WO2009036573A1

WO2009036573A1 - Apparatus and method for monitoring cell behaviour

Info

Publication number: WO2009036573A1
Application number: PCT/CA2008/001671
Authority: WO
Inventors: Frederic Charron; Patricia Yam
Original assignee: Institut De Recherches Cliniques De Montreal
Priority date: 2007-09-21
Filing date: 2008-09-22
Publication date: 2009-03-26

Abstract

An apparatus for observing behaviour of a cell in response to a chemical substance, the apparatus having a chamber in which can be formed a concentration gradient of the chemical substance, and a means arranged to moveably attach the cell to the apparatus to expose at least a free end of the cell to the chemical substance in the chamber, the free end being moveable. A method for observing behaviour of a cell in response to a chemical substance, the method comprising moveably attaching a cell to an apparatus comprising a chamber for housing a concentration gradient of the chemical substance such that a free end of the cell, which is moveable, is in contact with the concentration gradient in use; forming a concentration gradient in the chamber; and contacting at least the free end of the cell with the concentration gradient.

Description

APPARATUS AND METHOD FOR MONITORING CELL BEHAVIOUR

FIELD OF THE INVENTION

The present invention generally relates to an apparatus, a method and a system for monitoring or studying cell behaviour, particularly cell behaviour in response to chemical stimuli.

BACKGROUND OF THE INVENTION

The brain is composed of billions of neurons that must connect with an appropriate set of target cells to form the neuronal circuits that underlie its function. A neuron typically comprises a cell body and a long nerve fibre, called an axon, extending away from the cell body and having a growth cone at its leading edge. Neuronal connections form during embryonic development when neurons send out axons which migrate through the embryonic environment to their synaptic targets. Axons extend to the vicinity of their appropriate target regions in a highly stereotyped and directed manner by detecting a variety of attractive and repulsive molecular guidance cues presented in the environment. Typically, a molecular guidance cue that induces movement of an axon in the direction of its highest concentration is known as a chemoattractant and a molecular guidance cue that induces movement of an axon in the direction of its lowest concentration is known as a chemorepellent. This directional movement and growth of axons in response to attractive and repulsive molecular guidance cues will be referred to hereafter as axon guidance behaviour. The directional movement is also known as 'turning' and axon growth towards a certain direction is known as 'preferential growth' or 'preferential outgrowth' in the art.

Inappropriate wiring of these neuronal connections during development leads to abnormalities which can affect the sensory, motor and cognitive functions of the nervous system. Similarly, the failure of axons damaged by neurodegenerative diseases, stroke, or brain and spinal cord injuries to regenerate and reintegrate neuronal circuits seriously impacts on the life of affected individuals and causes death. Therefore, it is important to study, monitor or analyse the guidance behaviour of neurons in response to different guidance cues to aid the understanding of the complexity underlying the wiring of the nervous system, and ultimately to treat or prevent nervous system conditions. Various in vitro test methods exist for studying neuron guidance behaviour.

One of the most commonly used methods for measuring guidance behaviour is the "collagen gel" assay. This consists of culturing a piece of tissue ("an explant") containing neurons in a three-dimensional collagen matrix near or in a source of guidance cue e.g. a chemoattractant. However, these assays can not measure the turning of neuron axons toward the guidance cue source, but instead measure the preferential outgrowth towards the guidance cue source. These assays are also normally performed over a period of 12 to 48 hours which is too long to measure the initial, short-term effect of a guidance cue on an axon. Also, high-resolution real-time imaging of the axons is not possible with this assay. Furthermore, the guidance cue concentration gradient cannot be quantified with this assay. Moreover, due to the fact that the explants and the source of guidance cue need to be manually and quickly positioned in the collagen matrix before the collagen polymerizes, this assay has a very low throughput in terms of the number of neurons which can be studied at any one time.

Another method which has been used to study axon guidance behaviour and cues includes devices which "print" (i.e. attach chemically or passively absorb) guidance cues to a surface. One such device is known in the art as a microcontact printing device. This method is useful to study guidance cues which are membrane-bound and non-diffusible, such as ephrin molecules, but it is not adapted to study diffusible guidance cues such as Netrin, Slit, etc.. Therefore, only a subset of cues can be studied using this method. Additionally, because the cells must be plated directly onto the printed surface, preferential growth of axons, but not turning, is measured. This method also cannot be easily adapted to screen large numbers of guidance cues.

A third type of method that has been used to study axon guidance behaviour and cues are the "pipet turning" assays. This type of assay consists of culturing dissociated neurons on a glass coverslip. The coverslip is then placed on a microscope stage, and a micropipet is positioned close to the growth cone of an axon. The micropipet is connected to a pump which ejects a given volume of a guidance cue over time. This assay allows the visualization in real-time of the turning of an axon towards the source of guidance cue. Of the three methods, this is the only one that can measure axon turning rather than preferential growth of an axon.

However, as only one axon is measured at a time, one experimenter is limited to study a very small number of neurons per day, typically 6 to 12 neurons per day for an experienced user. Thus, this assay cannot be used for high-throughput analysis. Also, there are various limitations with this assay such as the pipette can block easily and the tip of the pipette must be monitored continuously so that it does not touch the growing neuron or break the fragile coverslip. Furthermore, the pipet-turning assays are arranged to be operated at room temperature and so typically can study only non- mammalian neurons, such as from frogs or chickens, which are more robust than mammalian neurons at room temperature. However, it is believed that mammalian and non-mammalian neurons will react differently to different guidance cues and so a method is required for studying the guidance behaviour, especially the turning behaviour, of mammalian neurons as well as non-mammalian neurons. Moreover, the reproducibility of the gradient established by the pipet assay has been questioned.

Therefore, it is desired to overcome or reduce at least some of the above-described problems.

SUMMARY OF THE INVENTION

The present invention reduces the difficulties and disadvantages of the aforesaid designs by providing an apparatus, a method and a system for observing the behaviour of cells in response to a stimuli. Preferably the stimuli is a chemical stimuli and the behaviour of the cells is observed in real-time. Advantageously, the invention can be applied to mammalian and non-mammalian neuron cells exposed to a guidance substance. The applicant has had the novel and inventive idea of studying neuron axons by providing apparatus arranged to maintain the neuron body relatively immobile whilst allowing the neuron axon to be able to move in response to stimuli. In this way, the turning behaviour of the neuron axon can be studied in response to stimuli such as guidance cues. Surprisingly, the applicant has found that an apparatus known in the art as a Chemotaxis Dunn Chamber may be adapted to study neuron axons in this way by establishing a stable concentration gradient of the stimuli substance in a chamber and by exposing the neurons to the stable concentration gradient. This is an unintuitive use of a Dunn chamber as this apparatus has previously only been used to study cell migration behaviour (see for example Zicha, D. et al (1991) A new direct-viewing chemotaxis chamber. J. Cell Biol. 99: 769-775, Zicha, D. et al. (1997) Analyzing chemotaxis using the Dunn direct-viewing chamber. Methods MoI. Biol. 75: 449-457 and Wells, C. M. and Ridley, A. J. (2005) Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy. Methods MoI. Biol. 294: 31-41). In contrast, in the present invention, the cells being studied do not migrate. They are moveably attached to the apparatus i.e. they remain in the same position with respect to the apparatus while a free end of the cell is able to move in response to stimuli (particularly neuron axons turning towards a guidance cue concentration gradient).

From one aspect, there is provided an apparatus for observing behaviour of a cell in response to a chemical substance (compound), the apparatus having a chamber in which can be formed a concentration gradient of the chemical substance, and a means arranged to moveably attach the cell to the apparatus to expose at least a free end of the cell to the chemical substance in the chamber, the free end being moveable. Preferably, the means is a plate which can be placed over an open end of the chamber and to which the cell can be moveably attached for contacting the concentration gradient. Alternatively, the means can be a wall of the chamber to which the cell can be moveably attached for contacting the concentration gradient. Advantageously, the means is transparent or translucent for observing movement of the free end of the cell in the chamber.

It will be understood that by moveably attaching a cell to the apparatus, it is meant that the cell has a portion, such as an anchor portion, which remains attached to the apparatus and another portion, such as a free end portion, which can move relative to the cell anchor portion or the apparatus. The cell may be a neuron and the chemical substance a guidance cue. In this way, the apparatus is arranged to present the neuron to the guidance substance in such a way that the neuron body is substantially fixed or immobile relative to the apparatus while the axon extending from the neuron body can move and the movement of the axon in response to the guidance substance can be observed and monitored.

In one embodiment, the chamber comprises a first and a second well in fluid communication with one another across a bridge region, the concentration gradient being formed in the bridge region in use. The first and second wells are concentric annular wells, the first well being an outer well and the second well being an inner well, the first well having an outer wall which is deeper than an outer wall of the second well. The apparatus can further comprise a temperature control means for controlling the temperature of the concentration gradient in the chamber. This means that mammalian cells can be studied at body temperature. The temperature control means can be a housing for the apparatus or a portion of the apparatus which can be heated or cooled.

From another aspect, there is provided an apparatus for observing behaviour of a cell in response to a chemical substance, the apparatus having a first well and a second well in fluid communication with one another across a bridge region, a concentration gradient of the chemical substance being formed in the bridge region in use, and a means arranged to moveably attach the cell to the apparatus to expose at least a free end of the cell to the concentration gradient in the bridge region, the free end being moveable.

Advantageously, the first and second wells are concentric annular wells formed in a support, the first well having an outer wall which is deeper than an outer wall of the inner well. Preferably, the first well is an outer well and the second well is an inner well. Advantageously, by means of this arrangement, a stable concentration gradient of the chemical substance can be established and cell behaviour in response to the chemical substance can be more accurately observed.

The means is a plate, such as a cover slip, which can be placed over an open end of the first and second wells in the support and to which the cell can be moveably attached for contacting the concentration gradient. The means could also be a wall of the chamber. Advantageously, the means is transparent or translucent for observing movement of the free end of the cell in the concentration gradient. In one embodiment, the cell is a mammalian cell such as a neuron and the chemical substance is a guidance cue.

The apparatus can further comprise a temperature control means for controlling the temperature of the concentration gradient in the chamber. This means that mammalian cells can be studied at body temperature. The temperature control means can be a housing for the apparatus or a portion of the apparatus which can be heated or cooled.

A plurality of the apparatus can be provided as an array on a support to study a number of different chemical substances, such as guidance cues, substantially simultaneously.

From yet another aspect, there is provided a method for observing behaviour of a cell in response to a chemical substance, the method comprising: moveably attaching a cell to an apparatus comprising a chamber for housing a concentration gradient of the chemical substance such that a free end of the cell, which is moveable, is in contact with the concentration gradient in use; forming the concentration gradient of the chemical substance in the chamber; and contacting at least the free end of the cell with the concentration gradient of the chemical substance.

The cell can be a mammalian cell such as a neuron and the chemical substance a guidance cue. The neuron has an anchor portion for remaining attached to the apparatus and a dynamic portion having the free end, the method further comprising allowing the dynamic portion of the cell to grow out before contacting the free end with the concentration gradient.

By means of embodiments of the present invention, a plurality of cells in contact with the concentration gradient can be observed and images of the movement of the free end of each of a plurality of cells in contact with the concentration gradient can be captured simultaneously. The concentration gradient of the chemical substance can be formed in the chamber before or after moveably attaching the cell to the apparatus. The movement of the free end can be a turning of the cell free end, a growth of the free end or a direction of growth of the free end.

Advantageously, the method may further comprise controlling the temperature of the concentration gradient, such as maintaining the temperature at 37°C. This is particularly advantageous in the study of mammalian cells

The method further comprises detecting a response of the cell to the concentration gradient. The detecting of the response can comprise observing the movement of the free end by optical microscopy or any other suitable means. Advantageously, the detecting of the response can comprise observing the movement of the free end along a length of the cell in order to observe free end turning.

Images of the movement of the free end can be captured, by photographic apparatus for example. The images may be a series of images which are captured as a function of time. Preferably, the capturing of the images of the movement of the free end is initiated at least on contact of the free end with the concentration gradient. In the examples provided herein, capturing of the images is initiated about ten minutes after chemical substance contact. However, the imaging or photographic apparatus can be arranged to start acquiring images on contact of the free end with the concentration gradient or at least substantially immediately soon thereafter.

By means of embodiments of the present invention, a plurality of cells in contact with the concentration gradient can be observed and images of the movement of the free end of each of a plurality of cells in contact with the concentration gradient can be captured simultaneously. Advantageously, the movement of the free end of the cell can be at least semi-quantified by tracking the relative co-ordinates of the free end with time.

The method may further comprise adding a compound to the concentration gradient or to the cell to assess whether the compound alters the cell behaviour or alters a function of the chemical substance of the concentration gradient. By alters it is meant that the cell behaviour or the function of the chemical substance in the concentration gradient changes in any way, such as increasing, decreasing, reversing or modulating its normal behaviour. The compound can be added to the concentration gradient or to the cell before, during or after contacting the cell with the concentration gradient.

The compound may comprise a plurality of compounds which are added sequentially to the concentration gradient or cell, or added substantially simultaneously to the concentration gradient or cell, before during or after contacting the cell with the concentration gradient. The first compound may be a guidance cue for neuron cells, having a chemoattractive or chemorepellent function, and the second compound may inhibit, enhance, reverse or modulate the function of the first compound, or prime the neuron cells. By priming the cells is meant rendering the cells more responsive to a compound i.e. increasing the neuron response. In this way, a first compound used in a concentration gradient and having an effect on neuronal guidance, for example, can be used in combination with a second compound to identify and characterize synergistic effects provided by the combination of the compounds.

From a further aspect, there is provided a method for identifying a compound that affects neuron cell behaviour, the method comprising: moveably attaching a neuron cell to an apparatus comprising a chamber for housing a concentration gradient of the compound such that a free end of the cell, which is moveable, is in contact with the concentration gradient in use; forming the concentration gradient of the compound in the chamber; and contacting at least the free end of the cell with the concentration gradient of the compound.

The method can further comprise detecting, and quantifying, a movement of the free end of the neuron cell in response to contacting the concentration gradient.

The compound may be a guidance cue which may already have been identified as such or be chemical substances having a guidance cue or a guidance cue-like function. Therefore, the method may be used for determining whether a compound is useful for neuronal guidance. The methods may be employed with either a single compound or a library (e.g. a combinatorial library) of compounds. Quantification can be performed by tracking the relative co-ordinates of the free end with time to identify parameters such as turning of the free end, growth of the free end or a direction of growth of the free end. Orientation of the turning of the free end of the neuron (growth cone) towards the highest concentration of the gradient will indicate a chemoattractive effect of the compound, whereas orientation of the turning of the free end of the neuron (growth cone) away from the highest concentration of the gradient will indicate a chemorepellent effect of the compound. Growth of the neuron without turning indicates that the compound has no effect on the neuron.

From a yet further aspect, there is provided a method for identifying a candidate compound that affects a function of a guidance cue or a neuron cell reaction to a guidance cue, the method comprising: moveably attaching a neuron cell to an apparatus comprising a chamber for housing a concentration gradient of the guidance cue such that a free end of the neuron cell, which is moveable, is in contact with the guidance cue concentration gradient in use; forming the guidance cue concentration gradient in the chamber; contacting at least the free end of the neuron cell with the guidance cue concentration gradient; and contacting the neuron cell or the guidance cue concentration gradient with the candidate compound.

Advantageously, this method can be used for screening candidate compounds which modulate the chemoattractive or chemorepellent functions of guidance cues or other substances. Generally, the presence of neuron cell activity or an increase in the neuron cell activity in the presence of the candidate compound indicates that the candidate compound is an activator. An absence of neuron cell activity in the presence of the candidate compound is indicative of the candidate compound being an inhibitor. In one example, the first compound is the Shh or NGF proteins and the candidate compound is SANT-I.

Advantageously, this method can be used to identify members of pathways in neuronal guidance. For example, the presence or increase of neuron cell activity in the presence of a candidate compound associated with a particular pathway, will be indicative that said pathway is involved in the activity of the first compound on neuronal guidance. Therefore, whether or not the activation of a signalling pathway is a molecular process required for a chemoattractive or chemorepellent effect of a guidance cue can be evaluated.

In one example, the guidance cue used was the Shh or NGF proteins and the candidate compound was PP2. PP2 was found to inhibit the chemoattractive effect of Shh. The pathway is the Shh-dependant Src pathway, the pathway members being SFKs, Src and Fyn. Thus, SFKs, Src and Fyn represent novel pharmaceutical targets in neuronal guidance, and modulators of their activities (e.g. PP2) could be used in neuronal guidance.

Advantageously, this method can also be used for characterizing cellular or molecular mechanisms affecting cell behaviour or a function of the guidance cue, and as such may be used to identify novel molecular targets in neuronal guidance. In one example, actinomycin D or DRB that inhibits RNA polymerase activity, was used in combination with a chemoattractive guidance cue (e.g. Shh) and the chemoattractive effect of Shh on neuronal guidance was shown to be independent of RNA transcription.

The method may further comprise detecting, and quantifying, a movement of the free end of the neuron cell in response to contacting the concentration gradient. Quantification can be performed by tracking the relative co-ordinates of the free end with time to identify parameters such as turning of the free end, growth of the free end or a direction of growth of the free end.

The neuron cell can be contacted with the candidate compound before, during or after contacting the guidance cue concentration. The candidate compound can be added to the guidance cue concentration gradient before, during or after the free end of the neuron cell is exposed to the guidance cue concentration gradient.

There may be provided a plurality of candidate compounds which contact the guidance cue concentration gradient or the neuron cell sequentially or substantially simultaneously. It will be appreciated that many possibilities for exposing the neuron cell to the guidance cue and to the candidate compound are possible. Neuron cells can be pre-treated with one or more of the candidate compounds before contacting the cells with the guidance cue concentration gradient. Alternatively, the candidate compound can be added to the guidance cue concentration gradient before or after the cell is exposed to the concentration gradient.

Instead of a guidance cue, the method may provide a compound which is altered into an active state (e.g. chemoattractant or chemorepellent) by the candidate compound. This combination of compounds may find application as a prodrug which is effectively activated at an appropriate site of treatment.

Any of the steps of the methods described above may be automated.

From a yet further aspect, there is provided a system for observing behaviour of a cell in response to a chemical substance, the system including an apparatus as hereinabove defined, an imaging apparatus for observing the movement of the free end of the cell and an image capturing apparatus for capturing images of the movement of the free end of the cell. The system may further comprise a processor for processing the captured images. Advantageously, by means of embodiments of the system, the behaviour of a number of cells can be observed and quantified automatically, or at least semi- automatically, and in real-time.

From a further aspect, there is provided a use of the apparatus, as defined above, for identifying a first compound that affects cell behaviour, and optionally a second compound that affects a function of the first compound or the function of the cell. The cell can be a mammalian cell such as a neuron. In other words, the present invention extends to use of the abovedescribed apparatus to screen and identify compounds having neuronal guidance activity, as well as compounds affecting the guidance activity of a known guidance compound or the function of the neurons. By affecting it is meant increasing, decreasing, reversing or modulating in any other way the function of the guidance compound or the neurons. The first compound can have a chemoattractive or chemorepellent effect on the cell, and the second compound can affect the activity or function of the first compound.

Advantageously, by means of aspects and embodiments of the invention, neuron axon guidance behaviour can be observed and measured in real-time and from the moment that the axon is exposed to a guidance cue. Furthermore, axons can be exposed to concentration gradients of guidance cues which are controllable, reproducible and measurable. Moreover, the axon guidance behaviour can be measured both qualitatively and quantitatively in terms of its turning behaviour and its preferential growth. Furthermore, greater numbers of axons can be studied at any one time without the need for more sophisticated equipment or more operators. Axon guidance behaviour in response to known and new axon guidance cues can be investigated to identify, test and screen existing and novel guidance cues (as well as testing chemical molecules for having guidance activity, even if they are not real guidance cues found in nature) and to characterize the molecular mechanism of action of guidance cues on axon guidance. Also, substances which may alter the function of the guidance cues or the cells can be studied e.g. by inhibiting, enhancing or reversing the presumed normal function of the guidance cue (i.e. a chemoattractant becomes a chemorepellent and vice versa). Libraries of such compounds may be established and investigated for their ability to alter the activity of a guidance cue. This in turn may help to identify novel strategies to promote the proper guidance and rewiring into neural circuits of regenerating axons damaged by genetic and neurodegenerative diseases, stroke, or brain and spinal cord injuries. It is thought that nerve regeneration therapy may involve the genetic reactivation of the normal embryo nerve function, or involve treatment using injections or patches.

Compared to other assays and methods for axon guidance, embodiments and aspects of the present invention allow for the monitoring and measuring of a large number of neurons (tens to hundreds) responding to a guidance cue. In addition, the short-term responses (in the order of tens of minutes) to guidance cues of the axons can be measured. Also mammalian neurons can be studied, a feature that has not been easy to implement with other known assays.

Therefore, all aspects of the invention described herein are an improvement of the devices and methods of the prior art, in that they provide the following advantages: an efficient method for the observation and measurement of cell, such as axon, movement in real-time; a simple method for the measurement of the axon guidance activity for a plurality of compounds; an inexpensive and/or disposable multiple-site axon guidance test apparatus; a multiple-site axon guidance test apparatus requiring very small volumes of guidance cues; and a high sensitivity multiple-site axon guidance test apparatus. Furthermore, the embodiments and aspects of the present invention may be applied to cells other than neurons for studying their behaviour to chemical gradients.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more readily understood, currently preferred embodiments will now be further described by way of example only with reference to the accompanying drawings in which:

Figure 1 is a cross-sectional view through a first embodiment of an apparatus according to the present invention;

Figure 2 is a perspective view of a second embodiment of the apparatus of the present invention;

Figure 3 is a cross-sectional view along the line A-A' of the apparatus of Figure 2;

Figures 4-7 illustrate the subsequent steps in forming a concentration gradient of a guidance cue in the apparatus of Figure 2;

Figure 8 is a plan view of a central portion of the apparatus of Figure 2 showing a bridge region between an outer well and an inner well;

Figure 9 illustrates a first embodiment of a system according to the present invention;

Figure 10 illustrates a chemoattractant gradient formation in the bridge region of Figure 8 according to Example 1 at (a) 0 to 1.5 hours, and (b) 1.5 to 9 hours;

Figures l la-c are optical micrographs of DRG neuron movement at 0, 0.75 and 1.5 hours in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to (a) a control gradient of buffer containing NSF, (b) NGF, and (c) NGF;

Figures 12a and b are trajectory plots of a sample of 19 DRG axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to (a) a control gradient, and (b) a 25 ng/ml NGF gradient;

Figure 13 illustrates the definition of an initial angle, α, the angle between an initial axon position and the gradient, and an angle turned, β, the angle between the vectors representing the initial and final position of the axon;

Figures 14a and 6 are scatter plots of the angle turned versus the initial angle for DRG axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to (a) a control, and (b) 25 ng/ml NGF gradient;

Figure 15 is a graph of mean angle turned for DRG axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to a control and 25 ng/ml NGF gradient for initial angles >20°;

Figures 16a-c are optical micrographs of commissural neuron movement at 0, 1 and 2 hours in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to (a) a control gradient of buffer containing BSA, (b) Shh, and (c) Shh;

Figures 17a and b are trajectory plots of a sample of 19 commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to (a) a control gradient, and (b) a 0.1 μg/ml Shh gradient;

Figures 18a-e are scatter plots of the angle turned versus the initial angle for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to (a) a control, (b) 25 ng/ml Shh gradient, (c) 0.1 μg/ml Shh gradient; (d) 0.4 μg/ml Shh gradient, and (e) 1.6 μg/ml Shh gradient;

Figure 19 is a graph of mean angle turned for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to a control, 0.025, 0.1, 0.4 and 1.6 μg/ml Shh gradient;

Figure 20 is a scatter plot of angle turned versus position across bridge region for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1;

Figure 21 is a scatter plot of the net extension of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to a control and 0.1 μg/ml Shh;

Figure 22 is a scatter plot of angle of extension and net extension of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to 0.1 μg/ml Shh;

Figure 23 is a scatter plot of angle turned versus net extension of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to 0.1 μg/ml Shh;

Figure 24 is a graph of the mean angle turned for initial angles >20° for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to a control gradient, and faster and slower axon populations;

Figure 25 is a scatter plot illustrating the time to commence turning of a commissural axon in a bridge region of the apparatus of Figure 2, according to Example 1 , in a gradient of 0.1 μg/ml Shh;

Figure 26 is a graph of transcriptional activity in commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, in a gradient of Shh;

Figure 27a-c are optical micrographs of commissural neuron movement at 0, 1 and 2 hours in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to a 0.1 μg/ml Shh gradient in the presence of (a) SANT-I, (b) DRB, and (c) Actinomycin;

Figures 28a-d are scatter plots of the angle turned versus initial angle for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to (a) a 0.1 μg/ml Shh gradient, (b) 0.1 μg/ml Shh gradient plus SANT-I, (c) 0.1 μg/ml Shh gradient plus DRB, and (d) 0.1 μg/ml Shh gradient plus Actinomycin;

Figure 29 is a graph of mean angle turned for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to a control gradient, 0.1 μg/ml Shh gradient, 0.1 μg/ml Shh gradient plus SANT-I, 0.1 μg/ml Shh gradient plus DRB, and 0.1 μg/ml Shh gradient plus Actinomycin;

Figure 30 illustrates immunoblots and a graph of relative phospo-SFK levels for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to 0.1 and 0.4 μg/ml Shh for 10 or 30 minutes in the presence or absence of SANT-I ;

Figures 31a and b are graphs of (a) SRC and (b) FYN kinase activity, for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to 0.4 μg/ml Shh for 30 minutes in the presence or absence of SANT-I;

Figures 32a and b are optical micrographs of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to 0.1 μg/ml Shh gradient in the presence of (a) PP2, and (b) PP3;

Figure 33 is a scatter plot of the angle turned versus initial angle of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when exposed to 0.1 μg/ml Shh gradient in the presence of PP2;

Figure 34 is a graph of mean angle turned for commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1, when exposed to a control gradient, a 0.1 μg/ml Shh gradient, a 0.1 μg/ml Shh gradient in the presence of PP2, and a 0.1 μg/ml Shh gradient in the presence of PP3; and Figure 35 is a graph of relative Gli-reporter activity of commissural axons in a bridge region of the apparatus of Figure 2, according to Example 1 , when pre-treated with PP2, PP3 or SANT-I prior to addition of 0.2 μg/ml Shh.

DETAILED DESCRIPTION OF THE INVENTION

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having", "containing", "involving" and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items.

Furthermore, although the present invention was primarily designed for studying the behaviour of the axon component of neurons in response to guidance cues, it may be used to study the behaviour of other types of cells in response to other types of stimulus. For this reason, expressions such as "neuron", "axon", "growth cone" and the like should not be taken as to limit the scope of the present invention and should be taken to include other kinds of cells and cell parts with which the present invention may be used and could be useful.

Moreover, in the context of the present invention, the expressions "guidance cue", "guidance substance", and any other equivalent expression known in the art used to designate a chemical substance or compound which induces or stimulates a reaction in the cell being studied, as well as any other equivalent expressions and/or compound words thereof, may be used interchangeably. Guidance cues may function as an inhibitor or as an activator i.e. they may have an inhibiting or activating function.

As used herein, the term "compound" or "chemical substance" includes natural or non- natural molecules such as proteins, antibodies, peptides, small chemical molecules etc. As used herein, the term "inhibitor" or "inhibition" refers to a compound which reduces axonal behaviour. The reduction may be at least from about 10% to about 100%, including all ranges therebetween, compared to normal activity or expression.

As used herein, the term "activator" or "activation" refers to a compound which increases axonal behaviour. The increase may be at least from about 10% to about 100%, including all ranges therebetween, compared to normal activity or expression.

In the following description, the same numerical references refer to similar elements. The embodiments shown in the figures are preferred.

Referring to Figure 1, there is provided a first embodiment of an apparatus 10 for exposing the axons of neurons 11 to a guidance substance 12 in order to be able to study the response behaviour of the axons exposed to the guidance substance.

Advantageously, by means of the apparatus 10, the axon behaviour can be observed in real-time. In its simplest embodiment, the apparatus 10 comprises a container 14 having at least one wall 16 defining a chamber 18, having an open face, in which can be formed a concentration gradient of the guidance substance 12 and a means of exposing axons to the guidance substance 12.

In the embodiment of Figure 1 , the means of exposing axons to the guidance substance 12 is a plate 20 onto which neurons 11 have been cultured in tissue culture plates (not shown) before the plate 20 is removed from the tissue culture plates and placed upside down over the open face of the chamber 18 such that the neuron axons can extend into the chamber 18. When the chamber 18 contains the guidance substance 12, the neuron axons will be in contact with or submerged in the guidance substance 12 whilst attached to the plate 20. The plate 20 also has the function of minimizing or avoiding evaporation of the guidance substance. The plate 20 also provides a sealed environment which can be temperature controlled to assist neuron growth. The plate 20 is preferably made of a translucent or transparent material such as glass, polymer, composite or blend, or any other suitable material. One suitable plate 20 is a cover slip of the type typically used in the art with microscope slides. The chamber 18 and plate 20 can be of any suitable shape, size or configuration. It will be appreciated that the guidance substance 12 may be placed into the chamber 18 before or after the plate 20 is placed in position. In this respect, the chamber may include a sealable or closable opening (not shown) through which the guidance substance may be caused to flow into the chamber 18. Also, the chamber 18 could be filled by flowing media containing the guidance cue through at least one microchannel (not shown). This could be performed on a large scale for many chambers arranged on an array and having microchannels by automated apparatus. Also, the concentration gradient of the guidance substance 12 may be formed in the chamber 18 before or after the plate 20 is placed in position.

A second embodiment (not shown) of the apparatus 10 differs from apparatus of the embodiment of Figure 1 in that the neurons may be cultured on the at least one chamber wall 16 before or after the guidance substance concentration gradient is formed within the chamber 18. For example, the chamber 18 may be defined by a bottom wall and at least one side wall, the neurons being cultured on the bottom wall to stick, fix or attach the neurons to the wall. In this embodiment, although the plate 20 is not required for providing the neurons, it can be used to minimize or prevent evaporation of the guidance substance.

In both the first and second embodiments of the apparatus 10, the apparatus 10 is arranged to allow the imaging in real-time of the axon behaviour in the presence of the guidance substance 12. In this respect, at least one wall of the apparatus 10, such as the chamber wall 16 or the plate 20, has optical properties suitable for imaging by optical microscopy to enable imaging of the neurons through the wall, preferably high resolution imaging. The neurons may also be imaged through the wall of the surface from which they extend into the guidance substance. A suitable optical property is translucency or transparency of the wall to allow for light transmission and observation by optical microscopy.

A third embodiment of the invention is shown in Figures 2 and 3. The apparatus 10 of this third embodiment differs from the apparatus 10 of the first embodiment in that the apparatus is arranged to form a stable concentration gradient of the guidance substance to which neurons attached to the plate 20 are exposed. In this embodiment, the apparatus 10 comprises an outer well 22 and an inner well 24, separated from one another by an inner wall 26 and a bridge region 36. The outer and inner wells 22, 24 are in fluid communication with one another across the inner wall 26. In this embodiment, the outer and inner wells 22, 24 are concentric circular troughs or channels formed in a base 28 such as a microscope slide. The inner wall 26 is therefore annular in shape. However, the wells 22, 24 and the base 28 may be any other suitable shape or configuration necessary to form a stable concentration gradient. The depth of the outer well 22 is defined by the height of an outer wall 30 and is deeper than the depth of the inner well 24 which is defined by the height of the inner wall 26. In this embodiment, the width of the inner wall 26 (i.e. the separation between the outer and inner wells 22, 24) is approximately 1 mm, and the difference in height between the outer and inner walls 30, 26 is approximately 20 μm. Figure 2 shows the apparatus 10 having an optional support pin 32 projecting from the centre of the inner well 24 which has been omitted from the remaining drawings for clarity.

A concentration gradient is formed between the outer and inner wells 22, 24, in the bridge region 36, in the manner illustrated in Figures 4 to 7. In this embodiment, the outer well 22 is filled with media containing the chemoattractant and the inner well 24 is filled with control media. The concentration gradient of the guidance substance 12 is formed after the neurons 11 are cultured and positioned for imaging. In a first step (Figure 4), the outer and inner wells 22, 24 are filled with tissue culture medium 33 or any other similar or suitable fluid. Meanwhile, neurons 11 are cultured onto the glass plate 20 (to moveably attach them to the plate 20) and axons are allowed to grow from the neurons 11. The plate 20 is preferably a cover slip. The plate 20, with the neurons attached, is then inverted over the outer and inner wells 22, 24, leaving a small opening 34 providing access to the outer well 22. The reverse is also possible. The tissue culture medium 33 is then removed from the outer well 22 through the opening 34 (Figure 5). Alternatively, any other means of removing the fluid 33 from the outer well 22 may be provided such as by draining the outer well 22 through another opening (not shown). The outer well 22 is then filled with the guidance substance 12 (Figure 6). The concentration difference between the outer and inner wells 22, 24 creates the bridge region 36 between the outer and inner wells 22, 24 having a stable concentration gradient. It will be appreciated that the stable concentration gradient region of the bridge region 36 of this embodiment is equivalent to the chamber 18 of the apparatus 10 of embodiments 1 and 2.

Alternatively, the inner well 24 can be filled with the media containing the chemoattractant, instead of the outer well 22 i.e. the direction of the concentration gradient reversed.

The axons of the neurons 1 1 can then be imaged in the bridge region 36 in real time using imaging apparatus and image recording apparatus such as a phase-contrast microscope fitted with a video camera connected to a computer with an image-grabber board (not shown). Multiple positions around the annular bridge region 36 are imaged to increase the number of axons observed. The microscope preferably has a moving stage to present different portions of the bridge region to the image recording apparatus. The apparatus 10 can be contained within an environmental control housing for controlling the temperature etc.

In an alternative embodiment, fluorescently tagged-proteins (e.g. with GFP) can be imaged using fluorescence microscopy. Alternatively, fluorescent dies such as calcium-sensitive or voltage sensitive dies could also be imaged by fluorescence.

Advantageously, since the outer and inner wells 22, 24 are concentric, the direction of the concentration gradient at any point in the annular bridge region 36 is perpendicular to the tangent along the annulus (i.e. radial) as shown in Figures 7 and 8. This orientation of the neuron axons allows them to be observed lengthwise when viewing them through the plate 20.

The present invention also extends to an array of a plurality of the apparatus 10 according to any of the embodiments described herein, the apparatus 10 being arranged as an array on a substrate to allow for the carrying out of parallel studies using the apparatus 10. In other words, a substrate or support comprising a plurality of the apparatus 10 of any of the embodiments described above, formed therein.

The present invention also extends to a system 40, shown in Figure 9, for monitoring neuron behaviour to guidance substances, the system 40 including any of the embodiments of the apparatus 10 as described above, together with imaging apparatus 42 for viewing the neurons. The system 40 may also include a image capture apparatus for recording the images from the apparatus 10. The system 40 may also include a processor (not shown) for processing the images captured from the apparatus. In one embodiment, the imaging apparatus is a microscope, the image capture apparatus 44 is a video recorder and the processor is a microcomputer.

The present invention also extends to a use of the different embodiments of the above- described apparatus 10 and arrays to observe the behaviour of cells to a stimuli, particularly neuron axons exposed to a guidance substance. Preferably, the neuron axons are observed in real-time in a concentration gradient of the guidance cue. Advantageously, mammalian neuron axons can be observed by means of the invention.

The present invention also extends to a method of observing the turning behaviour of an axon when exposed to a guidance substance. The method comprises forming a concentration gradient of a guidance substance in a chamber, moveably attaching the axon to one wall of the chamber and contacting axon with the guidance substance, and observing the axon behaviour. The method can further comprise the quantification of the axon behaviour (see Example 1). The quantification involves tracking the tip of an axon over time. The tracking can be performed manually on images of the axon or can also be automated. The method allows for tracking the behaviour of about 30 axons with time. In a preferred embodiment, images are captured every four minutes. This allows for a very early response of an axon to a guidance substance to be measured, thereby providing an insight into early and transient effects.

EXAMPLES

The following example is illustrative of the wide range of applicability of the present invention and are not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any method and material similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described. The example below describes in detail the method of assembling the apparatus 10 of Figures 2 and 3, which was an adapted Dunn chamber, and a method of measuring axon behaviour to guidance substance, according to the present invention.

Briefly, dissociated DRG or commissural neurons were cultured on a coverslip such that the axons were already outgrown, and the coverslip was then inverted over the wells 22, 24. A chemoattractant gradient was formed across the bridge region 36 as described with reference to Figures 4 to 7. Axons positioned over the bridge region 36 were directly imaged responding to the gradient. Multiple axons around the annular bridge 36 were imaged at the same time. Live cell imaging was performed at 37°C on a Leica DMIRE2 inverted microscope (Leica, Germany) equipped with a heated chamber and a MS-2000 XYZ automated stage (ASI, Eugene, OR). Time-lapse phase contrast and/or epifluorescent images were acquired using a 1OX fluotar or 2OX fluotar LD objective every 4 minutes for a minimum of 2 hours for DRG neurons and 2.5 hours for commissural neurons. For each apparatus 10, approximately 20-35 stage positions were imaged at various positions around the annular bridge. All images were collected on an Orca ER CCD camera (Hamamatsu) using Volocity (Improvision, Waltham, MA).

Chemoattractant gradient set up: The apparatus was pre-washed with conditioned media, and about 150 μl of conditioned media was added to fill the inner and outer wells 24, 22. A coverslip 20 with neurons was inverted over the wells 22, 24, leaving a narrow slit 34 at one edge for draining and refilling the outer well 22. The size of the slit was preferably, but not limited to, a third to a half of the width of the other well 22. Excess media was removed by blotting with filter paper, and three sides of the cover slip were sealed with hot paraffm:vaseline. Using a gel-loading tip or the like, all the liquid from the outer well 22 was removed through the filling slit 34, and the chemoattractant (diluted in conditioned media) was added to the outer well 22. The filling slit 34 was then sealed with hot paraffin:vaseline. The apparatus 10 was assembled rapidly (<10 minutes) to avoid changes in the pH of the media. After apparatus assembly, imaging was started within about 10 minutes. Only axons which had net growth over the analysis period (1.5 hours for DRG neurons and 2 hours for commissural neurons) were imaged because the turning of axons that are not growing cannot be measured. Only single non-fasciculated axons were analyzed. To avoid analyzing axons which may be responding to local endogenous cues, axons which touched another object, such as debris, another cell or its own cell body, were excluded from the analysis, even if it occurred within 30 minutes after the analysis period. Axons which formed branches at the leading growth cone or which had periods of retraction >10 μm from the initial position were also excluded from analysis.

For each axon, the distal 10 μm of the axon at the first time point was tracked, and this was defined as the initial position of the axon. To enable accurate measurement of the initial angle, only axons for which the distal 10 μm of the axon were straight were analyzed. Then the position of the base of the growth cone was tracked for each time point. All axon positions and trajectories were translated such that the axons started at (0,0). Since the absolute direction of the gradient in the apparatus 10 depends on the position around the annular bridge, all axon positions and trajectories were rotated such that the gradient always increased along the y-axis. The angle of rotation was determined from the coordinates of the stage position relative to the center of the wells 22, 24.

The initial angle was calculated as the angle between the initial position of the axon and the direction of the gradient. The angle turned was defined as the angle between the original direction of the axon and a straight line connecting the base of the growth cone from the first to the last time point of the assay period (Figure 13). The angle turned was defined as positive for turns towards the gradient, and negative for turns away from the gradient.

Gradient stability: The gradient formation and stability in the system was verified using tetramethylrhodamine-labeled 40 kDa dextran. It was shown that the gradient always monotonically increased across the bridge, from the inner to the outer well, rapidly became linear (-20-30 minutes), and remained stable for at least 9 hours (Figures 10a and b).

Neurons from dorsal root ganglia (DRG): Square #3D 18mm coverslips 20 for the DRG neuron cultures were acid- washed and sterilized, prior to coating with 10 μg/ml poly-L-lysine (PLL) for about 6-8 hours, followed by 3 μg/ml laminin in PBS at 37 ⁰C overnight. El 3.5 or E14 DRGs were dissected and quickly washed once in cold Ca²⁺/Mg²⁺-free HBSS. The DRGs were trypsinized with 0.25% trypsin in Ca²⁺/Mg²⁺- free HBSS for 30 minutes at 37 °C. DNAse was added for a further 2-3 minutes. The tissue fragments were then washed in warm Ca²⁺/Mg²⁺-free HBSS and triturated in Ca²⁺/Mg²⁺-free HBSS to yield a suspension of single cells. Cells were cultured in DMEM/F-12 supplemented with 2% B27, 10 rnM HEPES, and 1-10 ng/ml NGF. The DRG neurons were used for these experiments 22-29 hours after plating onto the glass coverslips 20. The primary neurons were grown on the cover slips 20 at a low density such that individual isolated neurons were present (about 50-90 000 cells/well in a 6 well plate).

Neurons isolated from dorsal root ganglia (DRG) were placed into the Dunn chamber as described above in order to test whether the Dunn chamber generated gradients of guidance cues that axons could respond to. Axons with net extension during the 1.5 h observation period were analysed only as it was not possible to measure turning of axons that were not growing. In a control gradient of buffer containing BSA (the vehicle for nerve growth factor (NGF)), the direction of axonal growth remained unchanged (Figure 1 Ia). In contrast, when the neurons were placed in a gradient of 25 ng/ml NGF, a concentration which induces biased axon outgrowth from collagen gel explants, the axons turned up the gradient (Figure l ib). Even axons which were initially oriented almost against the NGF gradient direction were able to turn towards the gradient (Figure 1 Ic).

To measure the extent of turning, the initial position (t = 0 h) of the distal 10 μm of the axon was tracked, followed by the position of the growth cone every 4 minutes over 1.5-2 h, to obtain the trajectory of the axon. All trajectories were translated and rotated such that the direction of the gradient always increased in the y-direction, and all initial axon segments began at (0,0). Visual examination of the trajectories showed that in the control gradient, the direction of axon growth did not deviate substantially from the initial direction, whereas in the NGF gradient, many trajectories showed a bias towards growing up the gradient (Figures 12a and b). To further quantify this, the initial angle, defined as the angle between the initial orientation of the axon and the gradient, and the angle turned, defined as the angle between the initial and final trajectories of the axon (positive for turns up the gradient; negative for turns down the gradient) were measured (Figure 13). Scatter plots of these angles showed that for the control gradient, no net turning occurred; the angle turned varied around 0° (-1.7±2.7°, mean+sem) (Figures 14a and 14b). In the NGF gradient, there was a significant bias towards positive angles turned, i.e. chemoattraction, for axons not already oriented towards the gradient, i.e. initial angle > 20° (17.9±5.6°, mean±sem, p=0.0041 for angles turned compared to the control, initial angle > 20°) (Figure 15). Not all DRG axons responded to the NGF gradient; this may be because some DRG axons extend to the PNS and some to the CNS, and they may not respond equally well to NGF. Nevertheless, these results provided us with proof of principle that the Dunn chamber can be used to observe and measure axon turning.

Neurons from commissural axons; Dissociated commissural neuron cultures were prepared generally as described in the art (see for example Bouchard, J.F. et al, 2004, J Neurosci 24, 3040-3050; and Shekarabi, M. et al, 2005, J Neurosci 25, 3132-3141), with some modifications. Briefly, tissue culture plates or acid-washed and sterilized glass coverslips were coated with PLL (100 μg/ml for 2 h). The dorsal fifth of El 3 rat neural tubes were microdissected and quickly washed once in cold Ca²⁺/Mg²⁺-free HBSS. The neural tube sections were trypsinized in 0.15% trypsin in Ca²⁺/Mg²⁺-free HBSS for 7 minutes at 37 °C. DNAse was added briefly. The tissue fragments were then washed in warm Ca²⁺/Mg²⁺-free HBSS and triturated in Ca²⁺/Mg²⁺-free HBSS to yield a suspension of single cells. Cells were plated in Neurobasal media supplemented with 10% heat-inactivated FBS and 2 mM L-glutamine. After -21 h, the medium was changed to Neurobasal supplemented with 2% B27 and 2 mM L-glutamine. Commissural neurons were used for experiments 30-58 hours after plating. The primary neurons were grown on the cover slips at a low density such that individual isolated neurons were present (120-180 000 cells/well in a 6 well plate).

The axon guidance method above was applied to dissociated commissural neurons in a gradient of Sonic hedgehog guidance cue (recombinant human sonic hedgehog (C24II), amino terminal peptide, R&D Systems, Minneapolis, MN) (Shh).

Commissural neurons exposed to a control gradient of buffer containing BSA (the vehicle for Shh) continued to grow with no change in direction (Figure 16a). The trajectories of axons in control gradient did not deviate substantially from their initial direction (Figure 17a). When placed in a gradient of Shh, the axons turned up the gradient (Figures 16a and 16b), and the axon trajectories exhibited a bias up the Shh gradient (Figure 17b).

Concentrations of Shh from 25 ng/ml to 1.6 μg/ml were applied to the outer well 22, and the response of commissural axons to these gradients was measured. With the control gradient, there was no net turning, with the angle turned varying around 0° (- 0.7+3.2°, mean±sem). Significant turning up the Shh gradient occurred with 0.1 μg/ml (17.6+2.9°, mean+sem) and 0.4 μg/ml Shh (16.5±6.2°, mean±sem) in the outer well (p=0.0001 and p=0.0125 respectively, for angles turned compared to the control, initial angles > 20°) , with a weak, but not statistically significant bias towards attraction at 25 ng/ml and 1.6 μg/ml Shh (Figures 18a - 18e, and 19). Turning up a Shh gradient was seen over a wide range of initial angles, and in most cases, turning was more robust at larger initial angles, i.e. when the initial orientation of the axon had a larger deviation from the applied gradient. Given the robust response of the axons to a gradient of 0.1 μg/ml Shh, this concentration was used for further studies.

At 0.1 μg/ml Shh, axon turning was robust over time, with significant turning observed after 1.5, 2 and 2.5 h. The experiments were performed for a 2 hour period, at which the axons had grown on average -20 μm. To investigate whether the variation in Shh concentration across the bridge region 36 (from the inner to the outer well) influenced the degree of turning in response to Shh, whether the position along the bridge correlated with the angle turned was investigated. It was found that the angle turned was independent of the position along the bridge (Figure 20).

To determine whether Shh had an effect on axon growth in addition to turning, the net axon extension over 2 hours in the 0.1 μg/ml Shh gradient was compared to the control gradient, and no difference was found between the two conditions (p=0.9968) (Figure 21). Furthermore, the amount of axon growth was independent of the orientation of the direction of growth relative to the Shh gradient (Figure 22). Hence a Shh gradient did not stimulate commissural axon growth in vitro, consistent with previous studies demonstrating that Shh is a guidance factor, but not a growth-promoting factor, for commissural axons. However, the rate of axon growth could still influence the degree of turning, the angle turned versus the net extension was analyzed. There was no correlation between the angle turned and the net extension (Figure 23). Furthermore, when the population was divided into two groups (faster 50% and slower 50%) around the median extension (14.6 μm), both populations had significant turning up the Shh gradient compared to the control (p=0.0022 and p=0.0008 for the faster and slower populations respectively), and they were not significantly different from each other (p=0.9510) (Figure 24). Together, these results demonstrate that commissural axons in the apparatus 10 of an embodiment of the present invention can respond to Shh over various positions along the gradient, at various incident and growth angles, and independently of growth rates.

Investigating molecular mechanism of axon guidance by Shh: The effect of Smo inhibition on the ability of the axons to respond to Shh was first tested. Addition of SANT-I, a Smo inhibitor, to the neurons prior to the application of the Shh gradient, abolished axon turning up the Shh gradient (Figure 27). Quantification of the angles turned showed that neurons in the presence of SANT-I, exposed to a Shh gradient, were not significantly different to the control (p=0.6652) (Figures 28 and 29) and showed no turning. This is consistent with previous in vitro and genetic in vivo evidence that Smo is required for Shh-mediated axon guidance, and provides further support that the present method for Shh-mediated axon guidance recapitulates what has been demonstrated using other assays.

Shh-mediated guidance of commissural axons is rapid and independent of transcriptional activity: Using the present method for Shh-mediated axon guidance, the time taken for commissural axons to begin turning towards the Shh gradient was measured. Since commissural axons, like most mammalian neurons, grow episodically, the time for an axon to begin to re-orient towards the gradient after the start of axon growth, was measured for those axons which turned >30°. The median time to begin turning was surprisingly short, only 8 minutes, and for some axons, there was no delay between the start of axon turning and the start of axon growth (Figure 25). This rapid induction of growth cone turning suggested that Shh-mediated axon guidance may indeed be independent of transcription. To test this hypothesis, transcriptional activity in commissural neurons was inhibited using two drugs, DRB (5,6- dichlorobenzimidazole riboside) and actinomycin D, which inhibit transcription via two distinct mechanisms: DRB by inhibiting RNA polymerase, and actinomycin D by forming a stable complex with double-stranded DNA. ) At the highest concentration of each drug that could be used without toxic effects, transcription was inhibited in primary neuron cultures by 93.6% for DRB (0.1 μM) and 97.6% for actinomycin D (0.5 μg/ml), as measured by a ³H-uridine incorporation assay (Figure 26).

Addition of the transcriptional inhibitors, at those concentrations, to the neurons 40 minutes prior to the application of the Shh gradient, did not inhibit the ability of the axons to turn up a Shh gradient (Figure 27). Quantitation of the response to Shh in the presence of DRB and actinomycin D (Figures 28 and 29) revealed significant turning for both DRB (p=0.024 compared to control) and actinomycin D (p=0.0013 compared to control). Furthermore, the turning observed in the presence of the transcriptional inhibitors was indistinguishable from that in 0.1 μg/ml Shh alone (p=0.9629 and p=0.2757 for DRB and actinomycin D, respectively, compared to 0.1 μg/ml Shh alone). Therefore, Shh-mediated axon guidance was maintained in the presence of transcriptional inhibition acting via two different mechanisms. That Shh-mediated axon guidance does not require gene transcription is consistent with the short response time of axon turning to Shh (Figure 25), and also implies that transcriptional activity through the Gli-family of transcription factors is not required for this process. Therefore, it is hypothesized that Shh must mediate its axon guidance effects through an alternative, non-canonical signaling pathway.

Shh activates Src family kinases: SFK activity has been implicated in the guidance of axons by ephrins/Ephs and netrin-1. SFKs mediate signaling between receptor activation and downstream cytoskeletal regulators involved in growth cone turning. In the case of netrin-1, another chemoattractant for commissural axons to the floorplate, binding of netrin-1 to its receptor, DCC, activates Fyn and/or Src, two SFK members expressed in commissural neurons. Activity of these SFKs is required for netrin-1 induced axon outgrowth and turning. To thest the hypothesis that SFKs might also play a role in commissural axon guidance by Shh, levels of active SFK members were assayed, which are characterized by phosphorylation at Y418. An antibody against phospho-SFK (Y418) was used to probe lysates of primary commissural neurons treated with Shh. Addition of Shh increased endogenous SFK phosphorylation at Y418 by -30% (Figure 30). This increase in SFK phosphorylation occurred with the same Shh concentrations which induced axon turning, and within a short time period (10 minutes). The increase in SFK phosphorylation was also abolished by SANT-I inhibition of Smo, indicating that Smo activity is required for Shh stimulation of SFK phosphorylation.

To directly measure the kinase activity of the SFKs, commissural neuron cultures were treated with Shh in the presence or absence of SANT-I, and immunoprecipitated Src or Fyn, which are highly expressed in commissural neurons, from the cell lysates. Measurement of the kinase activity of the immunoprecipitated proteins demonstrated that there was a significant (p<0.01) increase in the activity of both Src and Fyn when commissural neurons were stimulated by Shh (Figure 31). This was dependent on Smo activity because SANT-I eliminated the Shh-induced increase in Src and Fyn kinase activity (Figure 31 ). Therefore, the increase in SFK phosphorylation in response to Shh correlated with an increase in Src and Fyn kinase activity. Together, this demonstrates that Shh stimulates SFK activity in commissural neurons in a Smo-dependent manner.

Src family kinase activity is required for Shh-mediated axon guidance: To test whether SFK activity is required for Shh-mediated axon guidance, SFK activity was inhibited using PP2. Neurons in a bath of PP2, exposed to a Shh gradient, did not turn in response to Shh, maintaining a straight trajectory (Figure 32). Quantitation of the angles turned showed that SFK inhibition by PP2 abolished turning, and the angles turned were not significantly different from the control (p=0.541) (Figures 33 and 34). In contrast, addition of PP3, a chemically related analog of PP2 which does not inhibit SFK activity, had no effect on Shh-mediated guidance, with axons still able to turn towards Shh (p=0.8742 compared with 0.1 μg/ml Shh gradient alone) (Figures 32 and 34). These results indicate that in commissural neurons, Shh activates SFKs, and that SFK activity is required for Shh-mediated axon guidance. This identifies SFKs as downstream mediators of Shh signalling in axon guidance.

To determine whether SFKs are required for canonical Shh-dependent transcriptional activity, the effect of SFK inhibition on the activity of a GIi transcriptional reporter was tested. SFK inhibition by PP2 did not affect Shh induction of Gli-luciferase reporter activity (Figure 35), even at concentrations higher than that used for inhibition of Shh-mediated axon turning. As a control, Smo inhibition with SANT-I did prevent induction of Gli-luciferase reporter activity (Figure 35), consistent with the requirement for Smo in canonical Shh signaling. This suggests that SFKs do not play a role in canonical Shh signaling, consistent with the lack of canonical Shh signaling- related phenotypes in SFK mutant mice. Therefore, SFKs appear to act downstream of Shh in axon guidance, but not in canonical Shh signaling to the Gli-family of transcription factors.

³H-uridine transcriptional assay: To measure the effect of transcriptional inhibitors on RNA synthesis, primary commissural neuron cultures (75 000 cells) were incubated with various concentrations of DRB and actinomycin D for 40 minutes. [5,6-³H]- uridine (Perkin Elmer, Boston, MA) was added for 1 h at 37 °C. The cells were then washed three times on ice with cold Neurobasal supplemented with 0.5 mM non- radioactive uridine (Sigma, St. Louis, MO). The cells were lysed with 0.5 % sodium dodecyl sulfate (SDS), 10 mM EDTA in 50 mM Tris-HCl buffer pH 7.1, and macromolecules precipitated with an equal volume of 10% trichoroacetic acid (TCA) for 1 h on ice. The samples were centrifuged at 15 000 rpm for 15 minutes at 4 °C, and the pellets washed two times with 5% cold TCA. The pellets were resuspended in 0.1 N NaOH, mixed with scintillation fluid, and the incorporated radioactivity measured with a scintillation counter.

Western blotting: Cells were lysed in RIPA buffer (5OmM HEPES pH 7.4; 150 mM NaCl; 10% glycerol; 1.5 mM MgCl₂; 1% Triton, 1% SDS; 1 mM EDTA) and boiled in SDS sample buffer for 5 minutes. Protein samples were separated by SDS-PAGE and transferred to PVDF. The primary antibodies rabbit (polyclonal) anti-Src (pY⁴¹⁸) phosphospecific antibody (Biosource, Camarillo, CA) and anti-actin antibody (Sigma, St. Louis, MO) were used. Secondary antibodies were conjugated to horseradish peroxidase and western blots were visualized with chemiluminescence.

In vitro kinase assay: In vitro kinase assays were performed on primary commissural neuron cultures (6 x 10⁶ cells) -45 h after plating. Neurons were treated with Shh in the presence or absence of SANT-I and cells were then lysed. Src or Fyn was immunoprecipitated in IP buffer (50 niM Tris pH 8.0; 150 mM NaCl; 5 niM EDTA; 0.1% NP40) for 2 hours at 4°C. Src or Fyn kinase activity in the immunoprecipitates was measured with a Src kinase assay kit (17-131), which measures incorporation of [γ-³²P]ATP into a substrate peptide, according to the manufacturer's instructions (Upstate, Lake Placid, NY).

Luciferase assay: C3H 10T1/2 cells stably transfected with a Gli-luciferase reporter were cultured in DMEM supplemented with 10% fetal bovine serum and Pen/Strep, and seeded at 75 000 cells per well in a 24-well dish. 48 h later, the cells were pre- treated with PP2, PP3 or SANT-I for 45 minutes and stimulated with 10 nM Shh. 20 h later, the cells were lysed and luciferase activity measured.

With the apparatus, method and system of an embodiment of the present invention, many neurons could be imaged in parallel responding to a gradient in a short time period, and multiple conditions or treatments could be performed on the same day (>20 neurons per condition), a higher throughput than is usually achieved with existing assays and under more defined and controlled gradient conditions. Also, the turning of axons in response to the gradient could be measured, hence acute responses to Shh in a quantitative biological assay could be studied. Furthermore, the apparatus, method and system of an embodiment of the present invention does not require fabrication of microfluidic or micro/nanotechnology devices, making it easily accessible to researchers. However, it will be appreciated that the present apparatus, system and method adapted to be on include microfluidic or micro/nanotechnology aspects is also covered by the scope of the present invention.

In the example above, it was found that commissural neurons responded to a Shh gradient over a wide range of incident angles and were able to turn up Shh gradients with a slope between 1-10% per 10 μm, within the range of slopes generated by the pipette assay of 5-10% per 10 μm. This apparatus, method and system is also broadly applicable to mammalian neurons allowing the study of specific neuron types, such as those guided by Shh, in contrast to using a mixed population of Xenopus spinal neurons and should be widely applicable to the study of other neuronal types and other guidance molecules.

Using an embodiment of the present invention with primary commissural neuron cultures, it was demonstrated that Shh-mediated axon guidance does not require transcriptional activity, and that there exists a novel transcription-independent Shh signalling pathway that mediates axon guidance, which is proposed to act locally at the growth cone. This contrasts with the canonical mechanism of Shh in specifying cell fate, where binding of Shh to Ptc and Boc/Cdo activates Smo and leads to activation of the GH family of transcription factors and Gli-dependent transcription, resulting in global changes in the cell. In axon guidance, Shh acts by binding to its receptors Boc and Ptc, leading to activation of Smo. Smo activation is required for activation of Src and Fyn, which it is hypothesized induces changes in the growth cone cytoskeleton and turning of the axon up the Shh gradient. Through this novel pathway, Shh gradients can elicit a rapid and spatially polarized response within the growth cone. It was found that Shh-induced SFK activity required Smo activity. Smo has recently been shown to recruit and signal through β-arrestin which itself has been shown to act as a scaffold to recruit SFKs and induce their signalling. Thus, it is possible that Shh-induced activation of SFKs is mediated through β-arrestin.

Interestingly, similar concentrations of Shh elicit axon guidance and cell fate specification. In the above example, the Shh concentration in the outer well that gives the most robust turning response is 5 nM (0.1 μg/ml). At this concentration, -80 % of neurons in the apparatus are exposed to Shh concentrations between 0.5-4.5 nM. This is very similar to the optimal concentration range for inducing neural progenitor differentiation, which ranges from 0.5 nM (for VO interneuron induction) to 4 nM (for V3 interneuron induction). Thus, although the canonical and the non-canonical Shh signalling pathways use different signal transduction mechanisms, they can be activated by similar concentrations of Shh.

It was shown that members of this novel non-canonical Shh pathway are the SFKs, Src and Fyn, which are rapidly activated by Shh and are required for Shh-mediated axon guidance. Interestingly, screens for genes involved in vertebrate and invertebrate canonical Hh signalling, using Hh-responsive transcriptional reporters, have neither identified Src nor Fyn as components of the canonical Hh signalling pathway. That SFKs have not been identified by these screens as acting downstream of Shh is consistent with our data and probably reflects the assays which have been used to study Shh signaling, which are transcriptional reporter assays performed over 24-48 h. Together with the lack of canonical Shh signalling-related phenotypes in SFK mutant mice, this suggests that SFKs do not play a role in canonical Shh signaling. Therefore, SFKs appear to act downstream of Shh in axon guidance, but not in canonical Shh signalling.

SFKs can regulate axon guidance by stimulating cytoskeletal rearrangements and filopodia dynamics. Interestingly, SFKs are also required for netrin-1 guidance of commissural axons. That SFKs are required for commissural axon guidance by both

Shh and netrin-1 suggests that the signalling of these two different guidance cues may use common mechanisms to link their signalling to the cytoskeleton and converge at the level of SFKs. Such convergence points might be important to allow growth cones to integrate multiple guidance cues.

In addition to commissural axon guidance, Hedgehog (Hh) signaling also plays important roles in other motility processes such as Drosophila germ cell migration, oligodendrocyte precursor migration, and guidance of retinal ganglion cell axons. Although all these processes require Smo, they are unlikely to be explained by transcriptional effects of the Hh pathway and therefore might be mediated by a non- canonical Hh signalling pathway, possibly requiring SFK activity.

It has also been shown that DRG and commissural neurons can respond to guidance cues in the apparatus 10 of an embodiment of the present invention, thus showing that the apparatus, system and method of the present invention is versatile and can be used to study many neuron types and many guidance cues.

The present invention may be embodied in other specific forms without departing from its essential attributes as defined in the appended claims and other statements of invention herein. For example, embodiments of the apparatus, system and method of the invention may be used to study the behaviour of cells other than neurons. Also, the chemical forming the concentration gradient may not be a chemoattractant or guidance cue but have any other function on the cell being tested, whether that function is stimulating or otherwise.

Claims

1. An apparatus for observing behaviour of a cell in response to a chemical substance, the apparatus having: a chamber in which can be formed a concentration gradient of the chemical substance, and a means arranged to moveably attach the cell to the apparatus to expose at least a free end of the cell to the chemical substance in the chamber, the free end being moveable.

2. An apparatus according to claim 1 , wherein the means is a plate which can be placed over an open end of the chamber and to which the cell can be moveably attached for contacting the concentration gradient.

3. An apparatus according to claim 1, wherein the means is a wall of the chamber to which the cell can be moveably attached for contacting the concentration gradient.

4. An apparatus according to any one of claims 1 to 3, wherein the means is transparent or translucent for observing movement of the free end of the cell in the chamber.

5. An apparatus according to any one of claims 1 to 4, wherein the chamber comprises a first and second well in fluid communication with one another across a bridge region, the concentration gradient being formed in the bridge region in use.

6. An apparatus according to claim 5, wherein the first and second wells are concentric annular wells, the first well being an outer well and the second well being an inner well.

7. An apparatus according to claim 6, wherein the first well has an outer wall which is deeper than an outer wall of the second well.

8. An apparatus according to any one of claims 1 to 7, wherein the cell is a mammalian cell.

9. An apparatus according to claim 8, wherein the mammalian cell is a neuron and the chemical substance is a guidance cue.

10. An apparatus according to any one of claims 1 to 9, further comprising a temperature control means for controlling the temperature of the concentration gradient in the chamber.

11. An apparatus for observing behaviour of a cell in response to a chemical substance, the apparatus having: a first well and a second well in fluid communication with one another across a bridge region, a concentration gradient of the chemical substance being formed in the bridge region in use, and a means arranged to moveably attach the cell to the apparatus to expose at least a free end of the cell to the concentration gradient in the bridge region, the free end being moveable.

12. An apparatus according to claim 11, wherein the first and second wells are concentric annular wells formed in a support.

13. An apparatus according to claim 12, wherein the first well has an outer wall which is deeper than an outer wall of the inner well.

14. An apparatus according to claim 12 or claim 13, wherein the means is a plate which can be placed over an open end of the first and second wells in the support and to which the cell can be moveably attached for contacting the concentration gradient.

15. An apparatus according to claim 14, wherein the means is transparent or translucent for observing movement of the free end of the cell in the concentration gradient.

16. An apparatus according to any one of claims 11 to 15, wherein the cell is a mammalian cell.

17. An apparatus according to claim 16, wherein the mammalian cell is a neuron and the chemical substance is a guidance cue.

18. An apparatus according to any one of claims 11 to 17, further comprising a temperature control means for controlling the temperature of the concentration gradient in the chamber.

19. A method for observing behaviour of a cell in response to a chemical substance, the method comprising: moveably attaching a cell to an apparatus comprising a chamber for housing a concentration gradient of the chemical substance such that a free end of the cell, which is moveable, is in contact with the concentration gradient in use; forming the concentration gradient of the chemical substance in the chamber; and contacting at least the free end of the cell with the concentration gradient of the chemical substance.

20. A method according to claim 19, wherein the concentration gradient of the chemical substance is formed in the chamber before or after moveably attaching the cell to the apparatus.

21. A method according to claims 19 or claim 20, wherein the movement of the free end is a turning of the cell free end, a growth of the free end or a direction of growth of the free end.

22. A method according to any one of claims 19 to 21, further comprising controlling the temperature of the concentration gradient.

23. A method according to claim 22, wherein controlling the temperature comprises maintaining the temperature at 37°C.

24. A method according to any one of claims 19 to 23, further comprising detecting a response of the cell to the concentration gradient.

25. A method according to claim 24, wherein the detecting of the response comprises observing the movement of the free end by optical microscopy.

26. A method according to claim 24 or claim 25, wherein the detecting of the response comprises observing the movement of the free end along a length of the cell.

27. A method according to any one of claims 19 to 26, further comprising capturing images of the movement of the free end.

28. A method according to claim 27, wherein the images are captured as a series of images as a function of time.

29. A method according to claim 27 or claim 28, wherein the capturing of images of the movement of the free end is initiated at least on contact of the free end with the concentration gradient.

30. A method according to any one of claims 27 to 29, wherein the images of the movement of the free end of a plurality of cells in contact with the concentration gradient are captured simultaneously.

31. A method according to any one of claims 19 to 30, further comprising quantifying the movement of the free end of the cell by tracking the relative co-ordinates of the free end with time.

32. A method according to any one claims 19 to 30, wherein the cell is a mammalian cell.

33. A method according to claim 32, wherein the mammalian cell is a neuron and the chemical substance is a guidance cue.

34. A method according to any one of claims 19 to 33, wherein the cell has an anchor portion for remaining attached to the apparatus and a dynamic portion having the free end, the method further comprising allowing the dynamic portion of the cell to grow out before contacting the free end with the concentration gradient.

35. A method according to any one of claims 19 to 34, further comprising adding a compound to the concentration gradient or to the cell to assess whether the compound alters the cell behaviour or a function of the chemical substance of the concentration gradient.

36. A method according to claim 35, wherein the compound is added to the concentration gradient or to the cell before, during or after contacting the cell with the concentration gradient.

37. A method according to claim 35 or claim 36, wherein the compound comprises a plurality of compounds which are added sequentially to the concentration gradient or cell, or added substantially simultaneously to the concentration gradient or cell, before during or after contacting the cell with the concentration gradient.

38. A method for identifying a compound that affects neuron cell behaviour, the method comprising: moveably attaching a neuron cell to an apparatus comprising a chamber for housing a concentration gradient of the compound such that a free end of the cell, which is moveable, is in contact with the concentration gradient in use; forming the concentration gradient of the compound in the chamber; and contacting at least the free end of the cell with the concentration gradient of the compound.

39. A method according to claim 38, further comprising detecting a movement of the free end of the neuron cell in response to contacting the concentration gradient.

40. A method according to claim 39, further comprising quantifying the movement of the free end of the neuron cell in response to contacting the concentration gradient.

41. A method for identifying a candidate compound that affects a function of a guidance cue or a neuron cell reaction to a guidance cue, the method comprising:

moveably attaching a neuron cell to an apparatus comprising a chamber for housing a concentration gradient of the guidance cue such that a free end of the neuron cell, which is moveable, is in contact with the guidance cue concentration gradient in use; forming the guidance cue concentration gradient in the chamber; contacting at least the free end of the neuron cell with the guidance cue concentration gradient; and contacting the neuron cell or the guidance cue concentration gradient with the candidate compound.

42. A method according to claim 41, further comprising detecting a movement of the free end of the neuron cell in response to contacting the concentration gradient.

43. A method according to claim 42, further comprising quantifying the movement of the free end of the neuron cell in response to contacting the concentration gradient.

44. A method according to any one of claims 41 to 43, wherein the neuron cell is contacted with the candidate compound before, during or after contacting the guidance cue concentration.

45. A method according to any one of claims 41 to 44, wherein the candidate compound is added to the guidance cue concentration gradient before, during or after the free end of the neuron cell is contacting the guidance cue concentration gradient.

46. A method according to any one of claims 41 to 45, wherein there is provided a plurality of candidate compounds which contact the guidance cue concentration gradient or the neuron cell sequentially or substantially simultaneously.

47. A system for observing behaviour of a cell in response to a chemical substance, the system including an apparatus according to any one of claims 1 to 10 or 11 to 18, an imaging apparatus for observing the movement of the free end of the cell and an image capturing apparatus for capturing images of the movement of the free end of the cell.

48. A system according to claim 47, further comprising a processor for processing the captured images.

49. Use of the apparatus of any one of claims 1 to 10 or 11 to 18 for identifying a first compound that affects cell behaviour.

50. A use according to claim 49, wherein the cell is a neuron.

51. A use according to claim 49 or claim 50 for identifying a second compound that affects a function of the first compound or the function of the cell.