WO2003091694A2

WO2003091694A2 - Method for assay of cognition and memory based on low frequency stimulation

Info

Publication number: WO2003091694A2
Application number: PCT/US2003/012680
Authority: WO
Inventors: Geoffrey C. Tombaugh; Wayne B. Rowe; Gregory M. Rose
Original assignee: Memory Pharmaceuticals Corporation
Priority date: 2002-04-24
Filing date: 2003-04-24
Publication date: 2003-11-06
Also published as: AU2003234203A1; WO2003091694A3; AU2003234203A8

Abstract

The invention provides an in vitro method for eliciting long term potentiation (LTP) in the neuronal tissue of an animal using an electrical stimulation protocol that ranges from 4 to 12 Hz. The stimulation is applied over a short time course and yields an enhanced electrophysiological output which lasts for several hours. The method can be used to identify compounds, which can be used for enhancing LTP in an animal and ultimately cognitive performance and memory, as well as for developing pharmacological treatments for learning or memory impairments in e.g., aged humans with pathological memory impairments such as Alzheimer's disease.

Description

METHOD FOR ASSAY OF COGNITION AND MEMORY BASED ON LOW FREQUENCY STIMULATION

This application claims benefit of U.S. Provisional Application Serial No. 60/374,782, filed April 24, 2002.

FIELD OF THE INVENTION

The present invention relates generally to methods for eliciting long term potentiation in the brain of an animal comprising applying an electrical stimulation of 4 to 12 Hz. The invention further relates to methods for identifying compounds that can enhance long term potentiation, methods for distinguishing learning-impaired animals from unimpaired animals, methods for determining the extent of a cognitive disorder, a memory impairment, or a learning disability in an animal, and methods for treating a subject with a cognitive disorder, a memory loss, or a learning disability.

BACKGROUND OF THE INVENTION

Learning and memory impairments are among the most commonly recognized psychological features of humans. However, in the absence of pathological dementia, aging and cognitive decline are not obligatorily linked. Some aged individuals exhibit keen learning and memory into and beyond their eighth and ninth decades (McLearn (1997) Exp. Gerontol. 32:87-94 and Backman et al. (2000) In: The Handbook of Aging and Cognition (Craig FIM, Salthouse TA, eds.)). Like humans, aged rodents exhibit a marked heterogeneity in their ability to learn and remember. Spatial learning is particularly impaired in aged rats; however, some aged subjects show severe deficits in spatial learning, while others are indistinguishable from young controls (Gage et al. (1984) Ne robiol. Aging 5:43-48; Barnes and McNaughton (1985) Behav. Neurosci. 99(6): 1040-1048; Deupree et al. (1991) Neurobiol. Aging 14:249-258; Quirion et al. (1995) J. Neurosci. 15:1455-1462). The basis for the variability is unknown, but it is well documented that spatial learning in rodents is strongly hippocampus-dependent (Morris et al. (1982) Nature 297:681-683; Morris (1990) Ewr. J. Neurosci. 2:1016-1028, 1990; Hollup et al. (2001) J. Neurosci. 21:4504-4513). Hippocampal-dependent learning and memory deficits have been well documented in aging rodents. Lesions restricted to the dorsal hippocampus or the CAl cell field have been shown to impair spatial learning in rats (Davis et al. (1986) Physio. Behav. 37:387-392 and Moser et al. (1995) PNAS 92:9697-9701). However, age-related spatial learning deficits in rats do not arise from hippocampal neuron loss (Rapp and Gallagher (1996) PNAS USA 93:9926-9930), suggesting that more subtle changes in synaptic structure or function are involved (Rapp et al. (1999) J. Comp. Neurol. 403:459-470 and Smith et al. (2000) J. Neurosci. 20:6587- 6593). Long-term potentiation (LTP) has been prominently cast as a key factor in age- related cognitive decline (Foster (1999) Brain Res. Rev. 30:236-249).

Long term potentiation has been widely implicated in learning and memory. This link has been strengthened by studies in which hippocampal LTP in aged rats was found related to be related to individual differences in spatial learning (Barnes and McNaughton (1985) Behav. Neurosci. 99(6): 1040-1048; Deupree et al. (1991) Brain Res. 554:1-9). Such correlative studies reinforce the idea that the hippocampus plays a key role in spatial memory formation.

Numerous stimulation protocols can reliably elicit LTP in the hippocampus. However, the utility of these protocols in probing mnemonic mechanisms may not be equal. Many stimulus protocols trigger increases in synaptic responses (e.g. fΕPSPs) that are similar in amplitude and stability, yet arise from different biochemical cascades depending on the pattern of stimulation, age, species, and even strain of the animal (Shankar et al.(1998) J. Neurophysiol. 79:334-341 and Nguyen et al. (2000) Learn Mem. 7:170-179).

SUMMARY OF THE INVENTION The present invention relates to a method for eliciting long term potentiation in the brain of an animal comprising applying an electrical stimulation of 4 to 12 Hz, which yields an electrophysiological output with long term stability.

The present invention further relates to a method for identifying a compound, which can enhance long term potentiation in the brain of an animal, comprising applying an electrical stimulation of 4 to 12 Hz to the brain of the animal, measuring long term potentiation resulting from the application of said electrical stimulation in the presence of the compound, measuring long term potentiation resulting from the application of said electrical stimulation in the absence of the compound, and comparing the duration of long term potentiation in the presence of the compound and in the absence of the compound to determine if the compound enhances long term potentiation.

The invention relates to a method for distinguishing learning-impaired animals from unimpaired animals, comprising applying an electrical stimulation of 4 to 12 Hz to the brain of an animal, measuring long term potentiation, and comparing the measured long term potentiation to that exhibited by an unimpaired animal. The invention further relates to a method for determining the extent of a cognitive disorder, a memory impairment, or a learning disability in an animal, comprising stimulating the brain of the animal with memory impairment with an electrical stimulation of 4 to 12 Hz, and measuring the magnitude of long term potentiation.

The invention is drawn to a method for treating a subject with a cognitive disorder, a memory loss, or a learning disability, comprising administering to the subject a therapeutically effective amount of a compound that enhances long term potentiation and is identified by applying an electrical stimulation of 4 to 12 Hz to a brain of an animal, measuring long term potentiation resulting from the application of said electrical stimulation in the presence of the compound, measuring long term potentiation resulting from application of said electrical stimulation in the absence of the compound, and comparing the duration of long term potentiation in the presence of the compound and in the absence of the compound to determine if the compound enhances long term potentiation.

DESCRIPTION OF THE FIGURES

Figure 1 shows the results of Morris water maze testing for learning ability among aged F344 rats. (A) Mean escape latencies measured on training days 3-5 were grouped in 10s bins for both aged (24-26 mo) and young (4-6 mo) rats. Aged-unimpaired (AU) or impaired (Al) animals were identified as those whose mean performance differed by <0.5 or >3SD from the mean of young controls, respectively. The remaining rats (aged-other, AO) were excluded from further study. The acquistion curves during training are plotted for both mean escape latency (B) and pathlength (C). Error bars in B and C, when not visible are smaller than the symbol size.

Figure 2 shows probe measures that indicate that Al rats do not use a spatial strategy in the water maze. (A) With the platform removed, Al rats required significantly more time for their 1^st entry into the target area (annulus-40) than AU or young rats. Al and AU data were derived from those animals used for subsequent electrophysiological analysis in Figs. 4 and 5 (Y: n=36; AU:n=7; Al: n=12). *p < 0.05, ANOVA, compared to AU group. (B) Al rats spent less time within the target area than AU or Y rats. Dwell time for Al rats, unlike that for AU and Y rats, was unchanged when the extra-maze cues were obscured by a curtain (Y: n=12; AU:n= 1; Al: n=ll). ** pO.Ol, ***p<0.001, paired t- test, compared to matching "no curtain" control.

Figure 3 shows the muscarinic M2 receptor antagonist, BIBN-99, improves water maze performance in Al rats. Both the mean escape latency (A) and latency to first entry into the annulua-40 (B) were significantly reduced following a single injection of the antagonist given 45 min prior to each set of training trails. Baseline data refer to those collected on training day 5 prior to any injection. *p <0.05, unpaired t-test, compared to vehicle treatment. Figure 4 shows slices from AU and Al rats exhibit no differences in CAl synaptic transmission and short-term facilitation. (A) hiput-ouput curves reveal depressed synaptic transmission for aged rats relative to young controls, but were not different between AU and Al subgroups [Y, n=9(7)' AU, n=9(7); Al, n=19(8)]. (B) Paired-pulse facilitation in CAl was indistinguishable between young, AU and Al groups across a range of inter-stimulus intervals (ISI). Inset shows a representative trace (young rats). Scale bars: 2mN, 10ms.

Figure 5 shows 5Hz LTP distinguishes between slices from Al and AU rats. (A) Al slices subjected to 5Hz/30s stimulation exhibited significantly smaller synaptic potentiation in CAl than seen in either young or AU slices [Y:n=17(8); AU: n=20(7); Al: n=30(12)]. *** P <0.001, AΝOVA compared to young and AU. (B) All groups responded to 5 Hz stimulation with a similar time-course and number of complex spikes. Inset in B shows a representative set of traces collected during 30s of 5 Hz stimulation in a young slice. Scale bars: lmV, 10ms. (C) Slices taken from the contralateral hippocampus of some of the animals in A were subjected to 3 x 0.5s @ 30 Hz stimulation. The resulting LTP in both AU and Al slices were smaller than that in young controls, but no AU-AI difference was detected [Y:n=17(14); AU: n=ll(6); AI:n=8(5)] (D) 70 Hz stimulation in a separate set of rats induced robust, long-lasting and statistically equivalent LTP in all groups [Y:n=13(ll); AU: n=17(9); Al: n=8(7)] Data points in panels C and D are presented at 5 min intervals for clarity.

Figure 6 shows the deficit in 5 Hz LTP in Al rats is not linked to a loss of ΝMDA- receptor function. (A) Summary data from young slices [n=7(4)j demonstrates that 5 Hz LTP in partially ΝDMA-receptor dependent, as bath application of 50uM APN (hatched bar) depressed but did not completely block LTP triggered by 5 Hz/30s stimulation (delta). Complex spiking during 5 Hz stimulation in the presence of APN was abolished. Following washout, 5 Hz stimulation triggered complex spiking (total spikes = 98 +12) and robust LTP that was comparable to that typically seen in naϊve control slices (Figure 5). Representative pairs of traces (1^st and 150^th sweep) were collected during 5 Hz stimulation from the same slice in the presence of APN and after 50min of washout. (B) The NDMA-receptor mediated fEPSP, isolated in the presence of lOμM CNQX, 10 μM Glycine, and 0.2mM Mg2+, was depressed in both AU and Al rats by approximately 30% compared to young controls across a range of stimulus intensities [Y:n=21(7); AU:n=18(6); AI:n=13(6)]. When normalized to fiber volley amplitudes greater than 0.5mV, slope values for both AU and Al rats were significantly different from young controls (p<0.01 ANON A). No difference between AU and Al rats was detected. Inset depicts representative traces (average of 5 sweeps) recorded from an aged rat slice before and after exposure to 50μM APN. Scale bars: lmV,10ms.

Figure 7 shows 5 Hz LTP correlates with water maze learning in aged rats. (A) The average level of 5 Hz LTP calculated for each aged animal was strongly correlated to individual mean escape latency in the water maze (r^Λ2=0.52, p<0.001). In addition, 5 Hz LTP was significantly correlated to two probe measures: the latency to first entry into the annulus-40 (B;r^Λ2 =0.35, p <0.01); and total dwell time in the annulus-40 (C; r^A2=0.51, p<0.002). The fitted curve in panel C was generated by non-linear regression, hi each case, the data for young control rats are presented for comparative purposes only and were not included in the regression analysis.

Figure 8 shows the muscarininc antagonist (BIBΝ-99) enhances LTP in slices from Al rats. (A) Bath application of lμM BIBN-99 (heavy bar) significantly enhanced 5 Hz LTP in Al slices [n-=9(5)] to a level slightly smaller than that seen in young controls[n=17(8)]. PO.001, ANON A, AI+BIBΝ 99 compared to Al control. (B) BIBΝ-99 did not significantly affect the time-course or total number of complex spikes evoked during 5Hz stimulation. (C) BIBN-99 had no effect on LTP evoked by repeated trains of 70Hz stimulation in Al slices. Data in panel C are presented at 5 min intervals for clarity. Error bars were smaller than the symbol size.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to method for eliciting long term potentiation in the brain of an animal comprising applying an electrical stimulation of 4 to 12 Hz, which yields an electrophysiological output with long term stability.

hi one embodiment, the invention relates to a method for eliciting LTP in the brain of an animal comprising applying an electrical stimulation protocol which ranges from 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, or 10-12 Hz and yields a sustained electrophysiological output.

In a preferred embodiment of the above methods, the long term potentiation occurs in a brain tissue sample from the animal, hi a further preferred embodiment, the electrical stimulation and long term potentiation occurs in the hippocampus. In one embodiment, the animal is impaired, h a further preferred embodiment, the animal is a mammal, preferably a rat. Preferably, the method comprises applying an electrical stimulation of 5 Hz. Preferably, the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

In one preferred embodiment, the compound improves performance of an animal in a spatial learning task, h a further embodiment, the compound enhances memory in an animal, hi a further embodiment, the compound is a M2 receptor antagonist, a PDE4 inhibitor, or a nicotinic alpha-7 modulator. In another preferred embodiment, the invention relates to a method for identifying candidate drugs of different classes as cognitive enhancers in aging subjects comprising administering a 5Hz electrical stimulation to a section of neurological tissue (e.g. brain) from a animal and measuring the long term potentiation (LTP).

The invention relates to a method for distinguishing learning-impaired animals from unimpaired animals, comprising applying an electrical stimulation of 4 to 12 Hz to the brain of a learning-impaired animal, measuring long term potentiation, and comparing the measured long term potentiation to that exhibited by an unimpaired animal.

In one embodiment, the invention relates to a method for distinguishing learning- impaired from unimpaired animals comprising applying a 4-12 Hz electrical stimulation to the CAl region of the hippocampus of the animal and measuring LTP.

The invention further relates to a method for determining the extent of a cognitive disorder, a memory impairment, or a learning disability in an animal, comprising stimulating the brain of an animal with memory impairment with an electrical stimulation of 4 to 12 Hz, and measuring the magnitude of long term potentiation.

The invention further relates to a method for treating a subject with a cognitive disorder, a memory loss, or a learning disability, comprising administering to the subject a therapeutically effective amount of a compound that enhances long term potentiation and is identified by applying an electrical stimulation of 4 to 12 Hz to a brain of an animal, measuring long term potentiation resulting from the application of said electrical stimulation in the presence of the compound, measuring long term potentiation resulting from application of said electrical stimulation in the absence of the compound, and comparing the duration of long term potentiation in the presence of the compound and in the absence of the compound to determine if the compound enhances long term potentiation. In a preferred embodiment of the above methods, the long term potentiation occurs in a brain tissue sample from the animal. In a further preferred embodiment, the electrical stimulation and long term potentiation occurs in the hippocampus. In one embodiment, the animal is impaired. In a further preferred embodiment, the animal is a mammal, preferably a rat. Preferably, the method comprises applying an electrical stimulation of 5 Hz. Preferably, the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

In a preferred embodiment, the subject is a mammal, preferably a human.

The stimulation protocol of this invention which can be used to 1) link hippocampal LTP to hippocampal-based learning and 2) aid in the identification of compounds which can enhance LTP and thus cognition and spatial learning. The protocol provides a short time assay with extended stability compared to other stimulation protocols in the art.

Unless otherwise indicated herein, the protocol of this invention is conducted fully conventionally, e.g. as described in US patent 6,096,302.

As used herein, the term "long term potentiation (LTP)" refers to a persistent enhancement of an excitatory postsynaptic potential (EPSP) following brief high- frequency (tetanic) stimulation of afferent pathways (Shors TJ. and Matzel, L.D. (1997) Behavioral and Brain Sciences 20(4): 597-655).

As used herein, the term "theta-frequency stimulation" refers to 5 Hertz stimulation, a low frequency stimulation.

As used herein, the term "stimulation" includes an electrical stimulation to evoke an electrophysiological response from a population of neuronal cells. The neuronal cell population may be in a hippocampal slice in vitro, in a subject in vivo, or in other neuronal tissue.

As used herein, the term "cognitive disorder" includes a learning disability or a neurological disorder, which may be Alzheimer's disease, a degenerative disorder associated with learning, a learning disability, memory or cognitive dysfunction, cerebral senility, multi-infarct dementia and senile dementia, electric shock induced amnesia or amnesia.

As used herein, the term "learning disability" includes a hippocampal learning or memory deficit concurrent with an electrophysiological deficit. The term applies to young adult subjects (for example, 12 week old rat) and adult subjects (for example, 24 month old rat) that show behavioral impainnents and have a corresponding electrophysiological deficit.

The electrical stimulation protocol which can be used to stimulate LTP in the neuronal tissue of an animal may include electrical stimuli which range from 4-6 Hz, or 5-7 Hz, or 6-8 Hz, or 7-9 Hz, or 8-10 Hz, or 9-11 Hz, or 10-12 Hz. A particularly preferred stimulation is 5 Hz.

The present invention provides for a method for treating a subject with a cognitive disorder of memory or a learning disability which comprises administering to the subject a therapeutically effective amount of a compound identified using the 4-12 Hz electrical stimulation protocol of this invention. The compound may be associated with a suitable pharmaceutically acceptable carrier.

Compounds which may have use in treating memory disorders as identified by the electrical stimulation protocol include, but are not limited to M2 receptor antagonists, alpha7 nicotinic receptor agonists, phosphodiesterase 4 inhibitors and any other compounds that enhance LTP as tested in this assay. Novel compounds that selectively inhibit PDE4 enzymes have been previously described in U.S. application serial no. 10/051,309, filed January 22, 2002; U.S. application serial no. 10/067,996, filed February 8, 2002; and U.S. application serial no. 10/270,724, filed October 16, 2002.

The subject may be a mammal or a human subject. The administration may be intralesional, intraperitoneal, intramuscular or intravenous injection; infusion, liposome- mediated delivery, gene bombardment, topical, nasal, oral, anal, ocular or optic delivery.

In the practice of any of the methods of the invention or preparation of any pharmaceutical compositions a "therapeutically effective amount" is an amount which is capable of alleviating the symptoms of the cognitive disorder or memory or learning in the subject. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated. For the purposes of this invention, the methods of administration are to include, but are not limited to, administration cutaneously, subcutaneously, intravenously, parenterally, orally, topically, or by aerosol.

A "suitable pharmaceutically acceptable" carrier encompasses any of the standard pharmaceutically acceptable carriers, such as phosphate buffered saline solution, water, emulsion such as oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients.

The invention also provides for pharmaceutical compositions including therapeutically effective amounts of protein compositions and compounds capable of alleviating the symptoms of cognitive disorder or memory or learning in the subject together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in the treatment of deficits in LTP which can be identified with the 5 Hz stimulation protocol herein described. Such compositions are liquids or lyophilized or dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength additives such as albumin and gelatin, detergents, solubilizing agents, anti-oxidants, preservatives, bulking substances or tonicity modifiers.

Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Other compositions may include particulate compositions coated with polymers and the compound couples to antibodies directed against tissue-specific receptors, ligands, or antigens or coupled to ligands of tissue- specific receptors.

This invention is illustrated in the Experimental Details Section which follows. The sections are set forth to aid in understanding of the invention but are not intended and should not be construed to limit in any way the invention as set forth in the embodiments.

Experimental Details. Long term potentiation (LTP) has been examined in the CAl region of the hippocampus using theta-frequency stimulation (5Hz) in aged rats that had poor spatial learning in the Morris water maze. It was discovered that LTP induced in the CAl region using theta-frequency stimulation is selectively impaired in aged rats that show poor spatial learning. Further, 5Hz-LTP amplitude strongly correlated with individual learning performance among aged rats. No performance-related differences were observed when LTP was induced with 30 or 70 Hz LTP compared to 5 Hz LTP stimulation protocols. The 5Hz stimulation protocol can be applied over a short time(e.g. 15-60 seconds) and is stable long term (e.g. longer than 3 hours).

Young (4-6 months old) and aged (24-46 month old) male Fisher 344 rats were obtained from Hilltop Animals (Scottdale, PA). The animals were left undisturbed for a minimum of one month until the beginning of behavioral testing. Rats were housed in pairs in polycarbonate cages (45 x 30 x 18 cm) with corncob bedding and maintained on a 12:12 light-dark schedule (lights off at 1900 hours). Food (LabDiet 5001 rodent diet) and water were freely available. Animal health was monitored by a veterinarian. Animals showing overt signs of morbidity were removed from the study. All procedures were in accordance with local IACUC guidelines. Behavior Studies. Young and aged rats were tested on the Morris water maze (Rowe et al. (1998) Neuroscience 83:669-680). The maze consisted of a 1.6 cm diameter circular pool filled to within 15 cm of the rim with water (22 °C) made opaque by the addition of non-toxic white latex paint. A circular Plexiglas escape platform (14.5 cm in diameter) was located in the center of one of the quadrants of the pool. The animals were given 15 trials over 5 consecutive days with the platform submerged 2 cm below the surface of the water (3 trials/day; 120 sec maximum trial duration; 20-30 min. inter-trial interval). On the last training trial, a probe test was performed in which the retractable Plexiglass platform was pneumatically lowered out of reach of the rats for 30 seconds and then returned to its original position for the remainder of the trial. A 40cm diameter zone (annulus-40) around the platform center was used to assess probe performance, h some trials, an opaque curtain was placed around the pool perimeter to obscure the extra-maze cues. Latencies and swim distances to locate the hidden platform during training trials, time to first entry and dwell time in the annulus-40, and mean distance from the platform during probe trials, were all recorded and analyzed using a computer-based tracking system (San Diego Instruments, San Diego, CA). On day 6, rats not scheduled to receive any additional behavioral testing were given 60s trials in which the platform was raised 2.5 cm above the water level (visually cued condition) to test for visual, motivational or motor deficits that may have influenced performance. Rats which required more than 40 seconds to reach visual platform on any trial were excluded.

The cognitive status of the aged animals was defined on the basis of their latencies to find the submerged platform on days 3, 4 and 5 of testing relative to the mean latency of young controls (Figure 1). Aged impaired (Al) rats were defined as those animals whose mean latencies (across the 3 days of testing) differed by >3.0 standard deviations from that of young controls. Aged animals were considered unimpaired (AU) if their mean latencies were <0.5 standard deviations from young controls. Aged animals whose mean escape latencies fell between these values were not used in any further studies. One compound that was studied was BIBN-99, a selective muscarinic M2 receptor antagonist (Doods et al, (1993) Eur. J. Pharmacol. 242:23-30). The compound was synthesized and stored as a powder form in a dessicator. Al rats that were treated with BIBN-99 were given an additional three days of training (3 trials/day). On each of these days, a 0.5 mg/kg dose of BIBN-99 or vehicle was administered 1 hour before training began (Quirion et al (1995) J. Neurosci. 15:1455-1462). Performance during drug treatment was averaged for the entire 3-day testing period. On the last day of testing (day 8, trial 3), a 30 second probe trial was performed on all animals. The visually cued testing procedure was administered the following day. All statistical analyses, including those for electrophysiological data, were performed either with a t-test or a 2-way ANOVA, with subsequent pair-wise comparisons made using a Tukey-Krarner post hoc test.

Rats were anaesthetized with isoflurane and sacrificed by decapitation. Transverse hippocampal slices (400 μm) were prepared from young, adult and a subset of behaviorally characterized aged rats using a tissue chopper. Slices were maintained at 28°C in an interface chamber (Fine Science Tools) and perfused at l-2ml/min with artificial cerebral spinal fluid (ACSF) which had been pre-equilibrated with 95% O₂/5% CO₂. The ACSF composition was in mM: NaCl 124; KC1 4.5; NaH₂ PO₄ 1; NaHCO₃ 26; CaCl₂ 2.5; MgCl₂ 1.3; glucose 10. Bipolar stimulating electrodes (stainless steel, FHC) and glass recording electrodes (1-3 MΩ; filled with ACSF) were positioned in stratum radiatum of area Cl. Input-output curves were recorded and test stimulus intensity (0.017 Hz, 50 μs duration) was adjusted to evoke a fEPSP with an initial slope that was 40-50% of maximum. Following a stable baseline period (20-30 min), one of three stimulation protocols was applied at the test intensity: (A) a single 30 sec train at 5 Hz; (B) three 0.5 sec trains at 30Hz (5-min inter-train interval); or (C) three 0.5 sec trains at 70 Hz(5-min inter-train interval). After the induction protocol, single responses were evoked at the test intensity for either 60 min (A) or 180 min (B,C) to monitor LTP amplitude and stability. LTP was measured by 'comparing the mean fEPSP slope (averaged over 5 min) at 1 or 3 hours post-tetanus to that of the mean fEPSP slope recorded 5 min prior to either the tetanus or to drug application. The effect of NDMA receptor blockade on 5 Hz LTP was examined in slices from young rats by exposing the slices to 50 μM APV for 15 min and sequentially applying 5 Hz stimulation to the same slice, first in the presence of APN and then 60 min later after washout. BIBΝ-99 was dissolved in DMSO; stock solutions were diluted in ACSF immediately before use and bath-appplied to slices 30 min prior to LTP induction at a final concentration of 1.0 μM. The DMSO concentration never exceeded 0.05%). NDMA-receptor mediated fEPSPs were isolated by exposing slices for 50-60 min to modified ACSF containing 200 μM MgC12, 10 μM CNQX, and lOμM glycine.

Slices from aged rats subjected to 5 Hz or 30 Hz stimulation were prepared from different hippocampi of the same rat; electrophysiologists were blind to the behavioral status of the animal. Slices subjected to 70 Hz stimulation were taken from a separate group or rats. In all cases, recording from young and aged slices were interleaved. Recordings on a given day were typically made from multiple slices (2-4) from each animal. For LTP-behavior correlations and NDMA-R fEPSP measures, the datum for a given animal was represented by the average value from all slices examined. In experiments where BIBN-99 was applied, paired control recording were always performed in adjacent slices from the same animal. Complex spiking observed during 5 Hz stimulation was quantified by counting the number of negative spikes clearly resolved from the fEPSP (Thomas et al. (1998) J. Neurosci. 18:7118-7126). Slices that did not exhibit stable fEPSP baselines, or those in which the pre-synaptic fiber volley changed abruptly or shifted by more than 20% during the course of the experiment, were excluded from analysis. Input-output curves are plotted as fEPSP slope vs. fiber volley amplitude. All data are presented as mean + s.e.m. Sample size are given as the number of slices, with the number of animals shown in parantheses.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiment are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. The entire disclosure[s] of all applications, patents, and publications cited above are hereby incorporated by reference. EXAMPLE 1 Learning impairments observed in a subset of aged rats.

Aged rats were characterized as either impaired (Al) or unimpaired (AU) based on their mean escape latency calculated from training days 3-5 in the Morris water maze. As anticipated from earlier studies, aged animals showed significantly longer swim times and swim distances with a greatly increased variance, compared to young controls. Aged rats swan more slowly than young controls (Young: 27.9 + 0.6 cm/sec; AU:24.9 + 0.4 cm/sec; Al 23.1 + 0.5 cm sec). There was no significant difference in swim speed between AU and Al animals. Swim times and distances were highly correlated for all groups (r > 0.9 in all cases).

Swim times for aged rats exhibited a skewed, unimodal distribution (Figure 1A). Of 89 aged animals screened in the water maze for the present study, 40 (45%) were classified as cognitively impaired (Al) and 21 (24%) were classified as unimpaired (AU). The remainder of the animals did not fall clearly into either category and were excluded from further study. The acquisition curves for the AU and Al groups illustrate the dramatic impairment in the ability of Al animals to find the hidden platform (Figures IB and 1C). However, when the platform was made visible, all young and AU, and all but 4 Al, rats were able to achieve criterion performance. The mean swim times for young AU and Al rats that reached criterion in the visually cued trials were 5.0 + 0.3 sec, 8.5 + 1.0 sec and 8.8 + 0.8 seconds, respectively. These data suggest that the altered ability of the Al animals to locate the submerged platform was not due to a visual, motor or motivational deficit.

EXAMPLE 2 Al rats do not use a spatial strategy in the water maze.

At the end of the 5-day training period, each animal was given a 30-second probe test, during which the submerged escape platform was unavailable. The swim speeds of AU and Al rats did not differ during the probe trial (AU: 23.9 + 0.6 cm/sec; Al: 23.4 + 0.4 cm/sec) and were comparable to that of young controls (25.4 + 0.5 cm/sec). However, Al animals required significantly more time to make their first entry into the annulus-40 compared to either AU or young rats (p< 0.05; ANON A, Figure 2A). The Al animals also maintained a significantly greater mean distance from the platform during the probe trial (p< 0.05; AΝOVA not shown). For young and AU rats, the latency to their first entry into the annulus-40 was almost identical (Figure 2A).

Surprisingly, AU rats spent significantly less total time in the annulus-40 than young controls (Figure 2B; P<0.05: AΝOVA). This suggested that AU rats may have used a distinct (e.g. non-spatial) strategy to locate the platform. However, when a curtain was placed around the pool to obscure the extra-maze cues, dwell times for Y and AU rats fell to the level seen in Al rats, while the dwell time for the Al rats was unchanged (Figure 2B).

EXAMPLE 3 BIBΝ-99 improves spatial learning in Al rats.

Al rats treated with BIBN-99 one hour prior to re-testing in the water maze task exhibited significantly shorter escape latencies than the vehicle-treated controls (Figure 3A; p <0.05; ANOVA). Treatment with BIBN-99 did not significantly alter swim speed (Vehicle:20.3 + 0.5 cm/sec; BIBN-99: 20.4 + 0.6 cm sec). During the probe trial, latency to first annulus-40 entry was also significantly reduced in the drug-treated group (Figure 3B; p< 0.05, ANOVA).

EXAMPLE 4 Slices from young and aged rats respond similarly to electrical stimulation.

Input-output (stimulus-response) curves were generated from slices from young (Y) aged-unimpaired (AU) and aged-impaired (Al) rats by plotting the fEPSP slope as a function of fiber volley amplitude. For a given fiber volley amplitude, evoked responses from aged slices were consistently smaller than those from young controls (Figure 4A). However, input-output curves for AU and Al animals were not statistically different. In addition, no significant difference in paired-pulse facilitation was observed between any groups across a range of inter-stimulus intervals (Figure 4B).

EXAMPLE 5 Theta-frequency stimulation in vitro reveals an LTP deficit in Al rats.

Thirty seconds of 5Hz stimulation routinely induced robust LTP in slices taken from young rats. This form of potentiation rose gradually and stabilized within 10-15 minutes following the 5Hz train (Figure 5A). LTP slices from AU rats were comparable to that seen in young animals, but LTP elicited from Al slices was significantly smaller (Figure 5A; p <0.001), ANOVA). During 5Hz stimulation, complex spiking gradually emerged and was observed in all three groups (Figure 5B). No dramatic differences were seen in the average latency to spiking, as measured by the stimulus number at which half the slices experienced the first spike (Y: #54; AU:#50; AI:#53). No difference in the total number of spikes evoked was observed (Y:131 + 10; AU:120 + 11; A 131 ± 18).

Parallel experiments were run in which higher frequency stimulation was applied to slices taken from the opposite hippocampus of the same animals to determine whether the depressed LTP observed in Al rats was a function of the 5Hz stimulation protocol. In these slices, LTP was induced with 3 x 0.5s trains of 30 Hz stimulation, with a 5min inter-train interval. Under these conditions, LTP amplitude at 3 hr was significantly reduced in Al rats (p<0.01) and nearly so in AU rats (p=0.052) compared to young controls (Figure 5C); but no statistically significant AU-AI difference emerged. In slices from a separate set of animals, increasing the stimulation frequence to 70Hz generated a more robust but equivalent level of LTP in all three groups (Figure 5D).

EXAMPLE 6 LTP deficits in Al rats do not arise from reduced NMDA receptor function.

5Hz LTP in adult mice has previously been shown to be partially NMDA-receptor dependent (Thomas et al. (1998) J. Neurosci. 18:7118-7126). In agreement with this finding, bath applications of 50 μM APN significantly depressed, but did not completely block, 5Hz LTP in young adult rats (Figure 6A). Exposure to APV also completely prevented the appearance of complex spiking during 5Hz stimulation. In these experiments, a second train of 5Hz stimulation applied in the same slice 50 min after APV washout resulted in complex spiking (total spike # = 98 + 12) and robust LTP [158 +6, n=7(4)] that was comparable to that seen in naϊve young slices [162 + 7%, n=17(8)]. Because NMDA-R subunit expression is depressed in aged rats (Davis et al. (1993) Neurobiol. Aging 14:107-115 and Adams (2001) J. Comp. Neurol. 432: 230-243).The possibility that the selective impairment in 5Hz LTP in rats reflected a reduced capacity for LTP induction was considered. NMDA-R mediated responses in young, AU, and Al rats was examined. In the presence of CNQX, glycine and low Mg, fEPSPs evoked in all groups could be blocked with 50 μM APV (not shown). NMDA-receptor mediated fEPSPs recorded from both AU and Al slices were smaller on average (30%) than those seen in young controls over a range of stimulus intensities (Figure 6B). The NMD A fEPSPs recorded in slices from AU and Al animals were indistinguishable from each other.

EXAMPLE 7 5Hz LTP correlates with performance in the Morris water maze.

5 Hz LTP correlated with performance in the Morris water maze. LTP amplitude, measured at 1 hr after LTP induction, was plotted against various behavioral measures (Figure 7). Regression analysis revealed a strong negative correlation between 5Hz LTP and the average escape latency among aged rats (Figure 7A; p<0.001). Moreover, 5Hz LTP was also highly correlated with two measures or probe trial performance: 1) latency to the first entry into the annulus-40 (Figure 7B; p< 0.002); and 2) cumulative dwell time in the annulus-40 (Figure 7C; p<0.01).

EXAMPLE 8 BIBN-99 enhances 5Hz LTP Since BIBN-99 improved water maze performace in Al rats, we tested the possible linkage between 5Hz LTP and spatial learning by asking whether BIBN-99 could also enhance 5Hz LTP. Bath application of 1 μM BIBN-99 to Al slices for 30 minutes had no observable effects on baseline responses either during drug delivery or 60 min after washout [104 + 4%, n=5(2)]. hi contrast, BIBN-99 significantly enhanced the degree of synaptic potentiation in Al slices following 5Hz stimulation (control: 118 + 3%; BIBN-99: 139 + 4%; Figure 8A). This action was unrelated to any direct potentiating effect on the NMD A receptor itself, as a 30 min application of 1 μM BIBN-99 did not alter the slope of the isolated NMD A fEPSP recorded in Al slices (103 + 2% of baseline; n=6). In addition, BIBN-99 did not change the onset or total number of complex spikes evoked during the 5Hz train (Figure 8B). In contrast to its effect on 5Hz LTP, BIBN-99 (lμM) failed to enhance LTP in Al slices evoked by repeated 70 Hz trains (Figure 8C). Finally, the same concentration of BIBN-99 did not modify the 5Hz LTP recorded in slices from young rats (control: 152 + 8%; BIBN-99: 144 + 7).

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

While the invention has been illustrated with respect to a novel method for eliciting long term potentiation (LTP) in the brain of an animal and identifying compounds which can enhance LTP and thus cognition and spatial learning, it is apparent that variations and modifications of the invention can be made without departing from the spirit or scope of the invention.

Claims

What is claimed is:

1. A method for eliciting long term potentiation in the brain of an animal comprising applying an electrical stimulation of 4 to 12 Hz, which yields an electrophysiological output with long term stability.

2. A method of claim 1, wherein the long term potentiation occurs in a brain tissue sample from the animal.

3. A method of claim 1, wherein the long term potentiation occurs in the hippocampus.

4. A method of claim 1, wherein the animal is impaired.

5. A method of claim 1 , wherein the animal is a mammal.

6. A method of claim 5, wherein the animal is a rat.

7. A method of claim 1, comprising applying an electrical stimulation of 5

Hz.

8. A method of claim 7, wherein the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

9. A method for identifying a compound, which can enhance long term potentiation in the brain of an animal, comprising applying an electrical stimulation of 4 to 12 Hz to the brain of the animal, measuring long term potentiation resulting from the application of said electrical stimulation in the presence of the compound, measuring long term potentiation resulting from the application of said electrical stimulation in the absence of the compound, and comparing the duration of long term potentiation in the presence of the compound and in the absence of the compound to determine if the compound enhances long term potentiation.

10. A method of claim 9, wherein the electrical stimulation is applied to a brain tissue sample from the ammal.

11. A method of claim 9, wherein the electrical stimulation is applied to the hippocampus.

12. A method of claim 9, wherein the animal is impaired.

13. A method of claim 9, wherein the animal is a mammal.

14. A method of claim 13, wherein the animal is a rat.

15. A method of claim 9, comprising applying an electrical stimulation of 5 Hz.

16. A method of claim 15, wherein the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

17. A method of claim 9, wherein the compound improves performance of an animal in a spatial learning task.

18. A method of claim 9, wherein the compound enhances memory in an animal.

19. A method of claim 9, wherein the compound is a M2 receptor antagonist, a PDE4 inhibitor, or a nicotinic alpha-7 modulator.

20. A method for distinguishing learning-impaired animals from unimpaired animals, comprising applying an electrical stimulation of 4 to 12 Hz to the brain of a learning-impaired animal, measuring long term potentiation, and comparing the measured long term potentiation to that exhibited by an unimpaired ammal.

21. A method of claim 20, wherein the electrical stimulation is applied to a brain tissue sample from the animal.

22. A method of claim 20, wherein the electrical stimulation is applied to the hippocampus.

23. A method of claim 20, wherein the animal is a mammal.

24. A method of claim 23, wherein the animal is a rat.

25. A method of claim 20, comprising applying an electrical stimulation of 5 Hz.

26. A method of claim 25, wherein the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

27. A method for detennining the extent of a cognitive disorder, a memory impairment, or a learning disability in an animal, comprising stimulating the brain of the animal with memory impairment with an electrical stimulation of 4 to 12 Hz, and measuring the magnitude of long term potentiation.

28. A method of claim 27, wherein the electrical stimulation is applied to a brain tissue sample from the animal.

29. A method of claim 27, wherein the electrical stimulation is applied to the hippocampus.

30. A method of claim 27, wherein the animal is a mammal.

31. A method of claim 30, wherein the animal is a rat.

32. A method of claim 27, wherein the electrical stimulation is 5 Hz.

33. A method of claim 32, wherein the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

34. A method for treating a subject with a cognitive disorder, a memory loss, or a learning disability, comprising administering to the subject a therapeutically effective amount of a compound that enhances long term potentiation and is identified by applying an electrical stimulation of 4 to 12 Hz to a brain of an animal, measuring long term potentiation resulting from the application of said electrical stimulation in the presence of the compound, measuring long term potentiation resulting from application of said electrical stimulation in the absence of the compound, and comparing the duration of long term potentiation in the presence of the compound and in the absence of the compound to determine if the compound enhances long term potentiation.

35. A method of claim 34, wherein the electrical stimulation is applied to a brain tissue sample from the animal.

36. A method of claim 34, wherein the electrical stimulation is applied to the hippocampus.

37. A method of claim 34, wherein the animal is a mammal.

38. A method of claim 37, wherein the animal is a rat.

39. A method of claim 34, wherein the compound is identified using an electrical stimulation of 5 Hz.

40. A method of claim 39, wherein the electrical stimulation is applied over a 15 to 60 second time period and which yields a sustained electrophysiological output that lasts for at least 3 hours.

41. A method of claim 34, wherein the subject is a mammal.

42. A method of claim 41, wherein the subject is a human.