The Wellcome Trust for providing Open Access funding to Oxford University. Robert Deacon is a member of Oxford OXION group, funded by Wellcome Trust grant WT084655MA.

1. Introduction:
Mice and rats will initially consume only small amounts of a novel food. This is thought to be because they are liable to poisoning due to their inability to vomit. 
The only exception to this is if they have previously encountered another animal which has eaten that food; if 
they smell it on the exhaled air from their peers, they will classify it as safe13. This is because the food odour is paired 
with carbon disulphide on the other rat&#x2019;s breath12. Indeed, carbon disulphide itself increases food consumption in both 
mice and rats17. The phenomenon is called "social transmission of food preferences" and can be used as a simple olfactory 
learning paradigm22. This has been reported to be sensitive to lesions of the hippocampus3, but other studies5 
failed to replicate this finding. It has been suggested that the neocortical cholinergic system, particularly the orbitofrontal 
component19 may be more important than the hippocampal cholinergic system for social transmission. Social transmission has even 
been shown to track memory impairment in aged rats, probably mediated by decreased CREB (cAMP-response element-binding protein) transmission in the aged hippocampus7.
The ease with which rodents acquire a food preference by social transmission has strong implications for hyponeophagia testing. When the author was performing a hyponeophagia test with hippocampal lesioned and control rats, he accidentally put a tested hippocampal rat (which had eaten the sweet corn) into the cage of the next control rat due for testing8. It ate within 14 s, the only control not to have a latency of 300 s. Great care should therefore be taken to prevent such social transmission of food odours during hyponeophagia testing.
If the first hyponeophagia test does not result in a clear, unambiguous result, multiple further tests can be performed. Figures 1-4 illustrate examples of different apparatus. The number of test situations that can be devised is only limited by the imagination of the experimenter. Results are analysed by two-way repeated measures ANOVA. Since hyponophagia data is generally very variable, not conforming to a Gaussian distribution and often skewed, with some animals giving very long latencies, it should first be transformed; a square root or log transformation is particularly useful for truncating the extreme longer latencies. 
2. Apparatus:
Figures 1-4 are presented as examples of hyponeophagia test set-ups. The specific combinations of novel food and environment are not critical, though the food well shown in Figure 1 is obviously best suited to containing liquids, although a niblet of sweet corn could also be placed in it. 
In general, it is a good idea to have most of the floor of the apparatus covered with the food. Not only do animals 
appear to find this unusual surface aversive, this technique also eliminates confounds due to exploration before finding the food. Thus a 
sedative drug might appear anxiogenic because it increased the latency to find and eat the food because it depressed locomotion, not 
because it was anxiogenic. For example, one publication advised putting the food in the centre of an open field2. Not only 
does this result in the feeding latency being confounded by exploratory time, it also introduces an additional facet of anxiety, i.e. 
open field central area aversion, a classic measure of open field-mediated anxiety, which has been related to avoiding predation risk 
in natural habitats14. However, in some situations this might actually be a desired feature of the experiment. Another 
disadvantage of the experiment described in2 was that the bait was standard lab chow in the centre of the open field. This has the 
double disadvantage that it is not novel, and also is not extremely palatable, unlike foods such as sweet corn or sweetened condensed 
milk. We have always found that using baits of extreme palatability not only increases the conflict over whether to eat or avoid the novel 
food, but also the animals do not need to be excessively food deprived before the test; only mild deprivation is necessary, so this aspect 
of the protocols presented here reflects the "Refinement" aspect of the 3Rs of Russell and Burch20.
2.1 Procedure:
Ration the food of the mice overnight. Remove all food from the cage hopper the evening before testing then give 1 g/mouse of ordinary diet, which is approximately a third of what they would normally eat. If they are group housed make sure that the pieces are small so that all the mice can eat equal shares. Test them the next morning and replace their food as soon as testing is finished.
Set up the chosen hyponeophagia test apparatus, also a row of small cages in which mice from the group home cage are placed individually before and during testing. (This avoids the increase in anxiety in the remaining mice left in the group cage, which can occur if you progressively remove mice from a group, also social transmission of food preferences. It also assists in test standardisation (between group and singly housed animals) and ensures that the animals are optimally alert. 
Place a mouse in the test apparatus. Measure the latency to eat. This is defined as eating/drinking continuously for 2-3 s. Often the mice will sniff or lick the food briefly at first, but only later start to eat continuously without inhibition.
Other measures can also be taken. The time spent eating during a set period, e.g. 2 min after feeding first starts, and possibly also the amount of food consumed during this period, can also be measured. Brain lesions sometimes affect these measures differentially4.
2.2 Specific details of running a hyponeophagia test with the apparatus shown in Figure 1:
A translucent plastic jug (approximate volume 1.5 l, 15 cm diameter with a spout protruding a further 2 cm) is the test apparatus. Use full cream sweetened condensed milk (Nestle Carnation brand is excellent) diluted 50:50 with water (to facilitate handling it) as the novel food. To contain the mouse on the base and facilitate investigation of the milk, place the jug upside down with the spout forming a small alcove over the food well. This focuses the attention of the mouse on to the well and the milk, so minimising the effects of treatments that might affect perception of, or orientation to, the food source.
The mouse is placed facing away from the well and the jug is gently lowered into position, taking care not to trap the tail of the mouse. The latency from being placed in the apparatus to start properly drinking is taken. Sniffs at the milk do not count; at least two seconds of drinking should occur. Count any faecal boli and note any urination (but note that these measures will be influenced by the time in the apparatus). The cut-off time is 2 min. If a mouse has not drunk by then, remove it from the apparatus and test another one or two mice, then retest the one that failed to drink about 3 min later. This time is probably optimal for anxiety levels to decrease while still preserving a state of habituation to the apparatus. This test-rest-retest-rest-retest cycle can be continued until the mouse eats or a set number of trials have been performed. A maximum of 3 trials should result in most mice drinking without making the experiment too long. The test parameter is the (cumulative) latency to drink. 
As in other tests where odour could play a significant role, non-experimental mice should be placed in the apparatus before testing starts. Follow a standardised cleaning protocol between all mice. Remove urine/faeces, wipe the food well and the plastic base with moist followed by dry cloth/tissue. Refill the food well with milk (about 0.2 ml, to the rim of the well). 
<img src="/files/ftp_upload/2613/2613fig1.jpg" alt="Figure 1"/> Figure 1. A plastic domestic jug converted for use as a hyponeophagia testing unit. Sweetened condensed milk can be placed in the small food well located inside the spout, which conveniently directs the attention of the mouse straight to it. The lead weight glued to the inverted base of the jug helps to stabilise it against any movement of the mouse against the walls.
<img src="/files/ftp_upload/2613/2613fig2.jpg" alt="Figure 2"/> Figure 2. An observation chamber containing two white (anxiety-provoking) white inserts for hyponeophagia testing. Sweet corn could be used as the novel food.
<img src="/files/ftp_upload/2613/2613fig3.jpg" alt="Figure 3"/> Figure 3. Another hyponeophagia test unit. The novel food is monkey treat mix.
<img src="/files/ftp_upload/2613/2613fig4.jpg" alt="Figure 4"/> Figure 4. Another hyponeophagia test unit. The food presented is mixed chopped nuts.
3. Running multiple hyponophagia experiments:
The first hyponeophagia test may not provide an unequivocal result. This often happens and is frequently due to the baseline (control) values being too high or low, resulting in ceiling or floor effects. Floor effects were encountered when testing hippocampal lesioned mice10; it appeared that the control value had to be sufficiently high to observe reduced hyponeophagia. 
It can be difficult to judge the required aversiveness of the test situation. If the mice have been tested several times already (not necessarily on anxiety-related tasks) their emotionality level will be lower than if they are na ve to behavioural testing and a more anxiogenic test will be required. This can often be provided by an elevated test environment (as in Figure 4). Aversive noise could be added to the test situation, e.g. a radio playing at a loud volume or a white noise generator. Brighter lighting would also increase test environment aversiveness.
So, if the first hyponeophagia result is not significant, try several more test situations and perform a repeated measures ANOVA. This is a very powerful experimental design.
4. Expected results:
Hippocampal lesions produce a strong decrease in hyponeophagia in mice10 and recent unpublished work has shown that this is, as in the rat1, due to the ventral region of the hippocampus. Mice lacking the KATP channel subunit Kir6.2 showed increased hyponeophagia in one test but less in another9, a confusing result but one which serves to emphasis that differences in test environment can have a marked effect on the result, and also points out the usefulness of performing multiple tests. 129S2/SvHsd mice were more hyponeophagic than C57BL/6JolaHsd6. Indeed, a comparison of several inbred mouse strains showed marked inter-strain differences21.

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No conflicts of interest declared.

The latency figures in hyponeophagia testing are typically highly variable. The technique described here, of imposing a set time period for the animal to eat, then retesting later if it fails to eat, serves to minimise the risk of ultra-long latencies and saves the experimenter time. 
One advantage of hyponeophagia over anxiety tests such as the plus-maze, is that if the results of the first test fail to achieve statistical significance, another test can be run using a different novel test environment and food, as the type of anxiety being measured appears to be the same and not qualitatively changed by the previously run tests, unlike in the plus-maze16. Repeated measures ANOVA can then be used to assess the test series.
It is now known that several different aspects of emotionality are covered by the umbrella term "anxiety"11,15,18. Mice showing changes in hyponeophagia will not necessarily show changes in open field, elevated plus-maze or light-dark box tests. So the brain systems underlying performance on these tests must be at least somewhat different. For example, the hypothalamus and amygdale may well participate in hyponeophagia as they are concerned with (among other things) regulation of food intake.

hyponeophagia: a measure of anxiety in the mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Watch this Scientific Journal Video about Hyponeophagia: A Measure of Anxiety in the Mouse at JoVE.com

Hyponeophagia: A Measure of Anxiety in the Mouse

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

hyponeophagia-a-measure-of-anxiety-in-the-mouse

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21.7K Views.  University of Oxford. Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Video: Hyponeophagia: A Measure of Anxiety in the Mouse

A Simple Way to Measure Ethanol Sensitivity in Flies

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A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

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Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

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We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...