WO1993015184A1

WO1993015184A1 - Assay for neuro-excitatory agents

Info

Publication number: WO1993015184A1
Application number: PCT/US1993/000302
Authority: WO
Inventors: Richard H. Selinfreund
Original assignee: Yale University
Priority date: 1992-01-24
Filing date: 1993-01-25
Publication date: 1993-08-05
Also published as: AU3471793A

Abstract

An assay for identifying neuro-excitatory agents is provided. The assay comprises suppressing the electrical activity of neuronal cells by incubating the cells in a medium having a reduced concentration of serum. The electrically quiescent cells thus produced are then used to screen candidate materials. Neuro-excitatory agents comprise those materials which are able to restore electrical activity to the quiescent cells. Using the method, a pool of growth factors and a family of insulin-like growth factors have been identified as neuro-excitatory agents.

Description

Assay for Neuro-Excitatorv Agents

Technical Field

This invention relates to an assay for identifying materials which can restore electrical activity to neuronal cells which have become electrically quiescent. It also relates to a class of proteins which can be used to reactivate electrically quiescent cells.

Background Art

The electrical activity of neuronal cells is critical to, among other things, mental functioning and the control of muscle movement. In particular, by means of action potentials, neuronal cells communicate with and carry information to other neuronal cells, muscle cells, and glands.

In certain disease states and as a result of trauma or aging, neuronal cells can lose the ability to produce action potentials. Such a loss occurs, for example, in stroke, Alzheimer's disease, Parkinson's disease, and various neuropathies including Bell's palsy. As a result of these and similar diseases, there is a long felt and continuing need to identify materials which can restore action potentials to electrically quiescent cells. Such materials can serve as pharmaceutical agents for diseases of the foregoing types, as well as being used to maintain the electrical activity of neuronal cells employed in experiments designed to study the electrophysiology of the nervous system. For ease of reference, materials of these types shall be referred to herein as "neuro-excitatory agents," i.e., a neuro-excitatory agent is a material having the ability to restore action potential activity to electrically quiescent neuronal cells.

In the past, the process of screening materials to determine their effects on the nervous system has been time consuming, tedious, and expensive. For example, whole animal studies have been performed in which candidate materials have been injected into rats and the electrical activity of the rat's brain has been monitored using EEG electrodes. Alternatively, the rat's behavior after drug administration has been monitored.

Sections of spinal cord and/or brain have also been used to perform drug screenings. In these cases, either the gross electrical activity of the section or the activity of individual neurons has been monitored over time as candidate materials are added and/or removed from the solution bathing the section.

Single cell preparation have also been used in which electrodes have been introduced into a cell to assess action potential activity in the presence and absence of drugs. In addition, single cell preparations have been employed to assess the effects of drugs on the conductivity of the entire neuron or of individual ion channels by clamping the cell's transmembrane potential at particular values.

A variety of neurological drugs are known in the art. Significantly, most of these drugs act by suppressing the activity of neuronal cells rather than stimulating such activity. Accordingly, these drugs are of minimal value in treating the most common forms of neural disease, e.g., stroke, Alzheimer's disease, aging, and the like, all of which involve decreased neuronal activity.

Electrophysiological studies have been preformed in which the effects on ionic currents in normal neurons of various hormones, some of which have known growth factor activity, have been studied. See, for example, Peppelenbosch et al., 1991; Levitan and Kramer, 1990; Nussinovitch, 1988; and Dubinsky and Oxford, 1985. In general, these studies have shown that different hormones modulate different ionic currents and that their effects vary between cell types. None of this work has disclosed or suggested that proteins having growth factor activity can be used to restore electrical activity to quiescent cells. The process of serum arrest has been used in the past to control the proliferation of cells grown in tissue culture. See, for example, Baserga, 1985. Specifically, it has been found that cell division can be arrested by reducing the level of serum in the medium surrounding the cells to a level in the range 0 for example, 0.05 to 0.1 percent by volume. See Schubert et al., 1971. The understanding in the art is that the arrest of cell division occurs because of the removal f. m the medium of growth factors normally contributed by the serum. As with the studies on hormones, none of the serum arrest work has disclosed or suggested that proteins having growth factor activity can restore the electrical activity of quiescent neuronal cells.

Disclosure of the Invention

In view of the foregoing state of the art, it is an object of this invention to provide an improved assay for identifying neuro-excitatory agents, and optionally for also identifying agents which can block the activation of action potential firing in neuronal cells. More particularly, it is an object of the invention to provide an assay technique which is simpler to use than prior techniques and which can be performed rapidly using conventionally available equipment. It is also an object of the invention to provide a technique which gives a quantitative measure of the neuro-excitatory efficacy of tested materials. It is a further object of the invention to provide an assay wherein a neuro-excitatory agent and/or the agent which can block the activation potential firing in enuronal cells, can be tested against a range of neuronal cells of different types.

A further object of the invention is to provide methods and compositions for restoring electrical activity to neuronal cells which have lost such activity. In particular, it is an object of the invention to achieve such restoration using naturally-occurring compounds which can be accepted by the body without substantial i munological reaction.

To achieve the foregoing and other objects, the invention in accordance with certain of its aspects provides an assay for determining the neuro-excitatory effect of a selected material comprising the steps of:

(a) providing a neuronal cell in a medium containing a level of serum such that the cell is electrically quiescent;

(b) introducing the selected material into the medium; and

(c) monitoring the electrical activity of the neuronal cell the presence of the selected material.

In accordance with others of its aspects, the invention provides a method for restoring electrical activity to neuronal cells which have lost such activity comprising exposing the cells to selected proteins which have the ability to stimulate cell growth, i.e., by exposing the cells to selected growth factors. The growth factors are selected so that in the presence of the growth factors, the neuronal cells exhibit at least the following ionic currents: sodium or calcium current and potassium current.

In certain preferred embodiments of the invention, the selected growth factors comprise the combination of epidermal growth factor, platelet derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, nerve growth factor, transforming growth factor, S1003 dimer, insulin, insulin-like growth factor I, and insulin-like growth factor II. In other preferred embodiments, the selected growth factors comprise insulin, insulin-like growth factor I, and insulin-like growth factor II.

The accompanying figures, which are incorporated in and constitute part of the specification, illustrate preferred embodiments of the invention, and together with the description, serve to explain the principles of the invention. It is to be understood, of course, that both the figures and the description are explanatory only and are not restrictive of the invention.

Brief Description of the Drawings

Figure 1 shows action potential activity of neuronal cells when incubated in 10% fetal calf serum (Figure IA) , 0.1% fetal calf serum (Figure IB) , after reincubation in 10% fetal calf serum (Figure 1C) , and after incubation with a family of insulin growth factors (Figure ID) .

Figure 2 shows action potential activity of neuronal cells incubated in 0.1% fetal calf serum before and after addition of a pool of growth factors (Figures 2A and 2B, respectively) .

Figure 3 shows single ion channel activity in a patch of membrane on a neuronal cell incubated in 0.1% fetal calf serum before, during, and after addition of a pool of growth factors.

Figure 4 shows whole cell ionic currents for a neuronal cell incubated in 10% fetal calf serum (Figure 4A) and 0.1% fetal calf serum (Figure 4B) .

Best Mode for Carrying Out the Invention

As discussed above, the present invention relates to an assay for identifying neuro-excitatory agents. In the discussion of the invention, the following terms will be used:

1) "neuronal cell" means any neuron or neuron-like cell which is capable of producing an action potential and thus includes electrically active neurons, electrically active gland cells developmentally derived from the embryonic neural tube, and cell lines derived from neurons or gland cells developmentally derived from the embryonic neural tube;

2) an "electrically active cell" is a cell which can produce action potentials;

3) an "electrically quiescent cell" is a cell which either cannot produce any action potentials or which can produce action potentials only at a substantially subnormal rate and/or under substantially supernormal stimulatory conditions;

4) "serum" has its conventional meaning, i.e., the clear liquid which, in the clotting of blood, separates from the clot and the corpuscles. Since fetal calf serum is normally used in electrophysiological preparations, the serum levels which are controlled in the practice of the invention are typically fetal calf serum levels.

The assay of the invention employs neuronal cells which in their normal state produce action potentials. Preferably, the cells are of a type which can be grown in tissue culture. Various neuronal cells described in the literature can be used in the practice of the invention. A list of immortalized neuronal cells which can be used, along with their accession numbers from the American Type Culture Collection (Bethesda, Maryland) , are set forth in Table 1. In addition to these immortalized cells, primary cell lines can be established directly from, tissue samples, e.g. , samples taken from spinal cord or brain. See, for example, Bottenstein, 1983, and Crain and Peterson, 1964.

When it is desired to screen a material against neuronal. cells of different types, a bank of cells is used in the practice of the invention. Such a bank, for example, can include central neurons, peripheral neurons, and neuroblast cells.

Prior to their use in the assay, the neuronal cells are maintained in a suitable growth medium. Various such media are known in the art. In the examples discussed below, an α-modified essential medium containing 10% by volume of fetal calf serum was used.

In accordance with the invention, it has been surprisingly found that neuronal cells when incubated in a medium containing a low level of serum lose their electrical activity, but still remain alive. Moreover, it has been found that the application of neuro-excitatory agents to the dormant cells restores their electrical activity. Thus, the removal of serum produces a reversible change in the electrical activity of neuronal cells. Because the change is reversible, the quiescent cells can be used to determine whether or not a material of interest, e.g., a drug, exhibits neuro-excitatory activity.

The serum level needed to achieve a quiescent state is generally below about 0.1 percent by volume, although the exact level will vary somewhat with cell type. For example, lower levels of serum, e.g., 0.05 percent by volume, may be needed to suppress electrical activity of highly transformed cells. A suitable serum level for any particular preparation can be readily determined by persons of ordinary skill in the art from the disclosure herein by monitoring the electrical activity of the neuronal cells until the desired suppression of electrical activity is achieved.

The serum level should in general not be reduced to zero in order to ensure continued viability of the neuronal cells. Serum levels in the 0.05 to 0.1 percent by volume range are generally sufficient for this purpose. Alternatively, defined media can be used instead of serum to maintain cell life. Examples of such defined media include N-2 supplement and G-5 supplement (Gibco BRL, Grand Island, New York) .

Before assays for neuro-excitatory activity are performed, the neuronal cells must be incubated in the low serum medium for a time sufficient to suppress their electrical activity. Similarly, the assay must be performed before the cells lose their viability. For example, for pituitary GH4/C1 cells, it has been found that the beginning of the assay window is about 16 hours after exposure to the low serum medium and the end of the assay window is about 36 hours after exposure, thus giving an operating window of 20 hours which is more than sufficient to perform extensive screening of candidate materials. Similar windows for other cell types can be readily determined from the disclosure herein by monitoring the electrical activity of the cells after exposure to the low serum medium, and determining the times when restoration of electrical activity can still be achieved using, for example, the neuro-excitatory preparations disclosed herein.

The candidate materials which are to be tested for neuro-excitatory activity are added to the low serum medium using conventional sterile techniques such as pipetting. Generally, an incubation period is needed to allow diffusion and interaction of the candidate material with the test cells. Depending upon the material, the incubation can take less than a minute or several hours.

The electrical activity of the cells is monitored during the testing process. Most conveniently this is done by recording transmembrane potentials using standard intracellular recording techniques or by using whole cell patch clamp techniques in the current clamp mode. See Hamill et al. , 1981.

In a preferred embodiment, the method of detecting the action potential firing rate is changed, specifically from monitoring by standard electrophysiological methods to monitoring by voltage-sensitive dyes. In this technique, dye that is retained in the outer bilayer of the membrane and allows for optical measurements of transmembrane voltage changes in living cells can be used. Di-8-ANEPPS, which is a styrypnaphthal dye (i.e., a fluorescent probe) with a fast response time and that can be detected using a fluorescence microscope of plate reader, is illustrative of the dyes which can be used.

This embodiment may be carried out in accordance with the following illustrative protocol or a variation thereof. To incorporate the dye into the cells, electrically quiescent cells (GH4C1 pituitary cells) may be incubated for 1 minute with 40 μl of 0.1 mg/ml Di-8-ANEPPS diluted into 5 ml of extracellular buffer (NaCl, 150 mM; KC1, 4 mM; CaCl₂, 2 mM; MgCl₂, 1.3 mM, glucose, 10 mM; Na-HEPES, 5 mM; pH 7.2). Cells are then incubated with 20 ng/ml IGF-I, serum, or any factor or combination of factors that reactivate action potential firing in neuronal cells. Change in electrical activity is monitored through the change in fluorescence intensity using a Zeiss LSM (Laser Scanning Microscope) . However, any optical instrument capable of detecting changing fluorescence such as a standard fluorescence microscope or fluorescent plate reader can be used.

In practice, transmembrane potentials for a number of cells are recorded in the low serum medium prior to addition of the candidate material. The material is then added and the monitoring process is continued. For materials which have neuro-excitatory activity and which act relatively quickly, a change in electrical activity of the original set of cells is seen. Since the monitoring process of a given cell generally results in cell death in about 30 minutes, if a candidate material does not show activity within about 10-30 minutes, a second or if needed further sets of cells are monitored for later time periods until a conclusion is reached regarding the neuro-excitatory activity of the candidate material. Quantitative measures of neuro-excitatory activity can be obtained in this way by recording the frequency. of action potential generation and comparing that frequency with the action potential frequencies of normal neuronal cells and cells maintained in low levels of serum.

Using the foregoing method, a new class of neuro-excitatory agents has been identified. This class comprises proteins which 1) have growth factor activity, i.e., they will induce cellular division in at least some cell types, and 2) when applied to quiescent neuronal cells they cause such cells to exhibit at least sodium or calcium current and potassium current. Preferably, for pharmaceutical applications, the proteins should be naturally occurring in mammalian nervous tissue and most preferably in human nervous tissue.

As demonstrated by the examples presented below, members of this class of proteins include: 1) the combination of the growth factors epidermal growth factor (EGF) , platelet derived growth factor (PDGF) , acidic fibroblast growth factor (aFGF) , basic fibroblast growth factor (BFGF) , nerve growth factor (NGF) , transforming growth factor (TGF) , SlOO3 di er, insulin, insulin-like growth factor I (IGF-I) , and insulin-like growth factor II (IGF-II) , and 2) the combination of the growth factors insulin, insulin-like growth factor I, and insulin-like growth factor II.

As used herein the term "epidermal growth factor" is intended to include naturally occurring EGF, EGFα, and mixtures thereof (e.g., a 1:1 mixture of EGF and EGFα as used in the examples) , the term "platelet derived growth factor" is intended to include naturally occurring PDGF, PDGFAA, PDGFBB, PDGFAB, and mixtures thereof (e.g., a 1:1:1 mixture of PDGFAA, PDGFBB, and PDGFAB as used in the examples) , the term "nerve growth factor" is intended to include naturally occurring NGF, the various isolated forms thereof, and mixtures thereof (e.g., a 1:1 mixture of the 2.5S and 7.OS forms used in the examples) , and the term "transforming growth factor" is intended to include naturally occurring TGF, the various isolated forms thereof, and mixtures thereof (e.g., TGF-jS_x used in the examples) .

Processes for isolating and purifying these growth factors are known in the literature, and the factors are available as commercial products. Table 2 sets forth sources for the factors and citations to suitable techniques for their isolation and purification. The relevant portions of these references are incorporated herein by reference. With regard to S1003, the commercially available material is the monomer form of the protein. The biologically active dimer can be produced using the procedure set forth in Selinfreund et al., 1991, the relevant portions of which are also incorporated herein by reference.

When used to restore electrical activity to quiescent neuronal cells, the foregoing growth factors are combined in a physiological buffer at concentrations in the range of, for example, from about 0.5 to about 50.0 nanograms per milliliter for each of the factors. Lower concentrations can be used, if desired, but in general the effects of the agents will be diminished.

Among other applications, such growth factor preparations can be used as controls for the above assay for neuro-excitatory agents, i.e., the preparations allow the user to calibrate and standardize the assay prior to its use in identifying other neuro-excitatory materials.

The growth factor preparations can also be used pharmacologically. For example, the preparations can be administered to electrically quiescent cells in animals or humans using techniques of the type employed for administering drugs to neural tissues. Thus, cannula implantation, as used in cancer therapy, can be employed to administer the preparations to localized regions of the brain. For spinal neurons, the preparations can be introduced directly into the spinal fluid. Alternatively, the growth factors can be introduced using genetic engineering techniques such as gene transfer or by coupling the factors to carrier molecules which transport the factor across barrier membranes.

In another embodiment, the present invention provides a method for rapidly screening for agents capable of blocking the activation of action potential firing in neuronal cells (e.g. central nervous system-derived cells (pituitary GH4/CI cells) ) . This is achieved by employing a modification of the present method for assessing the ability of a material to electrically excite neuronal cells, in which the agent being screened for blocking activity is added to the electrically quiescent neuronal cells before a material known to electrically excites the quiescent neuronal cells is added thereto. More particularly, a neuronal cell is incubated in a medium containing a level of serum such that the cell becomes electrically quiescent. The electrically quiescent neuronal cell is then exposed to (a blocking amount) of the agent being screened for the ability to block the activation of action potential firing in the neuronal cell. This is followed by exposing the electrically quiescent neuronal cell to a material known to electrically excite neuronal cells, (e.g. as determined in accordance with the present invention) and the electrical activity of the neuronal cell is then monitored. Failure to detect electrical excitation of the neuronal cells is indicative of the agent's ability to block the activation of action potential firing.

Without intending to limit it in any manner, the present invention will be more fully described by the following examples. The materials and methods which are common to the examples are as follows.

Materials and Methods

GH4/C1 pituitary cells obtained from the Department of Pharmacology, Yale University, New Haven, Connecticut, were used to perform the experiments. Prior to use, the cells were synchronized by itotic shake. See Terasima and Tolmach, 1963.

After synchronization, the cells were grown for 16-22 hours in α-modified essential medium (α-MEM) containing 10% fetal calf serum at 37°C in a C0₂ incubator at 95%C0₂/5%0₂.

Quiescent cells were obtained by further incubation for 16 to 35 hours in a low serum medium comprising α-MEM containing 0.1% fetal calf serum. The same incubation conditions were used as for the initial growth procedure.

Growth factor stock solutions were prepared by mixing freeze dried protein with sterile water. The stock solutions were combined with α-MEM containing 0.1% fetal calf serum or were combined with electrophysiological extracellular assay saline (EEAS) comprising (in mM) 150, NaCl; 5, KC1; 2, CaCl₂; 1.3, MgCl₂; 10, glucose; 5, HEPES: pH 7.2. The final concentrations of the growth factors in the α-MEM and EEAS solutions are set forth in Table 3 for the pooled growth factor and the insulin family preparations.

The growth factor/α-MEM solution was used to test long term exposure of the quiescent cells to the growth factors; measurement of the electrical activity of the cells after exposure to growth factor was performed by transferring the cells to EEAS. For studies examining short term exposure to growth factors, the electrical activity of the cells was first monitored in EEAS, the growth factors were then added to the EEAS to achieve the concentrations of Table 3, and the electrical activity of the same cells were again monitored for another 5-30 minutes.

The effects of the growth factors on electrical activity were determined using patch recording techniques. See Harrill et al., 1981. EEAS was the bathing saline and the patch electrode contained (in mM) 130, K-aspartate; 20, KC1; 10, glucose; 5, HEPES; pH 7.2.

Example I

This example demonstrates that the electrical activity of neuronal cells can be reduced by incubating the cells in a medium containing a low level of serum and restored by re-introducing the serum to the medium, i.e. , the viability of the cells is not destroyed by the treatment with a low serum medium and thus the cells can be used to assay neuro-excitatory agents.

GH4/C1 cells were maintained in α-MEM containing 10% fetal calf serum for 16-22 hours. Their electrical activity was recorded using the patch clamp technique in the whole cell configuration, current clamp mode. Seven cells were tested. A representative result for one of the cells is shown in Figure IA.

As shown therein, cells maintained under these conditions exhibited frequent action potentials. In particular, 6 of the 7 cells exhibited a firing rate of more than 10 action potentials per minute. The seventh cell exhibited no action potentials. The behavior of the cells was thus similar to that reported in Dubinsky and Oxford, 1985.

Another set of cells was maintained in α-MEM containing 10% fetal calf serum for 16-22 hours and then in α-MEM containing 0.1% fetal calf serum for another 16-20 hours. The electrical activity of these cells was again recorded using the patch clamp technique described above. Thirty-two cells were tested. A representative result for one of the 32 cells is shown in Figure IB.

As shown therein, most of the cells maintained under low serum conditions exhibited no action potentials. In particular, 27 of the 32 cells failed to show action potential activity. The remaining five cells exhibited activity at a rate of more than 10 action potentials per minute.

Although most of the cells exhibited no action potentials, the cells were still alive as evidenced by the fact that they exhibited essentially normal resting electrophysiological properties, namely, they had resting potentials similar to those previously reported for GH4/C1 cells in normal media: -47+8 V (mean+SD) for cells in 0.1% serum; -50+14 V for cells in full serum (see Dubinsky and Oxford, 1984) .

A third set of cells was maintained in α-MEM containing 10% fetal calf serum for 16-22 hours, then in α-MEM containing 0.1% fetal calf serum for another 16-20 hours, and then transferred to α-MEM containing 10% fetal calf serum for a final 1-2 hours. Electrical activity was assessed as before for three cells. Two of the three cells exhibited a high rate of spontaneous action potential activity (greater than 10 actiόn potentials per minute) . The third cell produced some action potentials but at a lower rate (1-2 action potentials per minute) .

A trace for one of the two cells which exhibited a high rate of action potential activity is shown in Figure 1C.

As this data demonstrates, cells can be made electrically quiescent without killing them by reducing the amount of serum in the bathing medium for a period on the order of at least about 16 hours and can have their electrical activity restored within a much shorter time, e.g., 1-2 hours, by increasing the serum level.

Example 2 This example demonstrates that the insulin family of growth factors, i.e., the combination of insulin, IGF-I, and IGF-II, is a neuro-excitatory agent.

GH4/C1 cells were incubated in α-MEM containing 10% fetal calf serum for 16-22 hours, then in α-MEM containing 0.1% fetal calf serum for another 16-20 hours, and finally in α-MEM containing the insulin family of growth factors at a concentration for each factor of 10 nanogram per milliliter for 1-8 hours. Electrical activity was assessed as in Example 1 for ten cells.

Six of the ten cells exhibited a high rate of spontaneous action potential activity (greater than 10 action potentials per minute) . Three cells produced action potentials at a somewhat lower rate (1-9 action potentials per minute) . One cell was inactive.

A trace for one of the six cells which exhibited a high rate of action potential activity is shown in Figure ID.

As shown by this data, the assay of the present invention establishes that the insulin family of growth factors is a neuro-excitatory agent. Example 3

This example demonstrates that a pool of growth factors can serve as a neuro-excitatory agent and that the agent is able to restore electrical activity to quiescent neuronal cells in a period of time of less than about 5 minutes.

GH4/C1 cells were incubated in α-MEM containing 10% fetal calf serum for 16-22 hours, then in α-MEM containing 0.1% fetal calf serum for another 16-20 hours.

For a first set of three cells, after the incubation in the low serum medium, a further incubation was performed for either 1 hour or 24 hours in the low serum medium to which was added pooled growth factors at the concentrations given in Table 3. Electrical activity was assessed as in Example 1. All three cells exhibited at least some spontaneous activity after addition of the growth factors (greater than 2 action potentials per minute) .

For a second set of twelve cells, electrical activity was monitored as in Example 1 immediately after incubation in the low serum medium. All cells were electrically quiescent at this stage of the experiment. While continuing to monitor electrical activity, the pooled growth factors were added to the EEAS surrounding the cells, again at the concentrations of Table 3. Three of the twelve cells became highly active within 5 minutes of the addition of the pooled factors, i.e., they produced action potentials at a rate of above 10 action potentials per minute. Within this short time period, the other nine cells remained quiescent.

Figure 2 shows before and after traces for one of the cells which recovered activity within the 5 minute test period. Figure 2A shows the relatively quiescent state before the addition of the pooled growth factors; the rate of spontaneous firing during this period was about 1 action potential per minute. Within one minute of exposure to the pooled factors, the spontaneous firing frequency began to increase. Figure 2B shows the action potential activity 1 minute and 20 seconds after the addition of the factors. As shown therein, the activity was about 10 action potentials per minute at this point in the experiment.

The firing frequency continued to rise over the succeeding minutes and by 8 minutes after addition, the cell was firing at an average rate of 14 action potentials per minute as well as producing a number of smaller spikes.

Additional experiments were performed in which single ion channel activity was assessed in the presence of the pooled factors. Ion channel activity was monitored using patch clamp techniques in the on-cell recording configuration, voltage clamp mode. See Hamill et al., 1981. The cells were grown and incubated as described above in connection with Figure 2.

Figure 3 shows ion channel activity in a patch of membrane before, during and after application of the pooled factors. The patch of membrane from which the recording was made was still on the cell and, because of the presence of the patch electrode, was not bathed in EEAS and therefore did not come into direct contact with the pooled factors. As shown in Figure 3, prior to application of the factors, little ion channel activity was observed. An artifact appears on the trace at the time of addition of the factors (see bar on trace) . Immediately after application of the factors, no increase in activity was observed. However, with 1 minute of the addition, channel activity increased dramatically. Activity remained high for the duration of the recording, i.e. , 30 minutes.

As shown by this data, the assay of the present invention establishes that a pool of growth factors operates as a neuro-excitatory agent. Moreover, the pool can affect activity in a period of less than 5 minutes. Example 4

This example demonstrates that neuronal cells held in a medium containing 0.1% fetal calf serum exhibit reduced calcium and potassium currents when compared to cells maintained in 10% fetal calf serum. Electrical activity of these cells can thus be restored by treating them with an agent which causes them to exhibit these reduced or missing currents.

In these experiments macroscopic ionic currents (also known as whole cell currents) were assessed. Such currents underlie action potentials and arise from voltage-dependent ion channel activity.

Voltage-activated ionic currents were elicited by voltage jumps and monitored using the patch clamp technique in the whole-cell configuration, voltage clamp mode. See Hamill et al. 1981. Specifically, data were monitored with a List EPC 7 amplifier (List Electronics, Darmstadt, Germany) , filtered at 1 kHz, and digitally sampled and recorded using an INDEC-modified IBM personal computer (INDEC Systems, Sunnyvale, California) , and FAST-LAB stimulus and acquisition software (INDEC Systems, Sunnyvale, California) .

A first set of GH4/C1 cells was maintained in α-MEM containing 10% fetal calf serum for 16-22 hours, and a second set in α-MEM containing 10% fetal calf serum for 116-22 hours followed by incubation in α-MEM containing 0.1% fetal calf serum for another 16-20 hours. Typical voltage-activated currents for cells maintained under these conditions are shown in Figures 4A and 4B, respectively.

For these cells, voltage-activated currents were elicited by stepping the membrane potential from -80 mV to -40 mV (trace 1) , -20 mV (trace 2) , or +10 mV (trace 3) . Non-specific leak current has not been subtracted from these data and therefore each trace reflects both the specific activation of ionic currents and the non-specific leak current. The large spikes at the beginning and end of each depolarizing pulse reflect the capacitive current that was transiently induced by the change in membrane voltage.

As shown in Figure 4A, the cells incubated in 10% fetal calf serum exhibited a combination of inward calcium and outward potassium currents. In contrast, as shown in Figure 4B, the cells incubated in 0.1% fetal calf serum had smaller voltage-activatable currents and, in particular, the inward calcium current which underlies the depolarizing phase of the action potential was strongly reduced. The slow potassium currents responsible for repolarizing the cell membrane were also strongly depressed.

As shown by this data, restoration of action potential activity for these cells depends on restoration of the calcium and potassium currents.

A variety of modifications which do not depart from the scope and spirit of the invention will be evident to persons of ordinary skill in the art from the disclosure herein. The following claims are intended to cover the specific embodiments set forth herein as well as such modifications, variations, and equivalents.

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Johnsson, A., Heldin, C. , Wasteson, A., Westermark, B., Deuel, T. F. , Huang, J. , Seeburg, P., Gray, A., Ullrich, A., Scrace, G. , Stroobant, P., and Waterfield, M. D. 1984. The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor. EMBO J. 3, 921-928.

Levitan, E. S. and R. H. Kramer. 1990. Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration. Nature 348, 545-547. McKechan, W. L. , Barnes, D., Reed, L. , Stanbridge, E. , Murakami, H. and Sato, G. 1990. Frontiers In Mammalian Cell Culture. In Vitro Cell Dev. Biol. 26,9-23.

Nussinovitch, I. 1988. Growth hormone releasing factor evokes rhythmic hyperpolarizing currents in rat anterior pituitary cells. J. Physiol. 395, 303-318.

Peppelenbosch, M. P., L. G. J. Tertoolen and S. W. de Laat. 1991. Epidermal growth factor-activated calcium and potassium channels. J. Biol. Chem. 266, 19938-19944.

Schubert, D. , Humphreys, S., deVitry, F. and Jacobs, F. 1971. Induced Differentiation Of A Neuroblastoma. Develop. Biol. 25, 514-546.

Selinfreund, R. H. , S. W. Barger, W. J. Pledger and L. J. Van Eldik. 1991. Neurotrophic protein SlOOjS stimulates glial cell, proliferation. Proc. Natl. Acad. Sci. USA 88, 3554-3558.

Smith, M. C. , Cook J. A., Furman, T. C. , Occolowitz, J. L. 1989. Structure and activity dependence of recombinant human insulin¬ like growth factor II on disulfide bond paring. J. Biol. Chem. 264, 9314-9321.

Sporn, M. B. and Roberts, A. B. 1988. Transforming growth factor-beta: new chemical forms and new biological roles. Biofactors 1, 89-93.

Terasi a, T. and L. J. Tolmach. 1963. Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp. Cell Res. 30, 344-362. TABLE 1

Source Designation ATCC No.

Human neuroblastoma cells SK-N-MC HTB-10 Mouse neuroblastoma cells Neuro 2A CCL-131 Neuroblastoma cells IMR-32 CCL-127 Neuroblastoma cells NB41A3 CCL-147 Human brain cells A172 CRL-246.1 Human brain cells HS683 HTB-138 Human brain cells H4 HTB-148 Human brain cells TE-671 HTB-139 Human brain cells T98G CRL-1690 Human brain cells U-87MG HTB-14 Human brain cells U-138MG HTB-16 Human brain cells U-373MG HTB-17 Human brain cells BT-20 HTB-19

TABLE 2

Citation

Cohen, 1987

Barnes and Sato, 1980

Betsholtz et al., 1986

Johnsson et al., 1984

Deuel, 1987

Burgess and Maciag, 1989

Abraham, et al. , 1986

Bocchini and Angeletti, 1969

Gage et al. , 1987

Sporn and Roberts, 1988

Selinfreund et al. , 1991

McKechan et al., 1990

Froesch et al., 1985

Smith et al., 1989

¹ Collaborative Research Inc. , Bedford, MA

² Genzyme, Inc., Boston, MA

³ Sigma Chemical Co., Saint Louis, MO

⁴ Monomer form; Upstate Biologicals Inc., Lake Placid, NY TABLE 3

Concentration

Factor (nq/ml)

EGF 20

EGFα 20

PDGFAA 15

PDGFBB 15

PDGFAB 15 aFGF 20 bFGF 20

2.5S-NGF 20

7.OS-NGF 20

S1003 50

Insulin 10

IGF-I 10

IGF-II 10

Claims

1. A method for assessing the ability of a material to electrically excite neuronal cells comprising the steps of:

(a) incubating a neuronal cell in a medium containing a level of serum such that the cell becomes electrically quiescent;

(b) exposing the electrically quiescent neuronal cell to the material; and

(c) monitoring the electrical activity of the neuronal cell in the presence of the material.

2. The method of Claim 1 wherein the level of serum in the medium is in the range from about 0.05 percent by volume to about 0.1 percent by volume.

3. The method of Claim 2 wherein the level of serum in the medium is about 0.1 percent by volume.

4. A method for producing living neuronal cells for use in screening neuro-excitatory agents comprising incubating the cells in a medium having a level of serum such that the cells become electrically quiescent.

5. The method of Claim 4 wherein the level of serum in the medium is in the range from about 0.05 percent by volume to about 0.1 percent by volume.

6. The method of Claim 5 wherein the level of serum in the medium is about 0.1 percent by volume.

7. The method of Claim 4 wherein the incubation is carried out for a period of time of at least about sixteen hours.

8. Living neuronal cells in an electrically quiescent state produced by the method of Claim 4.

9. A method for restoring electrical activity to neuronal cells which have lost such activity comprising exposing the neuronal cells to a protein-containing preparation which (a) has the ability to stimulate cell growth and (b) causes the neuronal cells to exhibit at least sodium or calcium current and potassium current.

10. The method of Claim 9 wherein the protein-containing preparation comprises insulin, insulin-like growth factor I, and insulin-like growth factor II.

11. The method of Claim 9 wherein the protein-containing preparation comprises epidermal growth factor, platelet derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, nerve growth factor, transforming growth factor, SI00/3 dimer, insulin, insulin-like growth factor I, and insulin¬ like growth factor II.

12. A method for restoring electrical activity to neuronal cells which have lost such activity comprising exposing the cells to insulin, insulin-like growth factor I, and insulin-like growth factor II.

13. A method for restoring electrical activity to neuronal cells which have lost such activity comprising exposing the cells to epidermal growth factor, platelet derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, nerve growth factor, transforming growth factor, S100/3 dimer, insulin, insulin-like growth factor I, and insulin-like growth factor II.

14. A neuro-excitatory agent comprising a mixture of isolated and purified insulin, isolated and purified insulin-like growth factor I, and isolated and purified insulin-like growth factor II.

15. The neuro-excitatory agent of Claim 14 in a physiological buffer wherein the concentration of isolated and purified insulin is about 10 nanograms/milliliter, the concentration of isolated and purified insulin-like growth factor I is about 10 nanograms/milliliter, and the concentration of isolated and purified insulin-like growth factor II is about 10 nanograms/milliliter.

16. A neuro-excitatory agent comprising a mixture of isolated and purified epidermal growth factor, isolated and purified platelet derived growth factor, isolated and purified acidic fibroblast growth factor, isolated and purified basic fibroblast growth factor, isolated and purified nerve growth factor, isolated and purified transforming growth factor, isolated and purified S100/3 dimer, isolated and purified insulin, isolated and purified insulin-like growth factor I, and isolated and purified insulin-like growth factor II.

17. The neuro-excitatory agent of Claim 16 in a physiological buffer wherein the concentration of isolated and purified epidermal growth factor is about 40 nanograms/milliliter, the concentration of isolated and purified platelet derived growth factor is about 45 nanograms/milliliter, the concentration of isolated and purified acidic fibroblast growth factor is about 20 nanograms/milliliter, the concentration of isolated and purified basic fibroblast growth factor is about 20 nanograms/milliliter, the concentration of isolated and purified nerve growth factor is about 40 nanograms/milliliter, the concentration of isolated and purified transforming growth factor is about 10 nanograms/milliliter, the concentration of isolated and purified S100/3 dimer is about 50 nanograms/milliliter, the concentration of isolated and purified insulin is about 10 nanograms/milliliter, the concentration of isolated and purified insulin-like growth factor I is about 10 nanograms/milliliter, and the concentration of isolated and purified insulin-like growth factor II is about 10 nanograms/milliliter.

18. A method for assessing the ability of a material to block the activation of action potential firing in neuronal cells, comprising the steps of:

(a) incubating a neuronal cell in a medium containing a level of serum such that the cell becomes electrically quiescent, and exposing said neuronal cell to said material;

(b) exposing the electrically quiescent neuronal cells to a second material known to be able to electrically excite said neuronal cells when electrically quiescent; and

(c) monitoring the electrical activity of the neuronal cell in the presence of both materials.

19. A method for blocking the activation of action potential firing in neuronal cells, comprising exposing neuronal cells to a preparation containing a material whose ability to block the activation of action potential firing in neuronal cells has been determined in accordance with Claim 18.