|Publication number||WO2001056647 A1|
|Publication date||9 Aug 2001|
|Filing date||3 Feb 2000|
|Priority date||3 Feb 2000|
|Also published as||CA2398961A1, CN1450922A, EP1251904A1|
|Publication number||PCT/2000/2729, PCT/US/0/002729, PCT/US/0/02729, PCT/US/2000/002729, PCT/US/2000/02729, PCT/US0/002729, PCT/US0/02729, PCT/US0002729, PCT/US002729, PCT/US2000/002729, PCT/US2000/02729, PCT/US2000002729, PCT/US200002729, WO 0156647 A1, WO 0156647A1, WO 2001/056647 A1, WO 2001056647 A1, WO 2001056647A1, WO-A1-0156647, WO-A1-2001056647, WO0156647 A1, WO0156647A1, WO2001/056647A1, WO2001056647 A1, WO2001056647A1|
|Inventors||Gary Lynch, Richard H. Granger|
|Applicant||The Regents Of The University Of California|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (10), Referenced by (3), Classifications (8), Legal Events (15)|
|External Links: Patentscope, Espacenet|
ASSAYS FOR THE DETECTION OF AGENTS THAT AFFECT
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This work was supported, in part, by Grant No: N-00014-89-J-1255 from the Office of Naval Research. The Government of the United States of America may have some rights in this invention.
FIELD OF THE INVENTION
This invention relates to novel biosensors that detects and characterize alterations of cognitive function in response to various chemical agents. The biosensors thereby provide assay tools for the rapid screening of compounds (e.g. drugs) for psychoactive properties and the characterization of these properties.
BACKGROUND OF THE INVENTION
To date, there exists no sensitive, rapid, and replicable assay for the effect of various agents on cognitive function. The principal assays for agent (e.g., drug) activity on neurological activity typically rely on behavioral alterations (of live animals) or utilize tissue-based biosensors. Behavioral assays, however, are expensive, are time consuming, are not easily quantitated, and provide limited replicability. Tissue-based biosensors typically overcome some of these limitations and provide a more quantitative output. Such biosensors, however, have been of limited sensitivity and cannot detect subtle alterations in cognitive function. Tissue biosensors capable of detecting agents that impair or otherwise alter neuronal function typically consists of cultured neurons maintained on an array of electrodes that monitor passive membrane properties (e.g., input impedance) or spontaneous action potential activity. At best, these types of biosensors detect the catastrophic consequences of exposure to relatively high concentrations of toxic agents, i.e., conditions that lead to acute cell death. Moreover, most biosensors have provided only short-term data. Although some preparations have a relatively long survival period, there are little available data concerning their stability, reproducibility, sensitivity or selectivity. Several approaches have been pursued to maximize correspondence between the spatial distributions of cultured neurons and recording sites, but as yet, there are no systems providing the optimal solution of a dynamic reconfiguration of recording sites. Finally, no culture systems have been demonstrated to exhibit functional network properties (i.e., a population dynamic) characteristic of the intact brain and necessary to detect non- catastrophic decrements or, or other alterations, in higher cognitive functions such as memory, representational capacity and logical reasoning.
Thus, sensors for psychoactive agents suffer from one or more of three primary shortcomings: 1) While detector s exist for particular agents or classes of agents that are already known they cannot detect the presence of unsuspected or novel agents that might be psychoactive. 2) Rapidly acting agents can be detected, but agents that require many hours or more to have their effect cannot typically be detected with known methods. Finally, 3) agents whose activity e-affects the behavior of all neurons can be detected with a single-neuron detection device, however, if only some neurons are affected, it may be difficult or impossible to detect the effects in an assay that does not consist of intact adult neural circuits.
SUMMARY OF THE INVENTION
This invention provides assays and devices for the effective screening and characterization of agents (e.g. drugs) for psychoactive properties. The assays utilize intact neural circuitries exhibiting functional network properties to provide effective amplification of subtle alterations of neurological function. In a preferred embodiment, the assays utilize novel multi-element electrode arrays that interface with a portion of a mammalian brain (e.g. a hippocampal tissue slice, dissociated hippocampal neuron culture, or co-culture of septum and hippocampus slice).
Thus, in one embodiment, this invention provides methods of screening for agents that alter (e.g. impair, or enhance) brain function. The methods typically involve providing a portion of a mammalian brain contacted with a multi-electrode array. One or more electrodes (preferably 2 or more, 4 or more, or even 8, 16, or 32 or more) stimulate region(s) of the portion of mammalian brain with a time-varying input signal in the presence of at least two different concentrations of the "test" agent. A time-varying output signal from is preferably detected from the mammalian brain though two or more electrodes comprising a multi-electrode array. The output signal is typically a function of the input signal. Differences in the output signal produced by a given input signal in at least two different concentrations of that agent indicate that the agent acts to alter brain function. The two or more concentrations of the agent can include a negative control (zero agent concentration). This negative control need not be measured at the same time or in the same neural tissue sample. Thus, in one preferred embodiment, the output signal is compared to a "library" of such signals for the purpose of analysis, and/or identification, and/or classification.
The portion of a mammalian brain can be a portion of mammalian brain in culture (e.g. hippocampal brain slice with or without a septum input, a dissociated hippocampal neuron preparation, a co-culture of septum and hippocampus, a neocortical slice, a thalamocortical slice, a basal ganglia (striatal) slice, and/or a corticostriatal slice.). The portion of a mammalian brain can also be an acute preparation. Preferred hippocampal brain slices show myelination, and/or dendritic spines, and/or the capacity for long term potentiation. A preferred portion of a mammalian brain is a mammalian brain exhibits functional network properties. The portion of a mammalian brain can have transplanted therein one or more additional neural tissues. The neural tissue can be heterologous or autologous in origin and can be from an adult, juvenile, infant, or fetal animal. Where a neural tissue transplant is present the neural tissue transplant can itself be the agent (i.e., tissue-induced alteration in cognitive activity) or the tissue can release an agent (e.g. a neurotransmitter, a growth factor, etc.).
The time varying input signal can also be a spatially varying input signal. Preferred input signal(s) have a spatio-temporal pattern known to induce synaptic plasticity. Particularly preferred input signal(s) have a theta pattern. The input signal is preferably delivered to one or more of the following: a cell layer of dentate gyrus, a cell layer of CA3, a cell layer of CA1, a superficial cell layer of entorhinal cortex, a deep cell layer of entorhinal cortex, a cell layer of the subiculum, a cell layer of the presubiculum, a cell layer of the parasubiculum, a dendritic field of dentate gyrus, a dendritic field of CA3, a dendritic field of CA1, a dentritic field of entorhinal cortex, a dendritic field of subiculum, a dendritic field of presubiculum, or a dentritic field of parasubiculum.
In a preferred embodiment, the output signal (preferably a theta activity pattern) is recorded from one or more regions including, but not limited to, granule cells, the pyramidal cells of CA3, the pyramidal cells of CA1 deep layer cells of entorhinus, cells of subiculum, cells of presubiculum, cells of parasubiculum, a dendritic field of CA3, a dendritic field of CA1, a dendritic field of deep layer cells of entorhinus, a denedritic field of subiculum, a dendritic field of parasubiculum, or a dendritic field of presubiculum. Virtually any agent can be assayed for psychoactive properties using the assays of this invention. In one preferred embodiment, the agent is not previously known to have psychoactive properties. In another embodiment, the agent is known to be psychoactive, and the agent is present at a threshold concentration (e.g., less than about 100 mM, more preferably less than about 10 mM, most preferably less than about 1 mM) in the portion of a mammalian brain. Particularly preferred agents include anti-cholinergic agents. The methods can further involve identifying an activity signature for the agent and comparing the activity signature to members of a library of activity signatures. In one embodiment, the multi-electrode array preferably contains at least 64 electrodes preferably having a maximum inter-electrode spacing between adjacent electrodes of less than about 300 μm, more preferably less than about 200 μm, and most preferably less than about 100 μm. Particularly preferred arrays are fabricated from a silicon base. The silicon can be plated with a metal (e.g. gold, platinum, copper, or silver).
The signal detection and/or analysis can involve modeling the relationship between the input signal and the output signal using a hidden Markov model (HMM) or wavelets, or neural network methods of signal processing.
In another embodiment, this invention provides a hybrid biological-electronic biosensor for performing one or more of the assays described herein (e.g., screening for an agent that alters brain function). Preferred biosensors comprise a portion of a mammalian brain as described herein (e.g. in in vitro culture or acute preparation) contacted with a multi- electrode array as described herein, a device for stimulating the mammalian brain with a time-varying input signal through one or more electrodes comprising the multi-electrode array; a device for detecting a time- varying output signal from the mammalian brain though two or more electrodes comprising the multi-electrode array where the output signal is a function of said input signal. The device can further comprise a storage medium (e.g. a computer and readable media) containing a library of activity signatures. In some embodiment, the device (e.g. signal generator) that provides a time- varying input signal can also provides a spatially varying input signal in the electrode array. The device may provide an input signal having a spatio-temporal pattern known to induce synaptic plasticity. In certain embodiments, the device that provides a time-varying input signal provides a theta pattern.
The electrode array in the device can be positioned such that said time varying input signal is delivered to a cell layer of dentate gyrus, a cell layer of CA3, a cell layer of CA1, a superficial cell layer of entorhinal cortex, a deep cell layer of entorhinal cortex, a cell layer of the subiculum, a cell layer of the presubiculum, a cell layer of the parasubiculum, a dendritic field of dentate gyrus, a dendritic field of CA3, a dendritic field of CA1, a dentritic field of entorhinal cortex, a dendritic field of subiculum, a dendritic field of presubiculum, or a dentritic field of parasubiculum.
In one particularly preferred embodiment, the device for detecting a time- varying output signal recognizes a theta activity pattern. The device may be situated such that the output signal is recorded from one or more regions including, but not limited to granule cells, the pyramidal cells of CA3, the pyramidal cells of CA1 deep layer cells of entorhinus, cells of subiculum, cells of presubiculum, cells of parasubiculum, a dendritic field of CA3, a dendritic field of CA1, a dendritic field of deep layer cells of entorhinus, a denedritic field of subiculum, a dendritic field of parasubiculum, or a dendritic field of presubiculum. This invention also provides a biosensor in which the neural tissue (portion of a mammalian brain) is contacted with either an agent of unknown psychoactivity, or an agent known to be psychoactive where the agent is present at a thereshold concentration.
In still another embodiment, this invention provides an activity signature library. The library comprises a storage medium containing activity signatures for a plurality of compounds where each activity signature uniquely characterizes and distinguishes each compound in the library. In a preferred embodiment each of the activity signatures comprises at least a 0.5 second recording of the electrical potential at four or more sites in a cultured portion of a mammalian brain contacted with the compound for which the signature was generated. The activity signatures can further comprises at least a 0.5 second recording of the electrical potential at the same four or more sites in a portion of a mammalian brain in the absence of the compound for which the signature was generated. The activity signatures can be derived using any of the cultured neural tissue preparations described herein or an acute preparation. The signal library is preferably a component of a computer system that allows the sorting, searching, and retrieval of one or more activity signature members of the library. The library can further comprise connections to a biosensor described herein to facilitate the online analysis of various agents the uploading of new activity signatures, or the downloading of activity signature components to drive/stimulate the neural tissue of the biosensor.
The terms "agent" or "test agent" are used herein to denote a compound that is assayed for psychoactive activity in the methods of this invention. The test agent can be a compound not known to have psychoactive properties in which case, the assays both identify and characterize the properties of the compound. Alternatively, the test agent can be one that is known to have psychoactive properties in which case, the assays can further characterize the activity signature of the agent. The agent can be virtually any element, molecule, or chemical composition. Preferred agents are either biological molecules (e.g. proteins, glycoproteins, lipids, carbohydrates, nucleic acids, etc.) or small organic molecules. The agents can be derived from the environment (e.g. pollutants, breakdown products, waste, etc.), derived from various plants or animals, chemically synthesized (e.g. de novo) or known pharmaceuticals for human and/or animal use. Test agents will preferably not include known cytotoxins, materials typically provided as components of tissue culture media, buffers, etc. A "psychoactive agent or compound" or an agent or compound having
"psychoactive properties" is a compound or agent that when contacted to neural tissue, in particular when contacted to a portion of a mammalian brain, alters the neurological activity of that neural tissue. Preferred psychoactive agents or compounds alter the "cognitive activity" of the neural tissue. The term "cognitive activity", as used herein includes but is not limited to spatio-temporal patterns occurring within and across intact and healthy brain circuits as part of their natural operation.
The term "cognitive derangement" refers to an impairment or debilitation of cognitive function. Such impairment or debilitation occurs as changes to the normal spatio- temporal patterns outside the normal variance of those seen during normal cognitive function. Preferred cognitive function is a network property.
The term "functional network property" refers to the ability of a neural tissue sample to display an electrical activity (response) characteristic of that neural tissue in. an intact, preferably adult, mammalian brain. Network properties are therefore characteristic of a population dynamic within and among intact circuits, rather than individual neurons. "Intact or complete or substantially complete neural circuitry" refers to cultures of neural tissue that show functional network properties. Preferred neural circuitries are capable of displaying synaptic plasticity, e.g. as exemplified by long-term potentiation. The term "synaptic plasticity" refers to changes in the efficacy of cell-to-cell communication in which the ability of one or more neurons to activate target neurons to which they are synaptically connected is either increased or decreased.
The term "long-term potentiation" ("LTP") refers to the mechanisms underlying particular kinds of synaptic plasticity, typically those that occur rapidly and remain changed permanently or near permanently, as in normal long term memory. There are different forms of LTP underlying memory mechanisms of different types and durations. (An instance is one in which brief physiologically relevant stimulation to afferent axons causes a long-lasting increase in the efficacy of their synaptic junctions with target neurons via an influx of calcium to the postsynaptic membrane through NMDA-type glutamate receptors.) The phrase "a portion of a mammalian brain in culture" refers to brain-derived neural tissue in tissue culture (ex vivo). Preferred portions of mammalian brain are portions that show network properties and, more preferably, are capable of synaptic plasticity, in particular long-term potentiation. In a most preferred embodiment, the portion of a mammalian brain is a brain slice (e.g., hippocampal slice preparation), or a co-culture of septum and hippocampus).
The term "theta" refers to a naturally occurring brain rhythm of approximately 4-8 Hz appearing in many telencephalic areas including hippocampus when an animal is actively engaged in exploration or learning, but not typically when in non exploratory or unaroused states such as sleep. It is notable that this learning-dependent rhythm has been found to be the optimal physiological stimulation rate for inducing LTP.
The term "threshold concentration" when referring to a test agent, particular a test agent that is a drug, refers to the concentration of the agent found in neural tissue, most preferably in central neural tissue (brain), under a minimal treatment regimen (i.e., for a pharmacological composition, under the lowest typically prescribed therapeutic dosage for animal or human). A preferred threshold concentration is less than about 100 mM, preferably less than about lOμm, more preferably less than about lμm, and most preferably less than about O.lμm.
A "physiologically typical" concentration refers to the concentration (e.g. of a test agent) that is found in neural tissue, most preferably in central neural tissue (brain), in a normal healthy organism (animal or human), or under a standard (e.g. prescribed) therapeutic treatment regimen.
The term "input signal" and "stimulus" are used interchangeably to refer to an electrical signal applied to a neural tissue. The electrical signal (e.g. a. voltage and/or a current) can be time-varying and/or spatially varying. A "time-varying" signal is one in which the amplitude (voltage) of the signal varies with time. The variation of the signal can be continuous or a step function. Similarly, a spatially-varying signal is one in which one or more locations in which it is applied to the neural tissue varies with time. An "output signal" or "response" refers to the electrical activity detected in a neural tissue at one or more locations. The output signal can also be time-varying and/or spatially-varying.
An output signal is said to be a "function of an input signal" when the output signal varies in response to alterations and/or presence or absence of the input signal. The terms "activity signature" "cognitive signature", "activity profile" of an agent, refer to the alteration of electrical activity of a neural tissue, preferably a portion of a brain, brought about by contact of the neural tissue with that agent. The alteration can be the alteration of neural tissue activity in response to one or more stimuli (e.g., chemical or electrical) or the alteration of endogenous or spontaneous electrical activity of the neural tissue. Preferred activity signatures are comparative measures (e.g. at two different concentrations of one or more agents, or in the presence versus the absence of the agent). Particularly preferred activity signatures uniquely characterize or distinguish the agent among a group of activity signatures of different agents. Such signatures may be composites of the signatures occurring at different locales and under different conditions in a tissue sample, so that complete characterization of an agent may depend on multiple tests performed with different stimulation conditions, different medium conditions, etc.
An "activity signature library" is a collection of activity signatures for a multiplicity (e.g. 2 or more, more preferably more than 10, more preferably more than 100, most preferably more than 1000, 10,000 or even more than 1,000,000) of different agents. In a preferred library the activity signatures of any two different agents are distinguishable and thereby uniquely identify the agent with respect to the other agents in the library.
The term "storage medium" when used in reference to activity signatures or activity signature libraries refers to any information storage medium capable of storing one or more activity signatures. Storage media include, handwritten materials, printed materials, electronic and/or magnetic storage materials (e.g. computer memories, magnetic disks), optical storage (e.g. holographic storage and/or CD ROMS and/or DVD storage media), logical devices (e.g. programmable array logic, flash ROM, or other chip-based storage forms), or any other medium in which the signature(s) can be held over time and subsequently retrieved. A "multi-electrode" array refers to a collection of electrodes attached to each other in a manner that allows the manipulation of all of the electrodes in the array in together as a group. A "silicon-based multi-electrode array" refers to an array produced on a substrate using micro fabrication techniques typical of the microelectronics industry (e.g. microlithography, microdeposition, etc.). While the silicon-based array can be fabricated on a silicon substrate, it need not be so limited. Other substrates (e.g., gallium arsenide, quartz, various polymers, and the like that are well known to those of skill in the microelectronics art) are intended to be included by this description. Also included are arrays created by techniques other than lithography, e.g., other solid-state techniques such as vacuum deposition, microdeposition, molecular beam etching, laser etching, etc. The phrase "in at least two different concentrations" when referring to the different levels of test agent in the assays of this invention also includes zero concentrations (i.e., the absence of the test agent). Thus, the assays can be performed at two or more different concentrations of test agent where the test agent is present in both concentrations and/or where one concentration is a negative control (absent test agent). It is also recognized that the two or more concentrations need not be measured simultaneously, or even on the same individual neural tissue. Thus, in some embodiments, the two different concentrations can comprise the comparison of a measurement with a measurement made at an earlier time, i.e., the difference detected is between it (the current measurement) and a known or previously recorded pattern recognizing that the previously recorded pattern had to have been measured at some time usign the assays of the present invention.
The term "acute preparation" refers to a "freshly" excised sample of neural (preferably brain) tissue that has typically been held in cerebro-spinal fluid and preferably exposed to oxgenation. Acute preparations can typically be kept alive for 8 to 10 hours. The term "acute preparation" is used to distinguish a fresh preparation from one that has been held in culture media.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 schematically illustrates the test apparatus of this invention. In general terms, the apparatus comprises a signal generator that applies one or more time- varying and/or spatially varying signal(s) to a portion of a brain (e.g., in culture). The portion of a brain is contacted with a test agent and the signal is detected at one or, more preferably, at a plurality of locations on the portion of a brain. Changes in the signal due to the application of one or more agents having psychoactive properties are detected.
Figure 2 generally illustrates the complex network neural (input and output) circuitry available in a typical hippocampal slice, dg: dentate gyrus, sub: subiculum, pre: presubiculum, para: parasubiculum, II, III, IN, N, VI: respective layers of entorhinal cortex.
Figure 3 schematically illustrates a hippocampal slice preparation consisting of a complex neuronal network, with well-segregated layers. Figure 4 illustrates an experiment in which the effects of an AMP A receptor upmodulator on monosynaptic versus polysynaptic responses are explicitly compared to illustrate the significantly higher sensitivity of polysynaptic responses. The top middle graph shows responses to two different AMPA receptor upmodulators drugs where the tissue is stimulated at CA3 and recorded at CA1. This represents a monosynaptic circuit where the stimulus and output are separated by essentially a single synapse. In this instance the response to both drugs are essentially identical. The top right graph shows recordings from the same preparation at the same time, however the stimulation is in the perforant path from the entorrhinal cortex, and the measurement is made from the same CA1 cell. This forms a trisynaptic circuit and, as illustrated, the response to the two drugs differs widely. Thus network level properties reveal differences essentially undetectable in simple neural circuitry.
Figure 5 schematically illustrates a self-contained culture apparatus for use in the methods of this invention. The apparatus typically comprises one or more holding chambers (1) for support of acute or cultured neural tissue. I/O lines for making electrical connections to the neural tissue. Inlets and outlets for gases (e.g. O2, CO2, and atmosphere (ATMOS)), liquids (e.g. test agent solution(s)), maintainance fluid (e.g. cerebrospinal fluid (CSF) artificial cerebrospinal fluid (ACSF), or culture media, etc.), and an input port for a fixative should it be desired to preserve the tissue after use. While the signal source and data acquisition devices are illustrated separately it will be appreciated that the two functions can be incorporated into a single device.
Figures 6 A and 6B illustrate the orientation of an electrode array on a hipocampal slice preparation. Figure 6A shows a cultured hippocampal slice on a low density (450 μm electrode spacing) assay, while Figure 6B shows a cultured hippocampal slice on a high density (150 μm electrode spacing) array. Figure 7 shows the effects of kainate and diazepam on network properties in a hippocampal slice: the standard deviation of 20 consecutive responses was taken and time segments of 300-400 msec duration were analyzed as indicated by the time points at the bottom right of the panels. Shown are these activity measures on eight channels.
Figures 8A, 8B, and 8C illustrate the components of an assay system of this invention. Figure 8 A illustrates a multi-electrode dish (MED), a measuring unit comprising a switch box for electrode selection and amplifier and isolator, and a controlling unit comprising a computer and A/D and D/A converter(s). In one preferred embodiment, the multi-electrode array is provided in a culture dish with an edge connector to facilitate attachment of associated electronics (Figure 8B). Figure 8C provides a detailed view of microelectrodes comprising the multielectrode arrray..
Figure 9 illustrates the extremely dense associational feedback system of hippocampal field CA3 that allows events beginning at a discrete site to quickly recruit additional neurons (left illustration). The right illustration illustrates the role of the diverse population of feedforward and/or feedback GABAergic interneurons whose activity is expected to have effects on aggregate activity that are exaggerated relative to changes at individual synapses.
Figure 10 shows recordings from eight of 64 electrodes in the presence (lower traces) and absence (upper traces) of 10 μM diazepam.
I. Detecting psychoactive agents that alter cognitive function.
A) Screening and characterization of psychoactive agents.
This invention provides assays for detecting and/or characterizing pharmacological agents, environmental agents, and other substances that can act on the brain to alter brain activity. In one embodiment, the invention provides novel methods for identifying the presence of agents that induce derangement in the operation of brain circuitries for which no method of identification presently exists.
The methods preferably use intact circuitries from mammalian brain that operate both spontaneously and in response to artificial stimulation and enable s observation of the operation of these circuitries both in the presence and absence of substances to be assayed. In these methods the induction of circuit operation patterns indicative of cognitive derangement can be distinguished from normal non-deranged patterns. In addition or alternatively, various agents can be characterized and or compared by the alteration of normal patterns. This provides both an activity signature for the test agent(s) and reveals information regarding the detailed modes of action of the agents. The data provided by the methods of this invention further allows elucidation of specific modes of action of various psychoactive chemicals. For example, as described in Example 2, the activity pattern or "signature" obtained in response to diazepam suggests that the effect of diazepam is to synchronize the responses via interaction between excitation and feedback inhibition so that smaller excitatory response in the presence of diazepam nonetheless ehcits more coherent and thus larger field potentials. In one embodiment, the assays of this invention are achieved by extracting intact circuitries from mammalian brains and maintaining these in culture such that they attain normal adult characteristics and remain viable for long periods of time (e.g. up to months). The screening of suspected agents for cognitive derangement or cognitive activity "signatures" generally cannot be accomplished without intact (preferably adult) circuitries, since these, not individual neurons or other non-adult neuronal circuits, are the substrates of normal cognitive function.
Among other advantages, the methods and systems of the present invention allow the detection of subtle, relatively long term effects of various agents on functional network properties (e.g., higher cognitive properties such as are found in intact brain). Practical advantages of the methods of this invention include, but are not limited to 1) Behavioral prediction; 2) Improved sensitivity; 3) The detection of side effects of tested agents; and a 4) richness of description of the modality of agent activity previously unavailable.
With respect to the prediction of behavior, it is noted that networks, not single units generate behavior. Thus, assays that determine the response of neural networks are intrinsically more informative that individual synaptic effects in extrapolating the effects of various agents on the behavior of a subject brain.:
With respect to sensitivity, it is recognized that patterns of neurological activity arise from the coordinated activities of large numbers of neurons and, hence, are easily modified or disturbed. Even very modest drug effects can therefore have large consequences. However, it was a surprising discovery of this invention that, using the methods described herein, the effects of psychoactive agents can be identified at concentrations far lower than those permitted by previous methods.
Because networks contain several types of cells, receptor and transmitters, they operate over long periods of time and at varying levels of complexity. This complexity provides ample opportunity for potential undesired side-effects of various agents to manifest. The assays of this invention thereby permit sensitive detection of side effects that emerge over long time periods.
In addition, the diverse elements present in networks provide numerous sites that exhibit a wide variety of differences among related drugs or other compounds. The combination of this network complexity together with the dense sampling enabled by simultaneous recording from many sites makes it possible to develop "drug profiles or "activity signatures" of unprecedented specificity. This permits more precise classification of agents than was previously possible. B) Preferred assays.
The methods of this invention generally involve a hybrid biological-electronic biosensor consisting of a tissue culture model of memory function integrated with a multi- electrode input source and/or output to detect and assess debilitation or other alteration of cognitive function duce to acute or low level effects of agents applied to the culture. The test apparatus is generally illustrated in Figure 1. In its simplest terms, the invention utilizes a signal source that provides input (e.g. a time-varying and/or spatially-varying electrical potential, i.e. a stimulus) to a cultured neural tissue having functional network properties (e.g., comprising one or more complete neural circuitries). In one preferred embodiment, the neural tissue can be a portion of a mammalian brain. The response (i.e., electrical signal(s) of the neural tissue to the input signal(s) is then monitored at one or at a plurality of sites and the response of the neural tissue various input signals is detected. The neural tissue will produce characteristic response patterns (i.e. time-varying and/or spatially varying electrical potentials) in response to particular input patterns of stimuli.
The neural tissue can be contacted with one or more agents (e.g. drugs, therapeutic lead compounds, members of chemical libraries, potential toxins, environmental contaminants, food contaminants etc.) and the effect of the agent(s) on the response of the tissue to the input signal(s) can be determined. An alteration in the pattern of electrical responses by the neural tissue to the input signal as a consequence of the application of the agent indicates that the agent has psychoactive properties. It will be appreciated that an input signal or signal pattern is not always required. The mammalian brain circuitries utilized in the assays of this invention also exhibit spontaneous and/or endogenous activity. The effect of the agent on this spontaneous/endogenous activity can also be assayed.
1) Screening for potential drugs. The assays of this invention can be used in a wide variety of contexts. One major use is for rapidly and sensitively screening potential drugs. There is a constant need for "fast screens" by pharmaceutical companies, who typically libraries of potential therapeutic agents they are often screened by laborious (and not very revealing) behavioral tests. It was a discovery of this invention that physiologically relevant concentration (e.g. the amount present in a particular tissue under a prescribed dosage regimen) of a drug (e.g. valium) shows up in the assay with a recognizable or characteristic "signature". It is believed that this result has never before been achieved. There are no in vitro assays that will detect the effects of psychoactive agents such as valium in these normal low doses. Thus, in one embodiment the assay methods of this invention are used to identify an "activity profile" or "cognitive effect signature", also referred to herein as a signature, for known or potential therapeutic agents. The methods generally involve contacting the stimulated and/or unstimulated neural tissue with the agent to be tested/characterized and determining the effect of the agent on the activity of the neural tissue (e.g. changes in endogenous activity and/or changes in the response to particular stimuli). A summary of the changes in neural activity (e.g. response(s) to particular stimuli) provides a signature for the physiological activity of the agent (see, e.g., Example 2). The signature of an agent can then be compared against a preexisting compilation ("library") of signatures ("circuit prints") of the effects of known agents. This permits classification of the agent into one or more families of actual or potential pharmaceuticals. In addition, the signatures of similar drugs (e.g. Valium™ (diazepam) versus Halcion (triazolam)) will show slight and sometimes significant differences. This enables one of skill in the art to distinguish and thereby optimize treatment regimens for particular pharmaceuticals. In addition, the assay allows the identification of potential side effects (i.e. parts of their signature that is outside the desired profile) of new or known drugs.
2. Standard signatures for screening therapeutic or therapeutic lead ompounds,
In another embodiment, the methods of this invention can be used to prepare libraries of activity signatures. The profile libraries are basically collections of activity signatures for a multiplicity of agents having psychoactive properties.
Comparisons of the members of such a library will reveals patterns of activity for various classes of agents, and/or may be used to classify agents into groups of compounds having similar or different signatures (patterns of activity).
The signature(s) of agents having desirable physiological activities can then be used as benchmarks for screening for other agents having similar or different activity profiles.
Alternatively, a desirable signature can be produced de novo and serve as a benchmark in the search for compounds that exhibit that activity profile.
3. Screening for toxic agents. In one embodiment, the assay is used to detect cognitive derangement (e.g. a debilitation, aberration, or impairment of normal cognitive function) caused by various agents. In these assays, the test compound (e.g. chemicals found in a particular environment) is assayed primarily for its ability to impair normal cognitive function. However, even if the activity of the agent tested is not impairment perse, it will be appreciated that the identification of any activity on neurological tissue or, conversely, the lack of such activity is a useful result.
4. Screening for cognitive enhancers. The assays of this invention can also be used to screen for cognitive enhancers. Such enhancers can display a wide variety of effects including, but not limited to reduced stimuli necessary for long-term potentiation, increased synaptic plasticity, and the like.
5. Evaluating neural tissue transplant therapies. In another embodiment, this invention provides assays for evaluation of the effects of neural transplantation and/or the effects of various agents on neural transplants. Neural transplants offer, for the first time, potential clinical treatment of the damaged brain. In particular, autologous and heterologous grafting of neural tissue has been used in the treatment of Parkinson's disease. Parkinson's disease, however, has primarily served as a model for a more general strategy of "repair by cellular replacement." Parkinson's is known to result from the loss of a small population of cells that produce the essential neuromodulator, dopamine, for much of the brain. Initial studies suggest that heterologous and autologous transplants of central or peripheral neural tissue can, at least transiently, offset the loss of dopamine. The effect(s) of such transplants on cognitive activity, however, has not been analyzed at significant detail.
Thus, in one embodiment, the brain cultures of this invention can further comprise a transplanted neural tissue. The neural tissue can be derived from central or peripheral neural tissue and can be autologous or heterologous in source. Preferred neural tissue is embryonic in source. Cultured portions of a brain (e.g. cultured slices) can easily be kept alive long enough for the neural tissue transplant to manifest an effect (e.g. alter electrical activity) of the "brain". Methods of performing neural tissue transplants are well known to those of skill in the art (see, e.g., Redmond, et al. (1993) Ann N Y Acad Sci 695: 258-266 and Zager and Black (1988) Surg. Neurol. 29(5): 350-366 and references cited therein). Thus, in one embodiment, the assays described herein are performed with a
"cultured portion of a brain" that has been subjected to a neural tissue transplant. The cultured portion of a brain can be a portion of a "healthy" brain or one derived from a brain showing one or more pathologies, e.g. degenerative diseases such as Parkinson's disease or Alzheimer's disease etc.
The changes in activity can be ascertained relative to the same culture prior to the neural tissue transplant, with a control culture lacking such a transplant, simply measured for "absolute" changes (e.g. no comparison) or compared with one or more profiles in a library of activity signatures.
In addition, such transplants cultures can be used to evaluate the activity of various agents (e.g. psychoactive drugs) on the effect of the neural tissue transplants.
C) Other advantages of the assays.
The assays can be run over relatively long periods of time (e.g. up to 3 to 6 hours, preferably up to 1 day, more preferably from one day to one week most preferably up to 3, 4, or even 5 or 6 weeks, or even up to several months). This allows detection of agents that require many hours of action to have their effect. In addition, because activities are measured in a neural net, agents that effect only some neurons and act primarily in intact mature neural circuits can be detected. Thus, for example, the assay methods of this invention clearly distinguished the activity patterns of Valium and Halcion at physiologically relevant concentrations.
II. Biological Preparations.
A) Preferred preparations.
As explained above, in preferred embodiments, this invention utilizes intact circuitries from mammalian brains. The biological preparations thus comprise neurological cells and/or, neurological tissue (e.g. portions of a mammalian brain). Neurological cells and/or tissues can be obtained from virtually any mammal including, but not limited to rodents, lagomorphs, ungulates, ursines, bovines, primates including humans, canines, felines, and the like. However, in a preferred embodiment, the neurological cells and/or tissues are obtained from rodents, rabbits, felines, canines, or non-human primates.
Cortical systems of the mammalian brain, such as the hippocampus, have a large capacity for high-dimensionality representations that are remarkably robust with respect to noise, transformations, and partial completion. The cognitive function of the hippocampus is to encode the content of short-term memory (information about environmental stimuli and/or behaviors) so that it can be stored in long-term memory in a manner that minimizes interference with the hierarchy of existing long-term memories. The input to the hippocampus is from higher-order neocortical brain regions and consists of multiple sets of features that represent ongoing environmental stimuli and/or behaviors. The hippocampus transforms the multiple-featured representations that are the output of neocortex, and through mechanisms of synaptic plasticity, such as long-term potentiation (LTP), associates (combines into a single object) different subsets of those features into a new representation. Thus hippocampal preparations provide a particularly suitable neurological substrate for the methods of this invention.
Preferred biological preparations therefore include, but are not limited to: 1) cells and cultured hippocampal slices; 2) cultured hippocampal neurons; and 3) co-cultures of septum and hippocampus. Each culture system has its own advantages:
B) Hippocampal dissociated cell cultures.
In one preferred embodiment, the methods of this invention utilize hippocampal dissociated cells. The dissociated hippocampal neuron preparation has several advantages. One of these advantages is the ability to direct the output of processing neurons to identifiable sites within a multi-site electrode array. While the dissociated neuron preparation looses the intrinsic cellular arrangements seen in vivo and in the hippocampal slice culture preparation, synaptic contacts and neural circuits develop richly in the dissociated neuron culture preparation.
Our preliminary data show that high density cultures exhibit robust adherence to gold and aluminum-plated electrodes and to a number of silicon based substrates. Moreover, neuronal process extension is extensive; forming clear neural networks. In low- density cultures, the cell bodies and neuronal processes are easily observed. In other low- density cultures wherein the extensive axonal arbors of a single neuron have been confined to a single electrode surface. Our preliminary data and those of others (Kleinfeid et al. (1988) J. Neurosci., 8: 4098-4120) show that neural circuitry can be regulated by manipulating neuron number, hardware elements and the adhesive properties of the silicon and electrode substrates. A preferred culture chamber is a modification of that Gross and Schwalm (1994) Neurosci., 52: 815-827, modified to permit microscopic monitoring and electrophysiological recording from monolayer neuronal networks.
1. Preparation of Dissociated Cell Cultures. Cultures of hippocampal neurons are preferably prepared following the method described by Brinton et al. (1997) Neurochem. Res., 22: 1339-1351. Hippocampi are dissected from the brains of embryonic day 18 (El 8: with E0 as breeding day) Sprague- Dawley rat fetuses. The tissue is then treated with 0.05% trypsin in Hank's Balanced salt solution (50 mM KC1, 3 mM KH2PO4, 80 mM NaCl, 0.9 mM NaH2PO4(7H2O, 10 mM Dextrose, 0.3 M HEPES) for 5 minutes at 37°C. Following incubation, trypsin is inactivated with cold phenol red free Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10 mM NaHCO3, 10 % fetal bovine serum, 5 μg/ml penicillin and 5 μg/ml streptomycin, and 10% F12 nutrient medium for 3 minutes. Tissue is then washed with Hank's Balanced salt solution (2x) and dissociated by repeated passage through a series of fire polished constricted Pasteur pipettes.
Cells are plated either at a high density 1 x 106 cells/ml or a low density 20,000-30,000 cells/ml onto multi-site electrodes embedded in silicon dioxide masked as described below. Neurons are grown in the presence of phenol red containing Neurobasal Medium (which does not promote glial cell proliferation; Gibco), B27 medium supplement (Gibco), 25 μM glutamate, 0.5 mM glutamine, 5 μg/ml penicillin, and 5 μg/ml streptomycin and maintained in a 37°C 5% CO2 incubator.
C) Hippocampal slice preparation.
1. Hippocampal circuitry.
The hippocampal slice preparation preferably consists of a complex neuronal network, with approximately 90% of the input carried by fibers that originate from the entorhinal cortex. The hippocampal slice preparation consists of a complex neuronal network, but with well-segregated layers (Figure 3). These can be either acute or cultured preparations; the latter may be kept alive for periods of weeks without a significant degree of neuronal loss, and with good preservation of neuronal connectivity.
The network complexity available in hippocampal circuitry provided in a typical brain slice is illustrated in Figure 2. Even casual inspection of Figure 2 reveals that network integration is complex. For example, each granule cell in the dentate gyrus (dg) has about 10,000 synaptic contacts, and spatio-temporal patterns of activity in subsets of perforant path axons (e.g., from layer(s) of the entorhinal cortex) ("the input signals") generate spatio-temporal patterns of activity in subsets of granule cells. The granule cells in turn generate an axonal system, the mossy fibers, which innervate the proximal segment of the pyramidal cell of region CA3 of the hippocampus proper. In addition, granule cell axons also innervate polymoφhic neurons in the hilus of the dentate gyrus which project back onto the granule cells, thus providing a powerful recurrent excitatory loop. An illustration of one trisynaptic network circuit is provided in Figure 4. The terminal formed by the mossy fiber onto the CA3 pyramidal cell is extremely large, and the synchronized activation of a relatively small number of mossy fiber terminals is sufficient to fire a CA3 pyramidal neuron. Spatio-temporal patterns of activity in granule cells therefore generate spatio-temporal patterns of activity in a small number of CA3 pyramidal neurons. The axons of CA3 pyramidal neurons or Schaffer collaterals not only project to pyramidal neurons of CA1 of the hippocampus, but also form an extensive recurrent excitatory network within CA3. Thus, spatio-temporal patterns of activity in CA3 pyramidal neurons are transformed into new spatio-temporal patterns of activity in CA1 pyramidal neurons. In addition, perforant path axons also project to the distal apical domain of both CA3 and CA1 pyramidal neurons; in this way, the same input signals to the granule cells are delivered to the successive stages of the trisynaptic network.
Overall, the illustrated hippocampal circuit performs 3 successive transformations of input signals into output signals, with each stage of the circuit performing different types of computation and storage of processed signals via synaptic modifications. A typical cultured slice preparation represents about l/40th of the hippocampus and therefore has approximately 15,000 granule cells, 5,000 CA3 pyramidal neurons, and 10,000 CA1 pyramidal neurons, plus all the local circuit neurons. These preparations have been kept alive for periods of weeks without a significant degree of neuronal loss, and with good preservation of neuronal connectivity (Bahr et al. (1995) Hippocampus, 5: 425-439). Furthermore, such preparations are ideally suited for investigating the pharmacological effects of numerous classes of drugs because: 1) the large number and variety of synapses guarantees that most channels, neurotransmitter receptors and uptake systems are represented in the cultures; 2) the successive transformations of input signals taking place at different levels of the circuitry result in a large amplification of the drugs effects, thereby providing for an increased sensitivity of detection; and 3) the numerous plasticity mechanisms operating at different synapses also provide for the ability of detecting deleterious effects on cognitive processes (see below).
2. Preferred hippocampal slice preparations.
In particularly preferred embodiments, two different preparations of cultured hippocampal slices can be used, one without the septum input (H preparation), and one with the septum input (H/S preparation). In both preparations, input signals are preferably delivered to the granule cells of the dentate gyrus. In both cases, the output signals are preferably recorded at different levels of the network, i.e., the granule cells, the pyramidal cells of CA3 and the pyramidal cells of CA1.
Other sample preparations include but are not limited to: acute or cultured hippocampal slice preparations including dentate gyrus, CA3 and CA1, cut so as to include the plane of projection of the mossy fibers from dentate to CA3 (Staubli et al. (1990) Synapse 5: 333-335); cultured slice preparations including both hippocampus and parahippocampal regions such as subiculum, pre- and para-subiculum, and entorhinal cortex; or cortical slice preparations (e.g., neocortex or olfactory cortex (see, e.g., Jung et al (1990) Synapse 6: 279-283). Methods of preparing brain-slice cultures are well known to those of skill in the art (see, e.g., Staubli et al. (1990) supra, and Jung et al. (1990) supra.).
3. Preparation of brain slice culture.
Organotypic cultures of hippocampal slices are prepared according to the method described by Stoppini et al (1991) Neurosci. Meth., 37: 173-182. Briefly, hippocampi are harvested in sterile conditions from 11-12 day old Sprague-Dawley rat brains in chilled minimum essential medium (MEM) (Gibco Corp.no. 61100-061) containing: HEPES (25 mM), Tris-base (10 mM), D-glucose (10 mM) and MgCl2 (3 mM) and placed on a Teflon stage of a Mclllwain tissue chopper. Brain slices (e.g., about 100 μm to 600 μm, more preferably about 200 μm to about 500 μm, and most preferably about 400 μm) are cut and transferred onto multi-electrode arrays (e.g., the silicon-based multi-electrode arrays described herein) inside a well containing tissue culture medium. Any suitable culture medium for neurological tissues can be used. Such media are well known to those of skill in the art (see, e.g., Bahr et al. (1995). Hippocampus, 5: 425-39). Particularly preferred culture media consist of MEM medium (Gibco Coφ. no. 41200-072) containing: glutamine (3 mM), HEPES (30 mM), NaHCO3 (5 mM), D-glucose (30 mM), L-ascorbate (0.5 mM), CaCl2 (2), MgSO4 (2.5 mM), 1 μg insulin, 20 % horse serum, including penicillin, pH 7.2.
The brain tissue can then be cultured according to standard methods (see, e.g. U.S. Patent 5,766,948, Bahr et al. (1995) supra.). In a preferred embodiment, brain slices are kept in an incubator at 35 °C with a 5 % CO2-enriched atmosphere, with the medium being changed twice a week. During the initial ten days in culture, slices develop a variety of adult characteristics, including myelination, well-developed dendritic spines, and the capacity for long-term potentiation. Slices are preferably kept in culture at least 5 days, more preferably at least 10 days, and most preferably at least fourteen days before the start of the assays. O 01/56647 D) Co-cultures of septum and hippocampus
Co-cultures of septum and hippocampus allow testing of a forebrain activity pattern (e.g. theta) that is generally assumed to be vital for encoding of information and that has known links to synaptic plasticity. In addition, co-cultures provide an assessment of how test agents affect brain rhythms, a likely first target of any number of disruptive chemicals. Moreover, positive results in slices vis a vis rhythms can be used to predict effects in intact animals; third, the co-cultures introduce transmitter systems not found in the hippocampus and that are known to be susceptible to certain classes of toxins. As an example, much of the septo-hippocampal projection is cholinergic and thus sensitive to cholinesterase inhibitors. The connections also incoφorate GABA cell to GAB A cell connections of a type found throughout the brain's motor systems but are absent from the hippocampus. In all, the co- cultures increase the similarity of the proposed biosensor to the brain in situ and provide physiological activities directly related to higher order telencephalic processing.
1. Septo-hippocampal circuitry.
The septo-hippocampal system is the best understood example of how ascending modulatory systems regulate the physiology of cortical networks. The medial septum, and closely related vertical limb of the diagonal bands (DBB), send cholinergic and GABAergic projections into the three subdivisions of hippocampus. In considering the role played by these fibers, it is essential to recognize that, while numerous, they are vastly outnumbered by the glutamatergic connections arriving from cortex and generated by intrahippocampal pathways. Results from pathway tracing studies first suggested that septal inputs achieve an influence disproportionate to their relative size by targeting inhibitory interneurons, each of which innervates hundreds of pyramidal cells (Baisden et al. (1984) Brain Res., 290: 146-151; Brashear et al. (1986) Neurosci., 17: 439-451; Freund (1989) Brain Res., 478: 375-381; Kiss et al. (1990) J. Comp. Neurol, 298: 362-372; Nyakas et al. (1987) Brain Res. Bull, 18: 533-545). The GABAergic cells appear to be selective in this regard while the cholinergic septal projections also contact pyramidal neurons. Given that intemeurons in hippocampus — as elsewhere in cortical telencephalon — are GABAergic, one component of the septal projection sets up an inhibition to inhibition (GABA cell -> GABA cell) circuit of the type common to the basal ganglia. In this way, the medial septum/ DBB system is able to remove potent inhibitory influences from (disinhibit) the pyramidal cell population. The contributions of the cholinergic fibers are less clear. There is evidence that they act on presynaptic interneuron endings in hippocampus as well as on the so ata and dendrites of interneurons and pyramidal cells (Valentino & Dingledine (1981) J. Neurosci., 1 : 784-792; Ben-Ari et al. (1981) Neurosci., 6: 2475-2484; Kmjevic et al. (1981) Can. J. Phyusiol. Pharmacol, 59: 911-914). It has been proposed that the net effect of these arrangements is to reduce the release of GABA and to directly excite (via a suppression of potassium conductances) pyramidal neurons (Dodd et al. (1981) Brain Res., 207: 109-127; Bernardo and Prince, (1982) Brain Res., 249: 333-344; Cole & Nicoll (1984) Brain Res., 283-290). These two actions would be synergistic with each other as well as with the septal GABAergic projections in increasing the excitability of the pyramidal neurons.
2. Preparation of septo-hippocampal slices.
Previous studies have established that slices of septum collected from postnatal rats innervate the hippocampus and that cholinergic fibers are included in this connection (Heimrich et al. (1993) Neurosci., 52: 815-827; Baratta et al. (1996) Brain Res. Dev. Brain Res., 97: 143-147; Baratta et al. (1996) Neurosci., 72: 1117-1132). Preferred culture methods follow the general methods outlined in these publications.
Dissection is an important step in preparing successful slices and such methods are well described in the literature (see, e.g., Bahr et al, (1995) supra.). Co- cultures of septal and hippocampal slices have been described (see, e.g., Baratta et al. (1996) Brain Res. Dev. Brain Res., 97: 143-147; Baratta et al. (1996) Neurosci., 72: 1117-1132) It should be noted that the goal in the co-cultures is to arrive at co-cultures that exhibit characteristics of the mature septo-hippocampal system. Accordingly, it is possible, that hippocampal slices are collected from a different postnatal age than septal slices.
E) Other culture preparations.
Other portions of brain tissue suitable for the assays of this invention include, but are not limited to acute or cultured preparations of the following: neocortical preparations, including the five primary cell layers and superficial fiber layer, primary visual cortical slices; primary auditory cortical slices; acute or cultured primary somatosensory slices; acute or cultured secondary and associational cortical areas; acute or cultured slices containing primary and secondary areas; thalamocortical slices, co-cultures of thalamus and cortex, co-cultures of multiple cortical areas, striatal, including the caudate and putamen; including dopaminergic nuclei such as substantia nigra and ventral tegmental area; including pallidalareas (GPe, GPi); including thalamic target nuclei; corticostriatal slicesc, co-cultures of cortex and striatum; co-cultures of striatum and dopaminergic inputs, and the like.. F) Self-contained culture apparatus.
In one particularly preferred embodiment, the biological component of the basic assay device described above is a self-contained culture apparatus. Such a culture apparatus preferably incoφorates one or more electrodes or electrode arrays that can remain permanently affixed to the cultured tissue. The apparatus will be fluid-tight so that culture media can be held therein indefinitely. In a particularly preferred embodiment the culture apparatus will be equipped with electrical connectors to permit rapid connection to stimulation and detection apparatus, and/or ports for the convenient and rapid attachment and removal of perfusion pumps, gas cannisters, and the like.
The culture apparatus is preferably modular and seelable so that is can be rapidly and easily separated from the rest of the assay apparatus, stored (e.g. in a culture facility) and easily transported to distant sites for use. One self-contained culture apparatus for use in the methods of this invention is schematically illustrated in Figure 5.
m. Assay input.
While the neural circuitries (e.g. brain slice cultures) can be assayed utilizing spontaneous or endogenous activity, in a preferred embodiment, a signal is applied to the neural tissue. The signal source is preferably one or more potentials applied to discrete locations on the neural tissue. The potentials can be static (i.e. fixed voltages), however, in a preferred embodiment the potentials are time-varying. In addition the potentials can be applied to fixed locations or the locations can be varied (e.g. by selecting alternative electrodes) to provide a spatially-varying signal. In addition, the signal can be both time- varying and/or spatially-varying.
A) Input signal source.
Any of a wide variety of signal sources can be used as the input signal source in the methods of this invention. Essentially any device capable of delivering a voltage ranging from InV to 20mV more preferably from about lOnV to about 5mV V at 1 to about 200 μA is suitable. Preferred signal sources are capable of providing a time varying signal. Particularly preferred signal sources can provide regular or fully programmable time-varying signals. Most modem signal generators utilize a computer to create a stimulus profile that is optionally converted into an analogue signal using an analogue to digital converter that is then delivered to the electrode(s). Signal generation capability is often built into electrophysiological data acquisition systems. Commercial signal generators for use in electrophysiological work are well suited to the present methods. Such signal generators are well known to those of skill in the art and available from a wise number of commercial suppliers (e.g., Grass Instruments, Matsushita Electric Coφ., etc.).. The signal generator can be manually controlled, controlled by a computer, and controlled by a computer whereby the computer controller varies the input signal frequency and or amplitude and or location in response to the output signal from the neural tissue. In one embodiment, a multiplicity of input signals are delivered to the neural tissue in the assays of this invention. In this instance, each signal can be provided by a separate signal generator. Alternatively, a single signal can be divided or multiplied and the resulting multiple signals applied to different inputs. It is more convenient, however, to utilize signal generators capable of delivering a multiplicity of "independent" signals (e.g. multi-channel signal generators). Again, such signal generators are well known to those of skill in the art.
Virtually any number of input signals can be delivered to the neural tissue. The number being limited only by the number of electrodes available for administration of the signal. In preferred embodiments ,the number of input signals will vary from 1 to 8, more preferably from about 8 to about 64. In certain instances higher numbers of input signals will be used (e.g. up to 256, 512, 1024, or even more).
B Type of input signals.
A wide variety of input signals can be applied to the neurological tissue. Indeed virtually any signal can be so applied. However, in a preferred embodiment applied signals will include those known to be characteristic of neurological activity. Such signals include, but are not limited to alpha, gamma and theta. Preferred inputs are those spatio- temporal patterns known to induce synaptic plasticity including but not limited to "theta- burst stimulation" (TBS) consisting of groups of four pulses at 100 Hz, said groups separated by 200 msec intervals, such that the groups are occurring at the 5Hz theta rhythm. In a particularly preferred embodiment, the applied signal is a theta wave signal.
1. Preferred theta input.
Theta is a 5-10 Hz rhythmic pattern found in hippocampus during many kinds of behavior and during REM sleep. It is said to be the largest synchronous extracellular activity that can be recorded in the normal EEG (Vertes & Kocsis (1997) Neurosci., 81 : 893- 926). Pioneering studies by Petsche et al. (1962) Electroenceph. Clin. Neurophysiol, 14: 202-211, established that theta reflects the synchronized bursting activity of neurons in the medial septum/diagonal bands. Subsequent work showed that both GABAergic and cholinergic septal neurons generate high frequency bursts with a between burst interval corresponding to the period of the theta wave. Moreover, stimulation of the septum at the theta frequency generates normal appearing theta in the hippocampus (Apostol and Creutzfeldt, (1974) Brain Res., 15: 65-75; Wetzel et al. (1977) Behav. Biol, 19: 534-542; Kramis and Routtenberg, (1977) Brain Res., 125: 37-49). These points combined with evidence on the physiological effects of the septal projections within hippocampus, led to the hypothesis that theta arises from an inhibition/disinhibition cycle generated by the between burst/within burst activity of septal neurons (see Vertes and Kocsis (1997) supra, for review).
There has been considerable speculation about what theta contributes to hippocampal processing since Green and Arduini (1952) J. Neurophysiol, 17: 533-557, first identified the rhythm as a concomitant of hippocampal arousal. Recent thinking on the problem has been strongly influenced by the discovery of a deep relationship between theta and the long-term potentiation (LTP), a form of synaptic plasticity widely held to be critical to the encoding of memory. The initial studies showed that 30 msec stimulation bursts of four stimulation pulses are sufficient to induce potentiation when the bursts are administered repetitively at the theta frequency (Larson and Lynch (1986) Science, 232: 985-988; Staubli and Lynch (1987) Brain Res., 435: 227-234). Remarkably, moving the interval between the bursts outside the theta period resulted in progressively less LTP (Larson et al. (l986)Brain Res., 368: 347-350). Chronic recording work confirmed that theta bursting of the type ideal for inducing LTP is exhibited by hippocampal neurons during complex behavior including learning (Eichenbaum et al (1992) Behav. Neural. Biol, 57: 2-36; O'Keefe (1993) Curr. Opin. Neurobiol, 3: 917-924; Vanderwolf (1969) Electroencephalogr. Clin. Neurophysiol, 26: 407-418; Winson (1972) Behav. Biol, 7: 479-487; Winson (1974) Electroencephalogr. Clin. Neurophysiol, 36: 291-301). The mechanisms whereby theta patterning promotes LTP induction have been described. Briefly, afferent bursts within hippocampus activate synapses on feedforward inhibitory interneurons as well as target pyramidal neurons. The latter thus receive a monosynaptic excitatory input followed shortly by di synaptic EPSPs. Inhibition counteracts the initial EPSPs by hypeφolarizing the cell and shunting the inward excitatory current; thus, the composition response consists of 2 or 3 msec of depolarization followed by a much longer lasting hypeφolarization. Induction of LTP requires a depolarization of sufficient duration and degree to unblock voltage dependent NMDA type glutamate receptors. These conditions are not met by the response to a single burst. However, feedforward inhibitory synapses, once having been activated, enter a refractory period which reaches its peak after 150-200 msec, and then dissipates over the next second. Bursts arriving during the peak of the refractory period generate composite responses that are largely free of IPSPs and thus are able to generate a measurable degree of LTP. Pharmacological studies point to pre-synaptic GABAb type autoreceptor as being responsible for the refractory period (Mott and Lewis (1991) Science, 252: 1718-1720).
2. Spatially varying signals
In addition to varying in time, the applied signal(s) can vary in the location(s) to which they are applied. The location of signal application can be switched simply by switching the particular electrode attached to a particular signal source. Alternatively, signal sources attached to various electrodes and be turned on or off, or the amplitude can be increased or decreased thereby resulting in different spatial distributions of signal(s). The electrodes can be switched individually or in coordinated patterns. The switching can be achieved by mechanically making or breaking contacts of by various electronic switching techniques well known to those of skill in the art.
Preferred patterns of signal administration will include, but are not limited to, paired pulses of stimulation at intervals of 10 to 100 msec; repetitive stimulation at 10-100 Hz; alternating stimulation at two or more different sites.
C) Input/output array and variations.
In various preferred embodiments a multiplicity of input signals are applied to the neural tissue and/or a multiplicity of output signals (tissue response signals) are detected and optionally analyzed.
Generally each (different) signal, input or output, is administered to or detected from a single electrode. A multiplicity of single electrodes can be applied to the neural tissue. However, assay preparation is simplified by the use of one or more electrode arrays. An "electrode array" is a multiplicity of electrodes (at least two preferably up to 4, 8, 16, 64, 256, 512, or even 1024 electrodes or more) that are attached in a common unit, bundle, or surface. The association of the electrodes facilitates easy application to the neural tissue without requiring separate manipulation (application) of each individual electrode. In a particularly preferred embodiment, this invention utilizes one or more silicon-based multi-electrode array(s) such as those commercially from Matsushita Electric Co. (multi-channel-extracellular recording (MED) system). 1) Electrode array design.
Preferred multi-electrode arrays comprise at least eight different electrodes. However the number of electrodes can routinely range upwards to 16, 64, 128, 256, 512, 1024, and even higher. The electrode arrays can be provided in a wide variety of electrode densities (numbers of individual electrodes per unit area). In a preferred embodiment, "dense" or "high density" arrays preferably have an interelectrode spacing of about 300 μm or less, more preferably an interelectrode spacing of about 200 μm or less, and most preferably an interelectrode spacing of about 150 μm or less or even 100 μm or less. Broad (low density) arrays have an interelectrode spacing greater than about 300 μm, preferably ranging from about 300 μm to about 1 mm, more preferably from about 300 μm to about 300 μm to about 750 μm, and most preferably from about 300 μm to about 450 μm or 500 μm. A wide variety of electrode geometries are suitable for the assays of this invention. In one preferred embodiment, the electrodes are regularly distributed in an essentially uniform array (see, e.g. Figures 6A and 6B). In other embodiments, the electrode arrays are provided in single substrate and multichip-module packaging configurations. The arrays can include both one- and two-dimensional electrode arrays. Multi-chip module integration can be accomplished using high-density indium-bump-based flip-chip-bonding. Other preferred electrode geometries include conformal mappings of tissue cultures that incoφorate key configurational relationships among input layer and output layer neuron arrays. For example, to activate a subpopulation of hippocampal synapses that converge onto a common subpopulation of postsynaptic neurons through the trisynaptic circuit, input (stimulation) pathways are preferably focused (confined) to a relatively narrow region of the hippocampal slice, one that is consistent with the topographical projection of its pathways. This is accomplished by using probes with a high density of electrode sites, and having a spatial distribution matching the intrinsic neural circuitry. Similarly, the size of the microprobe elements recording electrical activity in CA3 and CAl in response to stimulation of the input side can be optimized. Thus, in some embodiments, it is preferable to use a smaller number of wider elements (e.g. ranging from about 50 μm to about lOOμm capable of recording the activity of groups of neighboring neurons rather than the individual activities of a spatially segregated group of neurons. The electrode arrays can additionally incoφorate switching (electrode- selecting) circuitry as well as preamplifiers (preferably isolated) for each active channel. Input lines into the electrode array can provide for downloading instruction sets to the array for local signal conditioning amplification as well as electrode selection. 2) Array fabrication.
The multi-electrode arrays used in the assays of this invention can be assembled according to any of a wide variety of methods well known to those of skill in the art. For example multiple individual electrodes can be used that are not joined together into an array. Alternatively, the multiple individual electrodes can be bonded together in a bundle using a mechanical support (e.g. a machined, or otherwise fabricated, support block with multiple "receptacles" for the electrodes). The electrodes can simply be bonded together (e.g. using a glue, epoxy, liquid plastic/resin, etc.). Where glass electrodes are used, they can be joined by heat fusing the glass.
In some embodiment, the electrodes are fabricated as a single "integrated" unit. In this instance, particularly where it is deired to achieve high probe density (small spatial resolution), preferred fabrication techniques include, but are not limited to integrated circuit fabrication methods. A detailed description of the use of such methods to produce an electrode array is described by Hubbard (U.S. Patent 5,388.577). The Hubbard patent describes an electrode array microchip fabricated, in one embodiment, using CMOS technology. The multi-electrode arrays described therein use standard fabrication line techniques and comprises metal regions on a substrate over which an overglassing material has been applied. The overglass is cut to expose metal thereby forming electrodes. The electrodes are electrically connected to wire bonding or probe pads or to integrated circuitry within the microchip. It is noted that multi-electrode arrays are also commercially available (see, e.g., Matsushita Electric Coφ., SAGC-5 and SAGC-10 multi-electrode dish (MED)). These electrode arrays provide 64 microelectrodes arranged in an 8 x 8 array covering an area of about 1 mm2 in the center of a glass plate with an inteφolar distance between electrodes of 150 μm. Each microelectrode is 50 x 50 μm and has an impedance of 50 kilohms or less.
D Positioning of array on slice.
1. Physical localization of electrode array.
The localization of the neural tissue (e.g. brain slice) on the electrode array (or vice versa) is preferably optimized to maximize the likelihood of suitable numbers of electrodes in the array contacting the region(s) of interest in the neural tissue. Dense electrode arrays offer more precise positioning/alignment at a reduced coverage area. In this instance, the coverage area can be increased by the use of larger arrays or multiple arrays. Conversely, low density arrays, can be utilized to record larger fields. Many placements of a slice on dense (e.g., 1mm x 1mm) arrays are possible.
For instance, a dense array can be placed such that most of field CA3 is included in the array along with portions of dentate gyrus and field CAl, or placed such that most of CAl is present along with portions of CA3. A broad broad array (e.g., 3mm x 3mm) can also be utilized for which optimal placement is such that all three primary regions of hippocampus (DG, CA3, CAl) are in contact with the array, as well as entorhinal cortex and all three subicular regions, if they are present on the slice. In general, it will be appreciated that array area, electrode density, and electrode array shape can be routinely altered to optimize contact with particular neural tissue preparations.
2. Selection of stimulus and/or recording sites. As indicated above input (stimulus) and/or recording sites can be selected in real-time simply by any one or more of the electrodes contacting the neural tissue. Electrode selection can be achieved using an "external switch box" or by commands to microcircuitry onboard the electrode array. Electrode selection can be fixed throughout the assay or can be dynamically reconfigured (e.g. manually, by a programmed system, or in response to particular neural output signals).
It is noted that a commercially available multi-electrode recording device provides a switch box that allows real-time re-selection of different electrodes at any time during an experiment (see, e.g., Matsushita ElectricCoφ., SACC-1 MED (multi-electrode dish) connector, that provides well-shielded switchable connections having a contact resistance of less than 30 mOhms for each 64 microelectrodes).
IV. Output signal(s).
In the assays of this invention the electrical output of the neural tissue (portion of a brain, intact brain circuitry, etc.) is detected at one or more locations. The output signal(s) are optionally amplified, conditioned, inteφreted immediately and/or stored for subsequent analysis as described below.
A) Orientation/nature of output connections (array, etc.)
Where the output signal is detected at one or only a few (e.g. 2-4) it is relatively straightforward to utilize discrete electrical probes. However, in a preferred embodiment, particularly where the signal is detected at a multiplicity of locations, the output signal is detected using an electrode (micro-electrode) array as described above. The electrode array can be the same array as the array used to administer an input signal (stimulus) or it can be a separate array. Where the same array is used for application of stimulus and detection of a response, in a preferred embodiment, different electrodes in the array are utilized for stimulation and detection. However, it is possible to utilize the same electrode for both activities, particularly where there is an anticipated lag between stimulus and signal production. The recording electrodes can be fixed within the array(s) or can be intermittently or continually varied as the array is dynamically reconfigured.
In various preferred embodiments, where input signals are delivered to the output signal is recorded from one or more regions selected from the group consisting of the granule cells, the pyramidal cells of CA3, the pyramidal cells of CAl deep layer cells of entorhinal, cells of subiculum, cells of pre or parasubiculum, and the dendritic fields of any of these regions.. In preferred embodiments, output signals are detected in the dendritic fields of CA3 and/or CAl.
B) Amplification of output signal^ and noise suppression.
Pre-amplification, amplification and signal noise suppression is accomplished according to standard methods well known in the electrophysiology art. Typically each signal channel is provided with a pre-amplification stage and this preamplifier is situated as close to the recording electrode(s) as possible. Thus, in one embodiment, preamplifiers are incoφorated into circuitry onboard the electrode array. Noise suppression can be accomplished electronically with appropriate filters and/or algorithmically during analysis of the acquired signal. Typically noise suppression is accomplished by the use of a one or more signal conditioner. Signal conditioners are typically computer controlled and can be used in conjunction with any laboratory A/D system. Signal conditioners provide a variety of filters and noise suppression manipulations including, but not limited to 4 or 8 pole Bessel low-pass filtering, high-pass filtering, AC/DC coupling, notch filtering, variable gain, baseline correction, and the like. Suitable preamplifiers, amplifiers, analogue to digital converters, data acquisition systems, signal conditioners and the like are commercially available (see, e.g., Matsushita Electric Coφ., Multi-channel Extracellular Recording System, and Axon Instruments Inc., Foster City, , preamplifiers, amplifiers, and signal conditioners, and the like).
A schematic illustration of multi-electrode dish (MED), switch box, amplifier, A/D and D/A converter and signal and analysis computer is illustrated in Figure 8 A, 8B, and 8C. V. Output signal analysis.
A) Simple recognition of alteration of cognitive function.
In one embodiment, output signal analysis can be as simple as recording and/or display of the output signal at each output site. Visual analysis of output signals so presented can be quite informative and is often sufficient to: 1) Identify compounds that alter cognitive activity; 2) Identify various modes of action of such compounds; 3) Provide characteristic signatures for such compounds; and 4) Facilitate comparison of such compounds (see, e.g., the Examples provided herein).
In one embodiment, the alteration in cognitive activity due to an applied agent is recognized by a wide variety of indications. These include changes in the signal amplitude, duration, frequency, or waveform(s) in response to particular input, changes in the correlation between output signals at different locations in the neural tissue, changes in the correlation between the input signal(s) and the output signal(s), etc.
Thus, for example, in one study examining the effect of diazepam on an acute hippocampal slice preparation, the response in a cell layer (shown as d, e, and fin Figure 10) as a function of a stimulus in the basal cell layer region of field CA3 was recorded. In addition the dependence of activity of another cell layer (shown as a-c, and g-h) on the activity in the first cell layer was monitored. Stimulation site in the basal cell layer region of field CA3 initiated responses at three sites in the cell layer (Figure 10, d,e,f) which in turn trigger further activity via recurrent contacts (Figure 10, a-c,g-h). Field potentials were larger in the presence of diazepam, a suφrising result in light of the GABA enhancing effect of diazepam. The output signals provided by the assays of this invention are also amenable to far more sophisticated analysis as described below.
B Advanced analytic methods.
Continuous time-varying signals such as the activity patterns arising from the cultured slices are traditionally analyzed using Hidden Markov Models (HMMs); indeed such analysis is so ubiquitous that entire government funding programs have been initiated with the sole puφose of identifying and developing alternative methods for the analysis of time- varying signals. One such method has been developed as a result of modeling of the auditory system (Aleksandrovsky et al., (1996) Proc. Intl. Conf. Pattern Recog. IEEE Comp. Soc. Press, 4: 550-554; Aleksandrovsky et al, (1997) Pages 104-115 in Biological and Artificial Computation: From Neuroscience to Technology., IWANN '97 Int'l Conf. on Artificial and Natural Neural Networks, Berlin: Springer Verlag.; Garzotto et al. (1997) Proc. Intl. Conf. Neural Networks, IEEE Press, 1 : 564-568). HMMs operate by the construction of statistical models of the sequence of features likely to be encountered as a time series progresses. For example, for voice, the pronunciation of a word ("Bill' or "ball" or "bald") is broken into time segments, each of which contains a particular set of dominant frequencies (e.g. labeled as aj, a2, etc.). The probability of occurrence of each possible feature (al 5 a2, etc.) is calculated over a set of samples of each utterance, for each time period, and a state-transition model is constructed corresponding to the probability of transition over time from one probability density function (PDF) of feature composition to the next.
HMMs are valuable for the analysis of signals as varied as sonar and speech, but suffer from two well-known and costly shortcomings: i) it is necessary to obtain a statistically significant sample of each possible utterance or trace in order to construct the feature composition PDF and the state transition model; and ii) the resulting model uses only the immediately preceding time point to calculate the probability of transition to the subsequent time point; no prior information or events can be used. This has the advantage of avoiding potential combinatorial explosion at the number of possible predictive transitions, but limits the predictive power of the method, especially for data that is sensitive to events in the past. The alternative sequence-processing model described by Aleksandrovsky et al. (1996 and 1997) supra., was constructed from models of the olfactory and auditory systems The storage and retrieval operations of the model in response to a stream of features presented to the input stage of the network have been studied.. In the example, a sequence of features comprises a version of the written number "3". The sequence consists of two ink spots followed by three in spots followed by two more ink spots, arranged in distance and thickness such that when adjacent they form one version of a "3". The network possesses two distinct input pathways: one topographic and one nontopographic. The topographic path passes the first feature vector (two vertically arranged spots) to the "middle layer" of the network, selectively activating the topographic region(s) of the network dedicated to the particular class of feature vectors.
The input is learned in the network's "superficial layer" via a clustering method described (Ambros-Ingerson et al. (1990) Science, 247: 1344-1348; Kilbom et al. (1996) J. Cog. Neurosci., 8: 338-353). The resulting learned cluster pattern is passed vertically to the "deep layer". This layer feeds back its response to the input structures as the second input is read. The intersection between the next input (three vertically arranged spots), and the feedback from the deep layer's response to the first input, is learned in the superficial layer, and the transition from the first cluster to the second is learned (via a sequence-learning rule described in Granger et al. (1994) J. Neurophysiol, 17: 533-557) in the deep layer. The process is repeated as inputs arrive, with successive transitions from one feature to the next being stored. The overall mechanism essentially encodes a sequence of random patterns along a collection of labeled recognition networks, with each successive pattern generated from its predecessor. The network described is the first in a hierarchically organized series of networks, such that the output of the network is input to a next network, whose output in turn is input to a following network. This cascade of primary, secondary, tertiary, etc., networks successively extracts feature generalizations from the input, enabling a form of "contextual" processing in which information about longer sequences (e.g., a word) is available to support the disambiguation of shorter sequences (e.g., a character in that word).
Implementation of this hierarchical cascade has been used in speech processing and in handwritten character recognition (Aleksandrovsky et al (1996 and 1997) supra.; Garzotto et al. (1996) supra.). Recently the network has been used for the analysis of evoked response potentials (ERPs) from human subjects and used to classify normal, schizophrenic and Alzheimer's subjects from their ERPs. In this study 28-elecrrode ERP data was collected from 24 normal and 24 schizophrenic subjects during presentation of tone pips in an "auditory oddball" paradigm, in which subjects were asked to respond when a rare high-frequency tone appeared in a string of low- frequency tones. The network was trained on 50%> of the data and tested on the remaining 50%. An HMM was compared to the model just described. The data were sufficiently difficult that the HMM performed at roughly 70% correct prediction, whereas the auditory model achieved a level of 90% correct.
These promising initial results suggest that the model is useful for predictive diagnosis of EEGs and ERPs. The marked similarity of such signals to the evoked synaptic responses in a slice preparation further indicated that the algorithm is useful to classify these signals.
VI. Application of agents to brain slice.
A) Typical kinds of agents.
Virtually any kind of agent can be tested and/or characterized utilizing the methods of this invention. Such agents include, but are not limited to psychoactive drugs whose activity it is desired to categorize (e.g., positive modulators of GABA receptors such as benzodiazepines, positive modulators of glutamate receptors such as ampakines, anti- cholinergic agents or cholinesterase inhibitors), compounds from carbohydrate, protein, nucleic acid, lipid, or small organic libraries, environmental agents, agents utilized in various manufacturing processes, breakdown by-products in various disposal processes, and the like. Agents also include, but are not limited to therapeutic agents and or lead compounds proposed or actually used in particular pathological conditions including but not limited to schizophrenia, Parkinson's disease, Alzheimer's disease, depression, anxiety, various drug addictions, and the like.
In preferred embodiments, the agents will not include known cytotoxins, typical buffers, salts, and components normally found in tissue culture media, and the like.
Recently, attention has focused on the use of combinatorial chemical libraries to assist in the generation of new chemical compound leads. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al. (1994) 37(9): 1233-1250). Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara et al(l992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a Beta- D- Glucose scaffolding (Hirschmann et al, (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidyl phosphonates (Campbell et al, (1994) J. Org. Chem. 59: 658). See, generally, Gordon et al, (1994) J. Med. Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, and U.S. Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines U.S. Patents 5,525, 735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate TJ, Zymark Coφoration, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
B) Applicant of the agent to the neural tissue.
The agent(s) can be applied to the neural tissue according to any of a number of standard methods well known to those of skill in the art. In the simplest embodiment, , particularly where the agent is water soluble, the agent can simply be added to the culture media. Where the agent is not soluble it may be complexed with one or more materials to render it compatible with the solution (e.g. as an emulsion or dispersion). Methods of solubilizing compounds are well known to those of skill in the art (see, e.g., Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980)). In addition to the above, or alternatively, , the agent can be applied
(contacted) directly to any exposed surface of the neural tissue. The agent can also be injected into the tissue and, particularly where long term administration is desired, the tissue can be cannulated and the agent can be perfused into the tissue using standard methods.
In another embodiment, the agent can itself be a tissue (e.g. a neural tissue) that is transplanted into the portion of a mammalian brain utilized in the assays of this invention. The tissue can be autologous or heterologous in origin and can be derived from adult, juvenile, infant, or fetal mammalian tissue sources. The transplanted tissue can itself alter cognitive function simply through cell contact or, more typically will alter cognitive function by the release of one or more psychoactive agents (e.g. serotonin, dopamine, etc.).
C) Other assay modalities (e.g. high-throughput modalities)
Any of the assays for compounds that alter cognitive function as described herein are amenable to high throughput screening. High throughput systems typically utilize robotics, information acquisition systems, computer control of experimental protocols, and interaction between automated synthesis of chemical libraries and screening systems to maximize test agent throughput.
Systems to facilitate high-throughput screening are commercially available (see, e.g., Zymark Coφ., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput modalities.
Vπ. Signature libraries. In still another embodiment, this invention provides for "activity signature libraries". An "activity signature library" is a collection of activity signatures for a multiplicity (e.g. 2 or more, more preferably more than 10, more preferably more than 100, most preferably more than 1000, 10,000 or even more than 1,000,000) of different activity signatures. In a preferred library the activity signatures of any two different agents are distinguishable and thereby uniquely identify the agent with respect to the other agents in the library. Each signature in the library preferably comprises at recording of sufficient length to distinguish the output signal at a particular location from the output signal at that location in the absence of the test agent (where the test agent produces a difference in output signal). Preferred signatures include at least about a 0.1 second, more preferably at least about a 0.5 second and most preferably at least about a 1 second, 10 second, or even 1 minute or more duration recording of the electrical potential at two or more, preferably four or more, more preferably 8 or more, and most preferably at 16, 64, 128, 256, or even 512 or more different sites in a cultured portion of a mammalian brain contacted with the compound for which the signature was generated. The signatures can optionally additionally include corresponding recordings of the electrical potential at each of the sites in the absence of the compound for which the signature is a record. Signatures are selected that uniquely identify every compound whose signature is present in the library.
The activity signature library provides a valuable resource for classifying and or characterizing agents. Agents having similar modes of activity will have similar activity signatures. At the same time, agents that appear to have similar or identical physiological effects on the organism can be revealed by their signature to have subtly or even grossly different modes of action. The activity profile(s) can thus be used to identify complementary or varying treatment regimens for different agents.
The activity signature libraries are particularly useful in the characterization of new psychoactive agents, in the evaluation of potential biohazards, or in the search for therapeutic lead compounds. For example, where an agent is newly identified as having psychoactive properties, a comparison of the activity signature of the agent with the library of activity signatures permits classification of the compound among other compounds simply by mode of action. Moreover, where the identity of a particular agent is unknown (i.e., in a complex extract from a plant) classification of the active compound in the library to identify agents having similar activity profile(s) may provide an indication of the chemical composition of the agent and thereby facilitate subsequent purification.
Where a psychoactive agent is a component of the environment (e.g. natural environment, work environment, waste-processing environment, etc.) classification of the agent by activity signature aids in assessment of the risk posed by that particular agent. The activity signature(s) of known compounds coupled with the physiological effects or side-effects provided in a therapeutic regimen allows prediction of the activity signature of a desired psychoactive agent. Complex chemical libraries can then be screened for agents that have or approximate the desired activity signature(s). These uses of the activity signature library are intended to be illustrative and not exhaustive. The activity signature library is most useful when activity signatures can be easily retrieved, sorted, classified, and/or otherwise organized. Thus, in a preferred embodiment, the signature libraries of this invention comprise a database, most preferably an electronic (e.g. computer-based) database. Thus, the libraries are typically components of a computer system. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems", mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like.
In another embodiment, this invention also provides kits for practice of the assay methods described herein. Preferred kits include a container containing one or more of the following: an electrode array, a culture device comprising an electrode array, a library of agent "activity signatures" in paper, electronic or optical storage form(s), a cultured neural tissue, electroencephalographic instrumentation (signal generator, preamplifier, amplifier, data acquisition system, etc.) buffers, templates for orientation and application of an electrode array to a neural tissue preparation, micromanipulators, VLSI logic components for post processing or analysis, and the like.
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. The kits can optionally include any other material, device, or instruction for practice of the methods of this invention.
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1: Cultured hippocampal slices. Two different preparations of cultured hippocampal slices were used, one without the septum input (H preparation), and one with the septum input (H/S preparation). In both preparations, input signals were delivered to the granule cells of the dentate gyrus. In both cases, the output signals were recorded at different levels of the network, i.e., the granule cells, the pyramidal cells of CA3 and the pyramidal cells of CAl .
A) Culture preparation.
Organotypic cultures of hippocampal slices were prepared according to the method described by Stoppini et al. (1991) Neurosci. Meth., 37: 173-182.. Briefly, hippocampi were harvested in sterile conditions from 11-12 day old Sprague-Dawley rat brains in chilled minimum essential medium (MEM) (Gibco Coφ.no. 61100-061) containing (in mM): HEPES (25), Tris-base (10), D-glucose (10) and MgC12 (3) and placed on a Teflon stage of a Mclllwain tissue chopper. Brain slices (400 μm) were cut and transferred onto the silicon-based multi-electrode arrays (see below) inside a well containing tissue culture medium consisting of MEM medium (Gibco Coφ. no. 41200-072) containing: glutamine (3 mM), HEPES (30 mM), NaHCO3 (5 mM), D-glucose (30 mM), L-ascorbate (0.5 mM), CaCl2 (2 mM), MgSO4 (2.5 mM), 1 μg insulin, 20 % horse serum, including penicillin, pH 7.2. Brain slices were then kept in an incubator at 35°C with a 5 % CO2-enriched atmosphere, with the medium being changed twice a week. During the initial ten days in culture, slices develop a variety of adult characteristics, including myelination, well- developed dendritic spines, and the capacity for long-term potentiation. Slices are maintained in culture at least ten days before the start of experiments.
B) Long term hippocampal slices generate complex spatio-temporal patterns of activity.
Initial experiments on cultured hippocampal slices maintained in a multi- electrode dish (MED; Matsushita Electronics) indicate the robust ability of these slices to generate extended activity (seconds) in response to brief (milliseconds) input stimulation. Hippocampal slices, prepared from Sprague-Dawley rats aged 11-13 days postnatal, were placed on the MED device and maintained at the interface between oxygen-rich atmosphere and artificial cerebrospinal fluid (ACSF), containing: NaCl (124 mM), KC1 (3 mM), KH2PO4 (1.25 mM), MgSO4 (1 mM), CaCl2 (4 mM), NaHCO3 (26 mM) and glucose (10 mM). All the experiments were carried out at room temperature.
Representative evoked synaptic responses were recorded from 4 different positions in field CAl in response to activation of Schaffer-commissural fibers. The response profiles of single and repetitive afferent stimulation were similar to those obtained with conventional recording methods. Paired-pulse facilitation was observed at inteφulse intervals of 40-80 ms.
C Patterned activity within cultured slices is sensitive to low doses of excitotoxins.
In this experiment, two different classes of upmodulators of the AMPA-type glutamate receptor were applied to a hippocampal slice. Hippocampal slices were positioned so that an 8x8 electrode array covered the majority of field CA3. Asynchronous synaptic activity was induced by activation of pyramidal cells in field CA3a-b and recorded from the cell body layers in CA3c. Field potentials were recorded for 300 msec after mild stimulation (50 μA) delivered at 20 sec intervals. The standard deviation of 20 consecutive responses was calculated for each channel and plotted before drug addition, after infusion of 50 μM GR120 (an ampakine) or 100 μM cyclothiazide (a benzothiadiazide), as a measure of the intensity and variability of neuronal activity. Both GR120 and cyclothiazide bind to AMPA- type glutamate receptors and allosterically increase the currents produced by these receptors. Both compounds increased neuronal activity at 6 of 8 electrode locations. These differential effects at different electrodes call attention to the fact that AMPA receptors are located on inhibitory as well as on excitatory neurons; thus drugs that enhance the activity of AMPA receptors may enhance or depress measured neuronal activity depending on the local circuitry being recorded.
It is notable that the effects seen are unexpectedly large for the concentrations of upmodulators used. A 100 μM concentration of cyclothiazide is at best a threshold dose for affecting monosynaptic responses. The size of the effects seen in the reported experiments is likely due to the amplifying effect of polysynaptic circuitry: each stage in the measured multisynaptic responses is affected by the presence of the drug, and it is apparent that the combined effects are at least additive, and perhaps nonlinearly so. This effect has also been reported in acute slices, with single recording electrodes. Figure 4 illustrates an experiment in which the effects of an AMPA receptor upmodulator on monosynaptic versus polysynaptic responses are explicitly compared. Stimulation of Shaffer-commissural afferents to hippocampal field CAl evoked a standard monosynaptic response. The increase in those monosynaptic responses to the infusion of 50 μM GR120 was barely perceptible. In the same experiment, the perforant path afferents to the hippocampus were stimulated and the downstream polysynaptic responses in CAl measured. These responses were seen to be greatly amplified by the 50 μM GR120, due to the additive effects of the drug at the intervening synapses (Sirvio et al. (1996) Neurosci., 74: 1025-1035). The use of the MED device makes this difficult experiment much more practicable, due to the presence of multiple recording electrodes that increase the likelihood of sensing polysynaptic responses in the slice, and the use of long-term cultured slices enables sensing of agents that may exert their disrupting effects only over extended time periods rather than immediately.
In one experiment, the effects of kainate and diazepam on reverberating activity in a hippocampal slice were measured through a single electrode of the multi- electrode array. The slice was stimulated at 50 μA every 20 sec and 20 consecutive responses were displayed with increasing vertical displacement. Kainate significantly increased recurrent activity in the slice, increasing both the amplitude of early responses to stimulation and the longevity of the reverberating activity that ensues. Diazepam prevented the effect, and dampens activity to levels lower, than those in medium without kainate.
Figure 7 shows the effects of kainate and diazepam recorded using a multichannel array. The standard deviation of 20 consecutive responses was taken and time segments of 300-400 msec duration were analyzed as indicated by the time points at the bottom right of the panels. Shown are these activity measures on eight channels of the multi- electrode array before drug addition, after infusion of 10 μM kainate, and after infusion of 10 μM kainate plus 10 μM diazepam. Kainate, which activates excitatory glutamate receptors, increased neuronal activity recorded at most recording locations, as in the experiments with upmodulators GR120 and cyclothiazide. The anxiolytic diazepam, which enhances inhibitory circuits by acting on GABA receptors, greatly suppressed neuronal activity at all eight recording sites.
Example 2: Additional Pharmaceutical Investigation. Hippocampal field CA3 has characteristics found throughout the cortical telencephalon. Its primary specialization is an extremely dense associational feedback system that allows events beginning at a discrete site to quickly recruit additional neurons (Figure 9 left). The process of activation and recruitment can continue through several cycles leading to a synchronized output. Inhibitory interneurons play a vital role in shaping the recurrent excitation that drives the network cells towards a coordinated response. Where there is too much inhibition the network "dampens down." Where there is too little inhibition the network becomes epileptic. Any change then, in the behavior of the diverse population of feedforward and/or feedback GABAergic interneurons (Figure 9 right) will likely have effects on aggregate activity that are exaggerated relative to changes at individual synapses. The above points are of interest to pharmacological studies because many psychoactive drugs have direct (benzodiazepines) or indirect (serotonin reuptake inhibitors) effects on interneurons.
Thus, in a second experiment, the effects of diazepam (Valium™) on an acute hippocampal semi-thin slice was investigated. Figure 10 shows recordings from eight of 64 electrodes in the presence (lower traces) and absence (upper traces) of 10 μM diazepam. The stimulation site in the basal cell layer region of field CA3 initiates responses at three sites in the cell layer (d, e, f) which, in turn, trigger further activity via recurrent contacts (a- c, g-h). field potentials are larger in the presence of diazepam, a surprising result in light of the GABA enhancing effect of diazepam. Analysis suggests that the effect of diazepam is to synchronize the responses via interaction between excitation and feedback inhibition so that the smaller excitatory response in the presence of diazepam nonetheless elicits more coherent, and thus larger, field potentials.
It is understood that the examples and embodiments described herein are for illustrative puφoses only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incoφorated by reference in their entirety for all puφoses.
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|International Classification||G01N33/15, A61N1/05, G01N37/00, A61N1/08, G01N33/50|
|Cooperative Classification||A61N1/08, A61B5/04001|
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