv, Swiss-Webster) led to the intriguing finding that all mice, independent from the genetic background strain, showed an 'anxious' phenotype compared to corresponding wild type mice. This is one of the most consistent behavioral findings in the mutant mouse literature (Ref 1, 2). It was also shown that the "anxiety" of 5-HT1A KO mice is benzodiazepine resistant in the Swiss-Webster strain, a characteristic described in certain forms of human anxiety, and that the phenotype could be related to molecular changes in GABAA receptor subunit expression.
References:
* Parks CL, Robinson PS, Sibille E, Shenk T, Toth M. (1998) Increased anxiety of mice lacking the serotonin1A receptor. Proc Natl Acad Sci U S A 95:10734-10749.
* Ramboz S, Oosting R, Amara DA, Kung HF, Blier P, Mendelsohn M, Mann JJ, Brunner D, Hen R. (1998) Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci U S A 95:14476-14481.

Spontaneous Alternation :

Introductions    Mice have an innate tendency to explore their environment in a systematic way. Successful exploration depends on the ability to avoid places recently visited, where food may have been depleted or is absent. Spontaneous alternation is an ethologically based test that does not involved reward delivery. Mice with compromised "working memory" cannot retain information regarding places just visited in memory; therefore they show decreased spontaneous alternation. At PsychoGenics, the Y-maze spontaneous alternation paradigm has been used to assess working memory. This paradigm measures the pattern of entrances into each of the three arms of the Y-maze. If the mouse visits each of the three arms during any three-arm visitation sequence, then it is considered to be displaying alternation.
A mouse with impaired working memory will not remember which arm it just visited and it will therefore be equally likely to visit each of the Y-maze arms, although the possibility of the use of a rule-of-thumb (i.e "exit-arm-and-turn-left") remains, and must be considering in the interpretation of the results. Impaired performance results in repeated entries into arms just visited. Thus, over a three-arm visitation sequence, one arm will be visited at least twice and all three arms will not be visited.
Method    The mice are placed in the Y-maze, with all three arms available for exploration, and their behavior is videotaped for an 8-minute period. The videotape is then scored for the number and sequence of visits to each arm. Global activity is reflected in the total number of visits to all three arms. A triple-alternation is defined as a visit to each of the three arms sequentially (e.g., ABC, ACB, BAC, BCA, CAB, or CBA). Percent alternation is calculated by dividing the number of alternations by the number of possible alternations multiplied by 100 (see figure above). Chance performance is at the 22.2% alternation level in this paradigm.
References: * Holcomb, L.A., Gordon, M.N., Jantzen, P., Hsiao, K., Duff, K., Morgan, D. (1999). Behavioral changes in transgenic mice expressing both amyloid precursor protein and presenilin-1 mutations: lack of association with amyloid deposits. Behav. Genet., 29, 177-85

Stress-Induced Hyperthermia:

Introductions    Mice, individually housed overnight, are subjected to two sequential rectal temperature measurements 10 minutes apart. The first measurement is the basal temperature (T1), the second one the stress-enhanced temperature (T2). The difference (delta T) is the stress-induced hyperthermia(1).
Anxiolytics such as benzodiazepines and 5-HT1A receptor agonists reduce delta T(2), whereas antidepressants do not (3)reduce delta T(2). Besides the effect of drugs on the stress-enhanced temperature (T2), this test also directly measures intrinsic effects of drugs on the core body temperature (T1).
Body temperature and emotional states are also closely related in humans(4,5), with changes in autonomic functioning considered when diagnosing generalized anxiety disorder. Stress-induced hyperthermia in mice is considered to have good face, predictive, and construct validity for certain anxiety/stress disorders in humans(3), with relatively few false positives/false negatives.
Method    Group-housed mice are isolated in an experimental room approximately one hour before lights off on the day before the test. On the day of testing, animals are taken quietly from the cage, held in a supine position, the rectal temperature is measured and the animal is placed back into the cage. The same procedure is repeated 10 minutes later. The first temperature (T1), the second temperature (T2) and the difference (delta T) are recorded. Drugs are administered 60 minutes before T1, regardless of the route of administration or drug, in order to prevent the stress of being injected from affecting the temperature measurements.
References:
* Van der Heyden JAM, Zethof TJJ, Tolboom JTBM, Olivier B(1997) Stress-induced hyperthermia in singly housed mice. Physiology and Behavior 463-470.
* Olivier B, Zethof TJJ, Ronken E, Van der Heyden JAM (1998) Anxiolytic effects of flesinoxan in the stress-induced hyperthermia paradigm in singly-housed mice are 5-HT1A receptor mediated. European Journal of Pharmacology 342:177-182.
* Zethof TJJ, Van der Heyden JAM, Tolboom J, Olivier B (1995) Stress induced hyperthermia as a putative anxiety model. European Journal of Pharmacology 294:125-135.
* Marazziti D, Di Muro A, Castrogiovanni P (1992) Psychological stress and body temperature changes in humans. Physiology and Behavior 52:393-395.
* Reeves DL, Levinson DM, Justesen DR, Lubin B (1985) Endogeneous hyperthermia in normal human subjects: Experimental study of emotional states(II).International Journal of Psychosomatics 32:18-23

Tail Suspension :

Introductions    Mice, suspended by the tail, rapidly adopt an immobile posture(1,2). It has been suggested that such immobility reflects a state of lowered 'mood' (depression) in which animals have given up hope of escaping. Porsolt(3) describes these kinds of behaviors as 'behavioral despair'. Tail-suspension immobility is reduced by a large variety of clinically active typical and atypical antidepressants(4). The test has a good predictive validity for antidepressant activity and works for most antidepressant classes including tricyclics, SSRIs, 5-HT1A receptor agonists, and MAOIs (specially in rats), but has some false positives (psychostimulants, anticholinergics). The test is sensitive to muscle-relaxant (benzodiazepines) and sedative (neuroleptics) effects, which lead to enhanced immobility(4). Strain differences in the tail suspension test have been found in mice(5). The tail suspension test has some face validity but its construct validity is rather weak.
Method    Mice are suspended by tape attached to the tail for 10 minutes on an electronic transducer device that transmits the movements of the mouse to a computer. The force of the movement, and therefore the absence of movement, is continuously recorded.
References:
* Porsolt RD,Chermat R, Simon P. Steru L (1986) The tail suspension test: computerized device for evaluating psychotropic activity profiles. Psychopharmacology 89:S28.
* Steru L. Chermat R, Thierry B, Simon P (1985) The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology 85:367-370.
* Porsolt RD, LePichon M, Jalfre M (1977) Depression: a new animal model sensitive to antidepressant treatment. Nature 266:730-732.
* Porsolt RD, Lenegre A, McArthur RA (1991) Pharmacological models of depression. In: Animal models in Psychopharmacology.
* B. Olivier, J. Mos. J.L. Slangen (eds) Birkhauser Verlag, Basel, pp.137-159.
* Trullas R, Skolnick P (1993) Differences in fear motivated behaviors among inbred mouse strains. Psychopharmacology 111:323-331.

Thermal Pain (plantar):

Introductions    Reaction to acute pain is a normal defensive function of an organism and is not associated with any pathological condition but signifies the integrity of the organism. Measurement of pain in animals is largely indirect and based on a nociceptive reflex(1). This reflex can be measured as the hind paw withdrawal or the tail flick reflex upon exposure to heat or pressure. The plantar analgesia test(2,3) uses infrared light as thermal stimulus to induce hind paw retraction. The method detects thermal hypo- and hyperalgesia and is able to characterize analgesic (e.g. opiates) and anesthetic drugs. In addition, changes in thresholds for nociceptive stimuli in mutant mice can be detected. This test is not sensitive for many common analgesics, e.g. non-steroidal anti-inflammatory drugs. Modulation of pain is essential for the homeostasis of an organism, and many neural systems in the brain and spinal cord are involved (e.g. opiates, neuropeptide Y, serotonin, NMDA, GABA, etc.). Subtle changes in any of these systems (either by drugs or by gene manipulations) may lead to changes in the sensitivity or threshold for nociceptive stimuli.
Method    Group-housed mice are taken in the home cage from the colony room to the dimly lit experimental room with white noise and placed into the plantar analgesia apparatus in groups of six. After a 30 minute acclimatization to the apparatus, the experimenter moves a fiber-optic heat source under an animal's hind paw and switches on the infrared beam. When the mouse moves its hind paw away from the heat source, the apparatus automatically records the movement and the latency time with a cutoff time of 10 seconds. Paw shakes and paw licks are noted by the experimenter. Each animal is tested 5 times with an average time interval of one minute, and the two extreme scores are discarded. Morphine and naloxone are used as standards to verify the opiate-sensitivity of the mice.
References:
* Walker W, Fox AJ, Urban LA (1999) Animal models for pain research. Molecular Medicine Today 5:319-321.
* Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988) A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32:77-88.
* Mansikka H, Shiotani M, Winchurch R, Raja SN (1999) Neurokinin-1 receptors are involved in behavioral responses to high-intensity heat stimuli and capsaicin-induced hyperalgesia in mice. Anesthesiology 90:1643.

Y Maze:

Introductions    The Y-maze is a two-trial recognition memory test in which performance does not depend on learning a new behavior or rule but relies on an innate tendency of a mouse to explore a novel environment. The Y-maze has three identical arms, which are symmetric to each other. Around the Y-maze visual cues enable the animal to locate itself spatially. In trial 1, one arm is blocked and one of the other arms functions as the 'start' arm. A mouse is left for 15 min in the apparatus to explore the maze (acquisition trial). After an inter trial interval (ITI) of 6 hours (or less), the mouse is placed in the start arm and the previously blocked arm is opened allowing the mouse to explore all arms for a 5 minute (recall trial). Because mice prefer novel environments, they spend most of the time in the previously 'blocked' arm. Several aspects of behavior can be measured in this test, including exploration, response to novelty, and spatial recognition memory. Additionally, as the test is run with dim illumination, there is little influence from other factors that may effect performance, like motivational or emotional states.(1,2)
Method    The Y-maze has three identical arms, 30 cm long, 9 cm wide and 10 cm high, and is made of transparent Plexiglas with a black low rim. Visual cues are placed around the maze. The test consists of two trials. During trial 1, one of the arms ('novel' arm) is blocked with black Plexiglas and referred to as the 'novel' arm. A mouse is placed in one of the two unblocked arms ('start' arm), and is allowed to explore the remaining arm ('familiar' arm). Familiar, novel and start arms are randomized between subjects to minimize spatial bias. At the start of testing the mouse is placed in the start arm and allowed to explore both arms for 15 minutes (acquisition trial). Entries are automatically recorded using infrared beams. At the end of trial 1 the animal is returned to its home cage. After an inter-trial interval of 4-6 hours, the mouse is placed in the same start arm as in trial 1, but with both the novel and the familiar arms available for exploration during a 5 minute recall trial. The first arm entered, the amount of time spent in each arm, and the number of entries made into each arm is recorded. The first choice and the time and entries into the novel arm reflect recognition of the novel arm (arm discrimination memory). The total number of entries also reflects locomotor and general exploration levels.
References:
* Dellu F, Mayo W, Cherkaoui J, LeMoal M, Simon H (1992) A two-trial memory task with automated recording: study in young and aged rats. Brain Research 588:132-139.
* Conrad CD, Lupien SJ, Thanasoulis LC, McEwen BS (1997) The effects of Type I and Type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze. Brain Research 759:76-83.