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Experimental Gerontology, Vol. 32, Nos. 4/5, pp. 451-469, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved

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EFFECT OF NEUROPEPTIDES ON COGNITIVE FUNCTION

G . W . BENNETr, T.M. BALLARD, C.D. WATSON and K.C.F. FONE

Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom

Abstract--Recent evidence indicates that, in addition to the involvement of cholinergic and other neurotransmitter systems, various neuropeptides that occur in cortical and subcortical brain regions have a role in cognitive behavior. This evidence results largely from behavioral studies in rodents and other animals, following peptide administration and only in a very few cases from similar studies in human subjects. Several neuropeptides studied appear to enhance or produce changes conducive to improvement in cognitive performance and these include vasopressin, corticotrophin-releasing hormone (CRH), somatostatin, substance P, neuropeptide Y, and thyrotrophin-releasing hormone (TRH), while one peptide, galanin, has been reported to inhibit cognitive processes. Of those neuropeptides that improve perfor- mance, only TRH has been shown recently to attenuate the memory impairment of human subjects and Alzheimer patients treated with an anticholinergic drug, and this review de- scribes a series of complimentary studies in adult and aged rodents that contribute to our understanding of the possible mechanisms involved in the role of TRH in cognition. © 1997 Elsevier Science Inc.

Key Words: TRH, neuropeptides, cognitive function, behavior

INTRODUCTION

IT :S WELL documented that the ability to perform cognitive tasks declines with age in animals (Hagan and Morris, 1988) and humans (Bartus et al., 1982), although the mechanisms respon- sible for age-related memory loss are not well understood. A number of authors have concluded previously that the normal aging process is associated with a reduction in brain cholinergic function consistent with the loss of cholinergic neurons, associated with memory loss in Alzheimer's disease (Rossor, 1988) and similar changes in aged animals (Decker, 1987; Fischer et al., 1989). This has been demonstrated by morphological and functional data (Fisher et al., 1989; Geula and Mesulam, 1989; Altavista et al., 1990) in aged nondemented humans and rodents, and these morphological changes are associated with a functional deficit that has been demonstrated directly in animals (Decker, 1987). At the previous First International Symposium

Correspondence to: G.W. Bennett

451

452 G.W. BENNETT et al

TABLE 1. THYROTROPIN RELEASING HORMONE [TRH] AND CENTRAL NERVOUS SYSTEM CHOL1NERGIC FUNCTION (A SUMMARY OF RECENT REPORTS)

Depolarization of spinal motorneurons by TRH Nicoll 1977 TRH distribution in rat spinal cord Kardon et al. 1977 TRH antagonism of pentobarbital narcosis Kalivas and Horita 1980 TRH and CNS cholinergic neurons (review) Yarbrough 1983 Analeptic location of action of TRH in brain Sharp et aL 1984b TRH enhances ChAT in cultured spinal neurons Schmidt-Achert et al. 1984 TRH increases spontaneous firing of septal-hippocampal neurons Lamour et al. 1985 TRH analog (MK771) reverses neurochemical and learning Horita et al. 1989

deficits in septal lesioned rat TRH and analogs increase acetylcholine release in viw~

TRH binding sites in infant and adult hippocampus TRH analogue (RX77368) reverses the anticognitive effects of

cholinergic antagonists

Hutson et al. 1990 Giovannini et al. 1991 Okada 1991 Eymin et al. 1993 Watson et al. 1990

1992 1994

on the Neurobiology and Neuroendocrinology of Aging, Pepeu et al. (1993) presented the evidence for a predominant cholinergic deficiency in humans and animals and indicated some of the attempts at correcting this in aging and senile dementia. Recent studies using selective neurotoxins to lesion specific groups of cholinergic neurons (Dunnett et al., 1991) have led to some debate whether all basal forebrain cholinergic neuron projections are associated with cognitive processing, but significant advances in our understanding of the underlying mecha- nisms has depended on this supposition and resulted in significant progress in our understanding of learning and memory. A further consideration, however, is what factors influence the function and survival of these neurons. For example, there are neuromodulators that may be paramount in maintaining the integrity of the cholinergic and other neurotransmitter systems. Many such neuromodulators appear to be neuropeptides. Various neuropeptides promote cholinergic func- tion and the release of acetylcholine, but one neuropeptide of particular interest in this respect is thyrotrophin releasing hormone (TRH) (Table 1), and it is this peptide that will be the major subject of this review.

NEUROPEPTIDES AND COGNITION

In addition to cholinergic neurons, there is evidence that various neurotransmitters are involved in cognition (Rossor, 1988), and these include a number of neuropeptides that have been implicated as modulators of cognitive processes. The evidence for this derives primarily from behavioral studies in animals using maze or choice tests, and to a lesser extent from similar studies in humans. In a number of cases these studies have been supported by evidence that the neuropeptide levels may change in aged animals or human dementia. The neuropeptides that have been studied in these ways include vasopressin (de Wied and van Ree, 1982), CRH, (Bissette et al., 1985), somatostatin (Rossor, 1988; Fitzgerald and Dokla, 1989), galanin (Givens et al., 1992; Ogren et al., 1992), substance P (Hasenrhrl et al., 1990) and neuropeptide Y (Flood et al., 1989). The aging of neuropeptide signals in rats with an emphasis on the latter peptide was also the subject of a presentation by Kalra et al. (1993) at the First International Symposium on Neurobiology and Neuroendocrinology of Aging. In addition, elevated dynorphin levels in

NEUROPEPTIDES, TRH. AND COGNITION 453

the hippocampus of aged rats has been associated with a learning deficit, and these studies thus implicate opioid peptides in the aging process (Jiang et al., 1989).

Some of the earliest animal studies on the potential role of vasopressin in learning and memory were reviewed a number of years previously (de Wied and van Ree, 1982). Some authors have criticized the interpretation of these earlier studies because the observed behavioral effects may involve performance improvement resulting from changes in motivation, attention, or vigilance and which can too easily and erroneously be interpreted as solely memory improvement. As will be seen later in this review, this may well be the mechanism by which a number of peptides in the CNS are able to enhance the learning process. Vasopressin analogs, however, were one of the earliest neuropeptides to be used in human subjects to attempt to improve cognitive behavior (de Wied and van Ree, 1982) with limited and variable success.

Postmortem examination of brains from Alzheimer patients has revealed elevated hypotha- lamic levels of vasopressin and neurotensin, a reduction of cortical and CSF levels of somatostatin, substance P, and CRH, but no change in other neuropeptides measured (Rossor et al., 1984, Bissette et al., 1985, Beal and Mazurek, 1987, Gottfries, 1990, Wallin et al., 1990). Further- more, the changes in CRH and somatostatin immunoreactivity in the cerebral cortex correlated with a reduction in choline acetyltransferase (CHAT) activity (an enzyme marker of cholinergic function), and it has been proposed that the degeneration of somatostatinergic neurons may trigger the well-described loss of cholinergic neurons characteristic of the disease (Rossor, 1988).

In the brain, the neuropeptide galanin inhibits the evoked release of acetylcholine (ACh), and other neurotransmitters (Fisone et al., 1987) and is thought to initiate inhibitory modulation of such systems. Based on the inhibitory action of galanin on cholinergic activity, it has been proposed that galanin may have an important role in learning and memory processes (Givens et al., 1992; Ogren et al., 1992). In Alzheimer's disease, the presence of galanin could enhance the inhibition of the surviving cholinergic system, so worsening the effects of the cholinergic loss, hence, negating the beneficial effects of drugs that increase cholinergic activity (Crawley, 1993). Based on these findings, it has been proposed that the administration of galanin antagonists could block the inhibitory effects of galanin on the cholinergic system and also those actions arising from other galanin containing pathways that innervate areas associated with cognitive processes (C~gren et al., 1992; Crawley, 1993). Consistent with this, the central administration of the galanin antagonist M35 (galanin(1-130-bradykinin(2-9)amide) was shown to facilitate both the acquisition and retention of the Morris water maze task in rats (Ogren et al., 1992, see below). Despite these findings, however, water maze studies with galanin in our laboratory have not confirmed these earlier behavioral findings and failed to find any change in ChAT activity (Aspley and Fone, 1993).

A potential role for somatostatin and galanin in dementia has been based on information largely obtained from animal studies. Our understanding of the role of neuropeptides in brain neuronal function is in its infancy, and coupled with the fact of the instability of these substances, the potential of peptides as therapeutic agents in Alzheimer's disease and other long-term neurodegenerative diseases is a long way from realization. However, one neuropep- tide, TRH, has been shown recently to improve cognitive performance in patients with Alzhei- mer's disease (Mellow et al., 1989, 1993) and other human volunteer subjects (Molchan et al., 1990, 1992) treated with the muscarinic cholinergic antagonist, scopolamine. The remainder of this review will concentrate, therefore, on a series of studies in animals carded out in our laboratory in Nottingham, which in general, support these findings in humans.

454 G.W. BENNETT et aL

THYROTROPHIN-RELEASING HORMONE (TRH) AND BEHAVIOR

TRH is a tripeptide, identified in 1969 as L-pyroglutamyl-L-histidyl-L-proline amide and was the first of the hypothalamic-releasing hormones to be isolated and characterized. The name TRH derives from it's action in the anterior pituitary, where it stimulates release of thyrotrophin as well as prolactin, and at times growth hormone (Jackson, 1982). TRH has been shown to be distributed throughout the extrahypothalamic nervous system (Hrkfelt et al., 1975), and early studies suggested that TRH may have a role in neurotransmission and that this function evolved prior to its thyrotrophin-releasing property (Morley, 1979). TRH has been shown to produce a variety of behavioral changes and neuropharmacological effects independent of its thyrotrophin- releasing properties by acting via an interaction with membrane receptors (Metcalf and Jackson, 1989). TRH is an established neurotransmitter/neuromodulator in the CNS (Bennett et al. , 1989) and such evidence has led to investigations into its use as a therapeutic agent in a variety of disorders, including Alzheimer's disease (Griffiths, 1987; Metcalf and Jackson, 1989; Kelly, 1995). In animals, previous studies have shown that TRH produces the following hyperactivity behaviors: enhanced locomotor activity and rearing (Sharp et al. , 1984a) and wet-dog shakes and forepaw licking (Sills et al., 1988). TRH also has an analeptic effect, because it reduces the duration of various depressant drugs (Sharp et al., 1984b; Yamamoto and Shimizu, 1989) and increases cerebral and peripheral blood flow (Koskinen, 1989).

TRH is rapidly metabolized in the body and slowly penetrates the blood-brain barrier, so when applied systemically in animals and humans, large concentrations are required to obtain behavioral effects. It has been shown that peak levels of TRH in the brain are reached five minutes after IP or IV infusion, 10 min after IM, or 30 min after oral administration, which overall corresponded to 0.02-0.20% of the total concentration of TRH administered (Mitsuma and Nogimori, 1983). However, there are a number of strategies to overcome the limitations of TRH as a therapeutic agent and these include the use of analogs of TRH, peptide mimetics, inhibitors of metabolic enzymes, and different methods of administration, such as injectable slow-releasing microspheres or intrathecal delivery (Kelly, 1995). Various analogs of TRH have been synthesized in order to overcome the problems of rapid degradation in therapeutic use, and these have also enabled the study of the behavioral and pharmacological properties of TRH in animals. If TRH-like compounds are to be used in the treatment of disorders, then a compound that retains its central effects, without its thyrotrophin-releasing property, is required. The enzymatically resistant TRH analog, RX77368 (L-pyroglytamyl-L-histidyl-L-3,3-dimethyl-pro- lineamide; Fig. 1), has been used in our laboratory at Nottingham because it has been shown to have a similar pharmacological profile to that of TRH but with a greater CNS potency (Metcalf, 1982) and, like TRH, shows positive effects on cognition in aged rats (Watson et al. , 1993) and scopolamine-treated rats (Watson et al. , 1994). These and other behavioural studies with RX77368 are described below in the present review.

Early studies suggested that TRH may be useful as an antidepressant (Kastin et al. , 1972), but such a potential has not been realised, even with the advent of stable analogs. In the previous decade, several other areas of possible therapeutic use have been identified, including the treatment of depression (Metcalf, 1982), epilepsy (Matsuishi et al., 1983), the reversal of anesthesia and narcotic overdose (Griffiths, 1987), spinal trauma and brain injury (Faden et al. ,

1988), motor neuron disease (Guiloff, 1989), cerebral ischemia (Shrewsbury-Gee et al., 1988), as well as Alzheimer's disease (Mellow, 1989, 1993). Evidence of the potential value of TRH and its analogs in the treatment of CNS disorders and trauma has been reviewed recently (Kelly, 1995). Faden and colleagues have shown in a number of studies of experimental spinal injury

NEUROPEPTIDES, TRH, AND COGNITION

H O

TRH . o c . 2

~NH CONH2

~ , , ~ 0 H 0 CH3 TRH analogue ~ ' ~ ' ~ N. /~

RX77368 H .~,HCH2 _X."E~....CH3 (~ "NH L;UINPI2

FIG. 1. The structure of thyrotrophin-releasing hormone (TRH) and the metabolism resistant analog of TRH, RX77368, used in the behavioral studies.

455

in the cat that TRH and certain analogs improve neurological recovery (Faden et al., 1988). TRH may also be useful for the treatment of anaphylactic shock, because it has been found to improve survival and attenuate the hyperglycemic effects of anaphylactic shock in mice (Holaday et al., 1989). There has been some experimental evidence that stable TRH analogs may be beneficial in stroke due to recovery effects following cerebral ischemia, which appear to relate to improvements in cerebral blood flow. The TRH analog, RX77368, when administered imme- diately, 5 min, and 24 h after middle cerebral artery occlusion in the rat caused a significant reduction in the loss of neuronal function, which ultimately resulted in a much reduced ischemic area compared with saline treated controls (Shrewsbury-Gee et al., 1988). Moreover, TRH has been shown to have neurotrophic effects in the treatment of amyotrophic lateral sclerosis (Williams and Windebank, 1991). Although the value of TRH analogs may be of little benefit in reducing the progression of this neurodegenerative disease, in one study a single dose of RX77368, improved bulbar function and reduced spasticity and some of these patients on repeated dosing maintained their improvements for up to two weeks (Guillof, 1989).

TRH AND COGNITION

The Morris Water Maze test, first described by Morris (1981) as a rodent test of spatial learning and memory, is advantageous over other tests because it is simple to learn and does not involve food deprivation or electric shock treatment. The rat has to learn to find a fixed hidden platform in an opaque water-filled pool by utilizing distal cues positioned around the maze. The water provides an intramaze environment, which eliminates olfactory cues. Extramaze cues have been shown to be used by the rats to orient spatially in this task, because reduction or loss of performance occurs when a curtain is used to surround the pool (Morris, 1984). The water maze has been widely used since its introduction to investigate different protocols and phar- macological or surgical intervention (McNamara and Skelton, 1993), and for these reasons we have used the test in our studies with TRH. The water maze used in our laboratory is a large circular, white fiber glass pool (2 m diameter and 0.7 m high) positioned at one end of a large room in the animal behavioral suite at Nottingham. The maze is filled with water (21°C) made opaque using a white artificial opacifier and a colorless perspex square platform (10 × l0 cm) is positioned at one of four possible locations in the maze, arbitrarily divided into four quadrants,

456 G.W. BENNETT et al.

equidistant from the walls and center of the maze and the maze was surrounded by visual cues, which included large black and white patterned posters. Previous evidence indicates that TRH and TRH analogs enhance performance in the Morris water maze, although this has only been shown following treatment that induces memory disruption (Yehuda, 1987; Watson et al., 1990; Khan et al., 1994).

The effect o f central pretreatment with the TRH analog (RX77368) on a) atropine-induced learning deficits on a water maze, and b) scopolamine-induced deficits in a radial maze in adult rats

Following the earlier demonstration that TRH and its analogs may be beneficial in the treatment of Alzheimer's disease (Mellow et al., 1989) we undertook a study to determine the effect of RX77368 on the spatial learning deficit of atropine-treated male Hooded Lister rats during the acquisition training to an unbiased platform position based on the method of Morris, 1981 (Watson et al., 1990). Four groups of intracerebroventricular (ICV) cannulated rats were injected 45 min prior to the first of two daily trials over five days with either intraperitoneal (IP) injections of saline or atropine (6 rag. kg l), which were infused with RX77368 (10 txg ICV) or saline 30 min later. The results clearly demonstrated that the time taken to find the platform significantly decreased over the 10 trials indicating learning, but compared with saline controls, learning in the saline/atropine group was significantly inhibited (NOVA F = 10.48, p = 0.006, comparing both within and between group differences) and this effect was significantly reversed (F = 11.9, p = 0.004) in the RX77368/atropine, but not the RX77368/saline group, indicating that RX77368 could counteract the cognitive deficit produced by the muscarinic antagonist, atropine. After the behavioral study, the animals were killed by decapitation and selected brain tissues were used to determine immunoreactive TRH levels and the activity of (CHAT). The TRH levels in hippocampus and septum were significantly elevated (51 and 75% p < 0.01, Student's t-test) in the atropine animals compared with controls, while ChAT activity in the septum was significantly (37% p < -0.05) reduced. These early behavioral and biochemical observations from our laboratory provided a) the clear indication that TRH modulates cholin- ergic function in the septal-hippocampal pathway, b) suggests a mechanism that could explain the involvement of this peptide in learning, and c) prompted the undertaking of further studies.

To assess whether the apparent cognitive enhancement produced by TRH is mediated by a cholinergic mechanism, the effect of a repeated ICV administration of RX77368 on a learning deficit induced by the brain selective, muscarinic antagonist, scopolamine was examined using an eight-arm radial maze paradigm (Watson et al., 1992, 1994). In essence, the results (not shown) demonstrated that scopolamine (0.3 mg" kg -~) pretreatment 30 rain prior to testing produced a significant deficit in the number of unrepeated arm entries and total arm entries on the maze and also increased the percentage of incorrect arm entries and the total time spent on the maze compared with saline-treated controls. The animals used in this study were also cannulated in one lateral ventricle prior to maze testing and placed on a 23 h food deprivation schedule eight days after recovery from surgery. In this way the effect of RX77368 (2 Ixg ICV) compared with saline given 10 rain prior to the scopolamine injections was evaluated. This prior treatment with the TRH analog produced a partial but significant attenuation of the scopol- amine-induced performance deficit on the maze during the first five days of daily testing for 15 days, as well as enhancing maze performance during this period when given alone. This study again suggested that the TRH analog is capable of improving a cholinergic drug-induced learning deficit. It is possible, however, that scopolamine may reduce the locomotion and

NEUROPEPTIDES, TRH, AND COGNITION 457

exploration behavior on the maze, and the apparent reversal with the RX77368 may have been due to an increase in arousal and exploratory behavior rather than a direct enhancement of an impairment of cognitive memory.

EFFECT OF TRH ON COGNITION IN AGED RATS

Although in human dementia disorders such as Alzheimer's disease the dysfunction of the cholinergic neurons in the forebrain is a predominant neurochemical abnormality, there is also a more widespread dysfunction of other neurotransmitters including noradrenergic and sero- tonergic systems (Rossor, 1988). Consequently, studies using selective pharmacological agents, such as scopolamine, or discrete lesion studies, using septal-hippocampal neurochemical lesions (see below), do not closely model the more widespread deficits characteristic of these neuro- degenerative disorders, although they do enable one to explore the specific role of cholinergic neurons. An alternative approach, which may more closely mimic late onset disorders, is to use aged rats. Such animals demonstrate a decline in cognitive performance, although, as shown by ourselves and others, this effect is variable. Aged rats have been used in a series of experiments in our laboratory to further study the effects of scopolamine on cognitive decline and to determine any possible attenuation of these effects following the administration of the TRH analog, RX77368.

The effect of peripheral pretreatment with the TRH analog, RX77368, on the cognitive behavior of aged rats tested by a) passive avoidance, and b) the water maze

The passive avoidance test, like the Morris water maze task, is a reinforcement behavior, but unlike the latter, which involves a positive-induced reinforcement, the test depends on memory of the stress of a shock and thus relies on a negative-induced reinforcement. Because a) aged rats exhibit a decline in cholinergic function analogous to human dementia disorders (Hagan and Morris, 1988), and b) previous studies in our laboratory suggested that pretreatment with a TRH analog improved cognitive performance in adult animals, it seemed reasonable to evaluate whether similar pretreatment might reverse an age-related learning deficit in rats.

In these studies the performance of aged (20-24 month) rats were compared with similar adult (four month) male Hooded Lister rats, in which groups of animals were pretreated either with RX77368 at 1 or 3 mg. kg -1 IP or saline vehicle as control 30 min prior to testing. The animals were tested over 11 days, using the passive avoidance test on the first two days followed by two days of testing for locomotor activity and then twice dally rats underwent an acquisition training on the water maze for seven days as described previously, including a final probe trial (Watson et al., 1993). Passive avoidance testing in a light-dark compartment, Skinner box consisted of an acquisition (training) trial involving 60 s habituation in the light compartment, followed by -~600 s latency measurement to enter the dark compartment and a scrambled footshock (0.5 m A for 0.5 s) three seconds after entry. Twenty-four hours later a retention (test) trial consisted of a similar time protocol, but no drug pretreatment or footshock in which the latency to enter the dark box was determined. The locomotor activity was determined for 600 s on days 3 and 4 using photocell activity cages and the water maze parameters were as described above.

The passive avoidance results (Fig. 2) demonstrated that all treatment groups showed a highly significant increase (p < 0.001, Kruskall-Wallis followed by multiple comparisons test) in crossover latency in the retention compared with the training trial but aged controls had a significantly (p < 0.05) shorter crossover latency in the training trial than adult controls. In the

458 G W. BENNETT et al.

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FIG. 2. Effect of pretreatment ( -30 rain) of aged (20-24 month) rats with RX77368 (1 and 3 mg • kg J IP) or saline ( 1 ml • kg ~), as indicated, on the latency to crossover (seconds; median and range, n = 9 each) from the light to dark compartment in a passive avoidance box. Latency was measured during a training (day l, drug pretreated followed by a 0.5 s, 0.5 mA footshock 3 s after entering the dark compartment) and a retention trial (day 2, no pretreatment or footshock). Crossover latency significantly increased (p < 0.01, nonparametric multiple comparisons test following sig- nificant Kruskal-Wallis, H --> 59.1, p < 0.001) in the retention compared with the training trial in all four groups, indicating acquisition learning in this behavioral test. In the training trial, all three aged rat groups tended to have a shorter crossover latency to the dark compartment than that of adult controls, but this only reached significance following RX77368 (1 and 3 mg. kg 1) pretreatment (*p < 0.05 and **p < 0.01, respectively). In the retention trial, control aged rats showed a significantly shorter (*p, 0.05) crossover latency than adult controls, consistent with an impairment of cognition while pretreatment with RX77368 (1, but not 3, mg. kg-~) reversed this impairment in retention.

retention trials, however (Fig. 2), aged rats treated with 1, but not 3, mg • k g - ~ RX77368 showed an increased crossover latency, which was comparable with adult controls and was significantly (p < 0.05) longer than that of the aged controls. This effect of RX77368 on the passive avoidance test is unl ikely to result from a change in locomotor activity to a novel env i ronment since photocell activity counts were comparable in all three aged groups on both subsequent days tested. Irrespective of age or treatment, all groups also showed a progressive and signif icant (p < 0.001, F re idman ' s A N O V A ) decrease in the t ime to reach the hidden platform in the water maze over the next seven days (Fig. 3A). However, compared with adult controls the perfor- mance of all three aged groups was highly significantly impaired (p < 0.001) even fol lowing either dose of RX77368 pretreatment. In the final probe test (not shown) aged rats treated with RX77368 (3 m g • kg ~), spent s ignif icantly (p < 0.01) less t ime (14 s) within 50 cm of the removed platform than adult (29 s), aged (19 s), control or 1 rag" k g - l RX77368 (19 s) groups. None of these deficits were associated with a visual or motor impairment , because visual acuity and swim speeds were similar for all groups (3B). The use of two re inforcement cogni t ive tests conf i rm that there is a decline in performance with age in the rat. However, the results of this study indicated that the TRH analog, RX77368, only improved performance of aged rats in the

NEUROPEPTIDES, TRH, AND COGNITION 459

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FIG. 3. Effect of pretreatment ( -30 min) of aged (20-24 month) rats (following the tests shown in Fig. 2) with RX77368 (1 and 3 mg" kg -1 IP) or saline (1 mg. kg 1), as indicated, on the latency (seconds; median, range not shown for clarity, n = 9 each) to reach; i) a submerged platform (A) on the water maze on days 1 to 6 and on day 7; ii) a dark-sided visible platform raised 1 cm above the water and located in a neutral area of the maze (B, visual acuity, upper histograms); and iii) the swim speed measured during final trial (B, lower histograms; measured by retrospective video-analysis by dividing the maze area into squares and counting the number of squares crossed. Performance of all groups significantly (A; chi-squared -> 28, p = 0.001, Freidman's test) improved over time, indicating that all groups of rats acquired the task. However, aged rats exhibited a significant (p < 0.01, nonparametric multiple comparisons tests following significant Kruskal-Wallis, H >-- 40.8, p > 0.001) deficit in maze performance compared with adult controls over the first eight training trials. Performance of all aged groups was comparable with that of adult controls by last trial, RX77368 pretreatment did not improve the performance of aged rats at either of the doses (1 and 3 mg. kg- l) used in this study, while visual acuity (B) and swim speed (B) measurements showed that neither age nor RX77368 pretreatment had any effect on the ability of the rat to identify or swim to the platform.

negat ive ly re inforced nonspat ial task and not in the pos i t ive ly re inforced spatial task. One

possible reason for this m a y be that the pept ide is more e f fec t ive in an acute reference m e m o r y

(short- term) task rather than a chronic spatial reference ( long-term) one.

T R H A N D W O R K I N G M E M O R Y

De layed nonmatch ing- to -pos i t ion ( D N M T P ) and de layed matching- to-pos i t ion (DMTP) tests

have been des igned to provide a sensi t ive measure o f work ing (short- term) m e m o r y (Dunnet t et al., 1989) and consis t o f discrete trials separated by a delay interval, which is r andomized f rom

trial to trial, thus vary ing the m e m o r y chal lenge. These tests require the i nvo lvemen t o f

reference m e m o r y a long with work ing m e m o r y in order to pe r fo rm the test successful ly. This

test, however , has a number o f advantages ove r o ther cogni t ive behaviora l tests such as pass ive

avoidance , which measures an act ive learning process as opposed to a measu remen t o f rate o f

forget t ing ove r t ime. D N M T P / D M T P tasks produce a basel ine o f normal forget t ing, which can

460 ( ; .w. BENNETT et al.

be used to assess the disruptive effect of drug treatment and/or lesions (see later). Any disruptive effects at short delays represent an effect on the ability to perform the task due to nonmnemonic factors (e.g., motor or motivation effects) but disruptive effects at longer delays, which increase as the delay increases, are considered to be an effect on working memory and a working memory curve can be obtained. We, therefore, chose the DNMTP procedure to further examine the cognitive effect of the TRH analog, because rats have a strong preference for novelty and thus acquire the nonmatching rule quicker than a matching one in this test (Sahgal, 1987).

The effect o f peripheral treatment with the TRH analog (RX77368) on a delayed nonmatching-to-position (DNMTP) behavioral test in adult rats

Initially, the acute effect of the TRH analog, RX77368 (1 mg. kg-~ IP) given 30 min prior to testing on the DNMTP test, was examined in pretrained rats, which were then treated 10 min later with scopolamine (0.03 mg .kg -I) or saline administered subcutaneously (sc). The DNMTP method used was modified from that of Dunnett (1985; Ballard et al., 1996b). The results of this study are shown in Fig. 4 in the form of percent correct responses (4A) and the number of missed trials (4B) over increasing delays from 0 to 16 s for the four treatment groups. Scopolamine significantly reduced the number of correct responses at 4 s but treatment with the TRH analog had no effect on this deficit. In contrast, the scopolamine significantly increased the number of missed trials at each delay interval, and RX77368 attenuated these increases. These results suggest that scopolamine may be affecting nonspecific measures, such as motivation and motor performance, which may mask any effects on working memory, but also that the TRH analog appears to reverse such deficits. Clearly, there was a need for a more selective method to that of using the administration of a muscarinic antagonist drug and one such approach is the use of selective procedures to lesion cholinergic neurons.

The use o f excitotoxins to selectively lesion cholinergic neurons in the rat brain

Although previous work based on selective lesioning techniques has provided evidence for cholinergic involvement in learning and memory processes, with the advent of improved toxin selectivity for cholinergic cell bodies, this view has been questioned (Dunnett et al., 1985, 1989, 1991; Fibiger, 1991; Page et al., 1991). Excitotoxins, such as ibotenic acid and kainic acid, produce a greater impairment in learning and memory tasks than the excitotoxins, AMPA, and quisqualic acid, which are more selective for cholinergic cell bodies and produce greater depletion of ChAT activity. It has been implicated from such studies that intact cholinergic projections from the basal forebrain are not required for the performance of all cognitive tasks. Despite this, there is still evidence that cholinergic neurons are required for performance of certain types of cognitive behavior. In particular, various septal-hippocampal lesioning proce- dures have been shown to disrupt acquisition of a hidden platform position in the water maze test. These include ibotenic acid-induced septal-hippocampal lesions (Hagan et al., 1988), or electrolytic-induced medial septial lesions (Kelsey and Landry, 1988) or ICV administration of the immunotoxin, 192 IgG-saporin conjugate (Nilsson et al., 1992), all of which showed a disruption of the acquisition of a fixed platform position in the maze. These studies provide further evidence that the septal-hippocampal pathway, and particularly the cholinergic projec- tion, are involved in spatial learning.

Other studies have shown that either fimbria-fornix lesions (Dunnett, 1985; Aggleton et al., 1991) or AMPA-induced medial-septal lesions (McAlonan et al., 1995b) or peripherally ad- ministered scopolamine (Cole et al., 1994) disrupt working memory in an operant DNMTP task,

NEUROPEPTIDES, TRH, A N D C O G N I T I O N 461

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FIG. 4. DNMTP pretrained rats were administered four acute treatments on separate test days in a balanced crossover design: saline (n = 12); RX77368 (1.0 mg" kg-1) (n = 12); scopolamine (0.03 mg. kg - t ) (n = 12); RX77368 and scopolamine (n = 12) and tested on the DNMTP task. Graphs show the percent correct choices against increasing time delays. (A) Shows that scopolamine significantly reduced percent correct responses at 4 s delay (*p < 0.05); RX77368 was unable to counteract the scopolamine-induced reduction in percent correct response at 2 and 4 s delay (+p < 0.05). (B) shows that scopolamine significantly increased the number of missed trials (**p < 0.01) at each delay interval, whereas RX77368 attenuated the scopolamine-induced increase in number of missed trials (+p < 0.05, + +p < 0.01). All data are expressed as mean --- SEM. Statistical analysis: ANOVA and Newman-Keuls.

i nd i ca t i ng the r e q u i r e m e n t o f cho l ine rg i c n e u r o n s in th is task. C h o l i n e r g i c - r i c h graf ts in the

h i p p o c a m p u s or n e o c o r t e x o f aged (24 m o n t h s ) ra ts s ign i f i can t ly a m e l i o r a t e d d e l a y - d e p e n d e n t

def ic i t s in a D N M T P task at i n t e r m e d i a t e de lays , w h i c h impl i ca tes the i n v o l v e m e n t o f cor t ica l

and h i p p o c a m p a l cho l ine rg i c p ro jec t ions ( D u n n e t t et al., 1988). R e c e n t s tudies u s ing A M P A -

462 G.W. BENNE~'etat.

induced septal-hippocampal projection lesions have been shown to disrupt aversive conditioned behavior (McAlonan et al., 1995a) and also delay-dependent memory impairment in a spatial delayed nonmatching-to-sample task (McAlonan et al., 1995b). Both these studies indicate that this cholinergic projection modulates working memory processes.

Effect o f peripheral treatment with the TRH analog, RX77368, on cognitive behavior in AMPA-induced septal-hippocampal-lesioned rats

Because the cholinergic enhancing effects of TRH appear to involve the cholinergic projec- tion in the septal-hippocampal pathway (see Table 1 ), it was of interest to determine the effects of selectively lesioning this pathway on the effect of TRH on cognitive behaviors, and a preliminary study (Horita et al., 1989) has also suggested the feasibility of this approach. Based on other recent studies, summarized above, it was decided to use the operant DNMTP task to determine the effect of the TRH analog, RX77368, on working memory following the AMPA- induced lesion of the septal-hippocampal pathway (Ballard et al., 1996b).

In this study, AMPA ((RS)-a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid; 1.8 IxL of 9 mM) or vehicle was infused into the medial septum/diagonal band area of male Lister Hooded rats, pretrained on an operant delayed nonmatching-to-position (DNMTP) test based on the method of Dunnett (1985). Following 10-14 days recovery, rats were tested once daily on DNMTP, 30 rain postadministration of RX77368 (1.0 mg" kg -~, IP) or saline using balanced groups of animals. The lesion reduced ChAT activity in hippocampi by 41%, which was not affected by treatment with the TRH analog. Locomotor activity was also measured one day before and one day after DNMTP testing and confirmed that, overall, none of the treatments affected this measure of motor performance.

The behavioral results of the DNMTP testing were determined in the form of percent correct choices shown by each of the treatment groups at increasing time delays, in which the results are combined over consecutive days of testing, namely 1-4, 6-7, 8-10, and 11-13. The most significant differences between the groups were observed in the latter period and are shown in Fig. 5. The lesioned group showed a significant reduction in the correct responses at 4, 8, and 16 s delays (**p < 0.01). However, treatment with the TRH analog, RX77368, resulted in a significant improvement compared with the lesioned rats at 8 s delay and partially so at 16 s delay. Similarly, when the percent correct responses on the first (day 1) and last day (day 13) of testing are compared for the AMPA lesioned animals (Fig. 6A) and the RX77368 treated lesioned animals (Fig. 6B), it can be seen that the lesioned animals injected with the TRH analog showed a significant improvement in their correct response at both 8 and 16 s delays, but the untreated lesioned rats showed no improvement.

An earlier study demonstrated that medial septal lesions disrupt spatial mapping in rats (Kelsey and Landry, 1988). In a further study in our laboratory, therefore, the action of the TRH analog, RX77368, on learning and memory was tested in AMPA-induced septal-hippocampal- lesioned rats using the Morris water maze test under three consecutive protocols. This study consisted of (1) acquisition learning; (2) reversal learning; and (3) working memory protocols and used a video-tracking, computer-driven procedure to monitor the position, swim speed, and latency to find the platform. All rats were given a pre- and postsurgery swim and cue trial (using a visible platform) of 120 s duration to assess that swim performance and visual acuity were normal. Rats were infused with AMPA (1.8 txL of 9 raM) or vehicle (0.2 M buffered saline) into the medial septum/diagonal band area under halothane (1.5%) anesthesia as described previ- ously (Ballard et al., 1995, 1996a) and allowed 10-14-day recovery prior to the main study. For

NEUROPEPT1DES, TRH, AND COGNITION 463

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FIG. 5. Graph showing the effect of AMPA-induced septal-hippocampal lesions and RX77368 (1 m g - k g - l , daily) on percent correct choices at each delay interval on the DNMTP task (see text) using the four treatment groups indicated. Data are combined for days 11 to 13 of testing and presented as mean percent correct responses _+ SEM. There was a significant effect of treatment (p < 0.01, ANOVA) and treatment × delay interaction (p < 0.01): the AMPA-SAL (AMPA-saline) group showed a significant reduction in percent correct responses at 4, 8, and 16 s delay (**p < 0.01). The AMPA-RX (AMPA-RX77368) group differed significantly from the AMPA-SAL (AMPA-saline) group at 8 s delay (+p < 0.05), but showed only partial improvement at 16 s delay.

the acquisition leaming stage of the study, the animals were given two trials per day (max 120 s) for 11 days. For each trial, the rat remained or was placed on the platform for 20 s. Twenty-four hours postacquisition the animals underwent a probe trial for 60 s to determine their swim pattern in the absence of a platform. For the reversal learning, the rats were trained to a new platform position using a similar protocol for eight days before a further probe trial. Then, finally, the working memory test consisted of again a repeated protocol for four days using a different platform position for each day. This was done to determine repeated daily learning in the form of latency differences between the two trials each day separated by 30 s using the same platform position. As in all lesion studies, the lesion effectiveness was confirmed by histological staining and determination of hippocampal ChAT activity. As before, groups of lesioned animals were tested with RX77368 (1 mg- kg-l IP) against saline controls and compared with equivalent groups in sham-lesioned animals, which underwent all surgical procedures without the AMPA injection.

Statistical analysis, using a two-factor ANOVA with repeated measures followed by posthoc Newman-Keuls test showed no difference between the four groups across all trials during acquisition and reversal learning (data not shown). However, during the acquisition and reversal probe trials, lesioned rats spent significantly (p < 0.05) less time in the platform area than sham rats. These results suggest that the rate of learning a platform position is unaffected in lesioned rats, yet the probe trial reveals impaired localization in these animals, although the TRH analog treatment did not affect this. In contrast, however, during the working memory study, the TRH analog altered the response of the lesioned rats. While the sham-lesioned rats showed a significantly reduced (p < 0.001, one-factor ANOVA) latency to find the platform in trial 2

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FIG. 6. Graphs showing a comparison between the first (day 1) and last (day 13) days of testing on the DNMTP test of AMPA-induced septal-hippocampal rats with and without treatment with RX77368 (1 mg. kg ~ daily). Data are presented as percent correct responses (mean _+ SEM) at each delay interval. (A) Shows the AMPA-SAL (AMPA-saline) group did not show significant improve- ment in percent correct responses from day 1 to day 13. (B) Shows the AMPA-RX (AMPA- RX77368) group, however, improved DNMTP performance on day 13, at delays 8 s (**p < 0.0l ) and 16 s (*p < 0.05).

compared with trial 1 (Fig. 7), the lesioned rats showed no difference in latency between the trials, but the lesioned rats treated with RX77368 did show a highly significant (p < 0.001) reduction in latency in trial 2. This suggests that the lesioned rats have an impaired working memory, which was significantly improved by treatment with the TRH analog. Consequently, consistent with the findings of the DNMPT study, these results further confirm that while the TRH analog did not improve long-term memory of a platform position, it significantly improved working (short-term) memory in septal-hippocampal-lesioned rats.

Taken together, the present lesion studies provide results that are consistent with the previous

NEUROPEPTIDES, TRH, A N D C O G N I T I O N 465

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findings of Horita et al., (1989), in which a different TRH analog, M K 771, was shown to reverse the behavioral deficits on a radial arm maze test of rats with septal-hippocampal lesions induced with ibotenic acid. The latter study also reported a recovery of cholinergic function in the form of restored ChAT activity, an effect which was not observed in the present study (data not shown). Nonetheless, it is possible to interpret the results of our own study as demonstrating an improvement in working memory processes due to the effect of the administration of the TRH analog. On the side of caution, however, a peptide effect on motivation or attentional processes again cannot be ruled out because the lesion did not produce a clear delay-dependent disruption of choice accuracy in this study.

CONCLUSIONS

The metabolical ly stable analog of TRH, RX77368, which has been shown to have potent CNS but little neuroendocrine activity, has been used in a series of behavioral studies in adult and aged rats to determine its involvement in cognitive processes, by using carefully designed

466 o.w. BENNETT et a l

tasks to examine different aspects of this behavior. Much ev idence suggests that this neuropep-

tide modulates various neurotransmit ter systems in the CNS, but the indicat ions are that its

i nvo lvemen t in early learning mechanisms probably are associated with its action on chol inergic

(and possibly other) neurons in the septa l -hippocampal pa thway where T R H and T R H receptors

are located (see Table 1). Furthermore, this pa thway is strongly impl ica ted with short- term

working m e m o r y as well as longer term acquisi t ion of spatial learning. The present studies

indicate that T R H has effects on the short- term behaviors , because partial or total recovery of

behavioral defici ts that may be interpreted as working m e m o r y have been observed. On the other

hand, some of this interpretation cannot to date exclude an effect on attentional or vigi lance and

mot ivat ion behaviors , and further studies, therefbre, are required to at tempt to elucidate and

clarify this situation. These nonspeci f ic behaviors are separate f rom truly cogni t ive mechanisms,

but equally, they are difficult to extract in test situations f rom the early learning process, and

moreover , they are also an important aspect o f the fai l ing mechan isms seen in aging and

dementia . The recent reported beneficial effects o f early t reatment o f A l z h e i m e r ' s disease, and

related dementias with the neuropeptide, T R H may well involve the enhancement or improve-

ment o f mot ivat ion and concentrat ion as well as working memory .

Acknowledgments--T.M. Ballard was supported by a BBSRC Quota Studentship, and C.D. Watson by a SERC Case Studentship in collaboration with Reckitt and Colman who also supplied the RX77368 and financial support for the aged rat studies. RX77368 for the lesion studies was supplied by Ferring Pharmaceuticals. The DNMTP studies were undertaken at the Department of Neurology Research, SmithKline Beecham Pharmaceuticals in collaboration with Dr. A.J. Hunter, who also provided the financial support for these studies.

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