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BASAL FOREBRAIN GLUTAMATERGIC MODULATION OF SUSTAINED ATTENTION PERFORMANCE IN THE RAT

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy m the Graduate

School of The Ohio State University

By

Janita N. Turchi, A 3.

*****

The Ohio State University 2000

Dissertation Committee: Approved W , Professor Martin Sarter, Advisor 'VoiA k/— Professor John Bruno Advisor Department ofNeuroscience Professor Norman Uretslty UMI Number 9982994

Copyright 2000 by Turchi, Janita N.

All rights reserved.

UMI’

UMI Microform9982994 Copyright 2001 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106.1346 Copyright by Janita Turchi 2000 ABSTRACT

Basal forebrain NMD A receptors have been hypothesized to be selectively involved in the performance of tasks associated with activation of cortical cholinergic inputs. For the first two experiments, rats were trained in either a sustained attention task requiring animals to discriminate between unpredictably occurring visual signals of varying lengths (25,50,500 msec) and non-signals, or in a cued visual

discrimination task. Chronic guide cannula were implanted bilaterally for the infusion

o f N-methyl-D-aspartate receptor ligands into the area of the substantia irmominata for

all experiments. The effects of administrations of N-methyl-D-aspartate (NMD A: 0.5,

1.0 & 3 nmol in 0.5 pl/hemisphere) and the NMD A receptor antagonist D-2-amino-5-

phosphonovalerate (APV: 6,10 & 20 nmol in 0.5 pl/hemisphere) were tested in

separate groups of animals. While neither drug affected performance accuracy among

animals trained in the cued discrimination task, administration of NMD A and APV

yielded contrasting impahments in the sustained attention task.

The effects of modulating basal forebrain glutamatergic function by suppression

of the relative number of NMDA-NRl subunits was tested in the second experiment

The effects o f administrations of an antisense oligodeoxynucleotide directed against the

NMDA-NRl subunit, or a missense oligodeoxynucleotide comprised o f the same base

ii pair propordons, into the area of the substantia innommata were tested in counter­ balanced fashion in the same group o f animals. Neither oligodeoTQmucIeotide affected per&rmance accuracy among animals trained in the cued discrimination task; however, administration of the antisense oligomer yielded decreases selectively in the relative number o f hits in anfmals tested in the sustained attention paradigm.

As accumulating data has suggested a significant role of basal forebrain NMDA receptor modulation m tasks taxing attentional processes, it was hypothesized that positive

NMDA receptor modulation via the glycine site might attenuate the substantive

impairments of sustained attentional processing observed following specific lesions of corticopetal cholinergic neurons. The effects of intrabasalis administrations of D- cycloserine were tested in these two groups of animals (sham- and 192 IgG-saporin-

lesionetQ. Administration of the higher dose of D-cycIoserine tested partly attenuated the

lesion-induced decrease in bits in the sustained attention paradigm. The findings fiom the

final experiment suggest that unpanments of sustained attention incurred by damage to the

basal forebrain cholinergic system may be effectively ameliorated by positive NMDA

receptor modulation via the partial agonism of the glycine site; such findings may have

implications for the neurodegenerative disorder of Alzheimer’s disease, which is

characterized in part by loss of basal forebrain cholinergic projection neurons. Taken

together, the data generated firom these three experiments supports the role of basal

forebrain NMDA receptor stimulation exclusively in the performance of tasks which

explicitly tax attentional procKses.

m To V.EJL & F E .T

IV ACKNOWLEDGMENTS

I would like to thank my advisor» Martin Sarter, for extraordinary support of this thesis project I also thank my committee members, John Bruno and Norman

Uretsky, for their helpful commentary regarding both the design of the experiments and the document wherem these experiments are discussed. I am grateful to John Oberdick for lyophilizing the oligodeoxynucleotides and to Susan Travers for generous provision of microtome use for the hnmunohistochemistry. VTTA

1991...... AJB, Anthropology, Princeton University.

1993-present ...... Graduate Research, Assistant, Department of Neuroscience, The Ohio State University

PUBLICATIONS

Research Publications

Sarter, M., Bruno, SJB. and Turchi, J. (1999). Ventral striatal-basal forebrain mechanisms modulating cortical acetylcholine, attention, and implications for neuropsychiatrie disorders. Annals ofthe New York Academy o fSciences, 877,368- 382.

Turchi, J. and Sarter, M. (1997). Cortical acetylcholine and processing capacity: effects of cortical cholinergic deafferentation on crossmodal divided attention in rats. Cognitive Brain Research, 6147-158. ,

Turchi, J., Holley, Lj A and Sarter, M. (1996). Effects of benzodiazepine receptor inverse agonists and nicotine on behavioral vigilance in senescent rats. Journal o f Gerontology: Biological Sciences, SIB225-231.A,

Holley, LA., Turchi, J., Apple, C. and Sarter, M. (1995). Dissociation between the attentional effects o f mfttsions o f a benzodiazepine receptor agonist and an inverse agonist into the basal forebrain. Psychopharmacology, 120,99-108.

Turchi, J., Holley, LA . and Sarter, M. (1995). Effects of nicotinic acetylcholine receptor ligands on behavioral vigilance in rats. Psychopharmacology, 118, 195-205.

McGaughy, J., Turchi, J. and Sarter, M. (1994). Crossmodal divided attention in rats: effects o f chlordiazepoxide and scopolamine. Psychopharmacology, 115,213- 220.

vi FIELDS OF STUDY

Major Field: Nearoscience

vu TABLE OF CONTENTS

Page Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita...... vi

List of Tables ...... x

List of Figures ...... xi

Chapters:

1. hitroduction ...... 1

lA . GLU/ACh interactions in BF ...... 4 IJB. Effects of NMD AR and NMD A/GS antagonism ...... 7 1 .C. Viability of using antisense oligodeoxynucleotides for the NMDA-NRl subunit ...... 9 ID . NMDAR and NMDAR/GS: changes with aging and Alzheimer’s disease...... 12 1.E. Specific aims ...... 13

2. General Methods ...... 17

2A- Subjects ...... 17 2D. Behavioral training and measures ...... 18 2.C. Surgical protocols ...... -21 2D. Intracranial mfiisions ...... 2 3 2D. Histological assessments ...... 24 2D. Statistical analysis ...... 25

vni 3. Basal forebram glutamatergic modulatiou of sustamed attention performance in rats: effects of APV and NMDA ...... 2 8

3 A . Introduction ...... 28 33. Specific methods ...... 30 3.C. Results...... 34 33. Discussion ...... 38

4. Antisense oligodeoxynucleotide-induced suppression of basal forebrain NMDA-NRl receptor subunits: sustamed attentional consequences ...... 41

4A . hitroduction ...... 41 43. Specific methods ...... 43 4.C. Results...... 50 43. Discussion ...... 55

5. Partial attenuation of impairments of sustained attentional processes following 192 IgG-saporin induced cholinergic lesions of the SI/NB: effects of mtrabasalis mfiisions of the NMD A/GS partial agonist D-cycloserine ...... 59

5 A . hitroduction ...... 59 53. Specific methods ...... 61 5.C. Results...... 65 53. Discussion ...... 71

6. General discussion ...... 74

Tables and Figures ...... 81

Tables ...... 82

Figures...... 85

Bibliography ...... 128

IX LIST OF TABLES

Table Bags

4.1 Experimental design for oligodeoxynucleotide infusions ...... 82

5.1 Cortical AChE - positive fiber density: group data ...... 83

5.2 Cortical AChE - positive fiber density: individual data and ranking ...... 83 USX OF FIGURES

Figure Page

2.1 Sustained, attention task schematic ...... 85

22. Cued visual discrimination task schematic ...... 87

23 Cannula target placements in basal forebrain ...... 89

3.1 Effects of mtrabasalis infusion of APV on accuracy in animals performing cued visual discrimination task...... 91

3 2 Effects of intrabasalis APV infusions upon sustained attention performance...93

3 3 ESects of intrabasalis infusion of NMDA on accuracy in animals performing cued visual discrimination task ...... 95

3.4 Effects of intrabasalis NMDA infusions upon sustained attention performance ...... 97

3.5 Lack of neurotoxicity associated with NMDA doses tested in behaving animals: Fluoro-Jade assessment ...... 99

4.1 Effects of intrabasalis infusions of antisense or missense oligodeoxynucleotides in animals performing cued visual discrimination task ...... 101

43 Comparison of effects of mtrabasalis mfiisions of antisense or missense oligo­ deoxynucleotides upon sustained attention performance 12 hours after second infiision ...... 103

43 Comparison of effects of intrabasalis infusions of antisense or missense oligo­ deoxynucleotides upon sustained attention performance 24 hours after final infiision ...... 105

4.4 Comparison o f effects of mtrabasalis infusions of antisense or missense oligo­ deoxynucleotides upon sustained attention performance 48 hours after final mfiision ...... 107 xi 4.5 Effects of intrabasalis antisense oligodeoxynucleotide (8.0 nmol) administrations upon sustamed attention perfetmance: temporal gradient of impairments ...... 109

4.6 Fluoro-Jade assessments of the effects associated with antisense or missense doses tested in behaving animals ...... I l l

4.7 Lack of neurotoxicity in the SI following infiision of antisense (8.0 nmol)...113

4.8 Antisense oligodeoxynucleotide-induced suppression of NMDA-NRl subunit immunoreactivity: comparison with missense infusion effects ...... 115

4.9 High magnification evidence of antisense-induced suppression of NRl- immunoreactivity ...... 117

5.1 Effects o f sham- or 192 IgG-saporin lesions on the relative number of hits and correct rejections ...... 119

5.2 Effects of intrabasalis infusions of D-cycloserine upon sustained attention performance in sham-lesioned animals ...... 121

53 Effects of intrabasalis infusions of D- cycloserine upon sustained attention performance in 192 IgG-saporin lesioned animals ...... 123

5.4 Comparison of AChE—positive fiber density in sham-lesioned and lesioned rats: cortical areas ...... 125

5.5 Post hoc correlation analysis of relationship between AChE - positive fiber density and increases in the relative number of hits following intrabasalis o- cycloserine infusions...... 127

XII CHAPTER 1

INTRODUCTION

The BFCN provides cholinergic innervation of neocortical, ailocortical and mesocortical regions as well as the basolateral amygdala (BLA) and thalamic targets in the rat (Zaborszky 1992; Kordoweret al., 1989; Eckenstein et al., 1988; Grove, 1988;

Mesulam and Geula, 1988; Schwaber et al., 1987; Struble et al., 1986; Lamour et al.,

1984; Mesulam et al., 1983; Bigl et al., 1982; Sofîraniew et al., 1982); there are also non-cholinergic projections arising from the basal forebrain (including GABAergic neurons: Pang et al., 1998; Gritti et al., 1997; Freund et al., 1992; Rye et al., 1984).

Further, changes in cortical release of ACh have been demonstrated to be similar in

nature across different cortical areas (Himmelheber et al., 1998). It has been

extensively documented that the BFCN receives input from diverse sources including

glutamatergic projections from the prefrontal and, to a lesser extent, insular and

piriform cortices [though these cortical sources have not been proven directly apposed

to cholinergic neurons: see below], as well as from the basolateral amygdala,

GABAergic projections frnm the nucleus accumbens, noradrenergic input from the

locus coeruleus, serotonergic projections from the dorsal raphe, cholinergic projections

1 &om the pedunculopoatme tegmental nucleus and DÂergîc fîrom the ventral tegmentum (Smiley and Mesulam, 1999; Khateb et al., 1998; Waters and Allen, 1998;

Dincpoulos et al^ 1997; Zaborszky et al., 1997; Fort et al., 1995; Baskerville et al.,

1993; hosier and Semba, 1993; Bertorelli et al., 1991; Gaykem aet al., 1991; Zafaorszlqr et al., 1991; Zilles et al., 1991; Consolo et al. 1990; Jones and Cuello, 1989; Walker et al., 1989; Grove, 1988; Semba et ai, 1988; Haring and Wang, 1986; Mogenson et ai.,

1983).

The BFCN has been suggested to play a prominent role in attentional processes

(Muir et al., 1994), likely serving to enhance the processing of behaviorally salient stimuli. Acetylcholine in the cortex has been shown to enhance the responsivity of neurons to sensory stimulation, thus cortical ACh does not itself induce signal, rather it has been posited to enhance the signal to noise ratio ( Kimura et al., 1999; Hars et al.,

1993; Pirch et al., 1991; Metherate and Ashe, 1991; Tremblay et al., 1990; Metherate et al., 1987). This modulation of neuronal responsivity by ACh has been noted to persist beyond the direct presence of ACh in the vicinity examined; such persistence has led to the notion of cholinergic modification/organization of cortical receptive fields among multiple cortical areas (visual, auditory, somatosensory: Sillito and Kemp, 1983;

Dhnyan and Weinberger, 1990; Sachdev et al., 1998; Howard lEt and Simons, 1994;

Metherate and Weinberger, 1999; Jiménez-Capdeville et al., 1997; Tremblay et al.,

1990; Bear and Singer, 1986; Hassehno and Barkai, 1995; Rasmusson et al., 1992;

Bakin and Wemberger, 1996; Wilson and Rolls, 1990; Richardson and DeLong, 1990).

Additionally, it has been demonstrated that the plasticity o f cortical field representation

2 afforded by BF cholinergic input is impeded in the absence of these cholinergic mpnts

(Baskerville et al., 1997; Sato et aL, 1987). hicreases in cortical acetylcholine efflux have been found following exploratory behavior, conditioning/behavioral training, lever pressing (Giovannini et al., 1998; Rigdon and Pirch, 1986; Acquas et al., 1996;

Orsetti et al., 1996; Masuda et al., 1997; Thiel et al., 1998; Izaki et al., 1998; hioue et ai., 1983; Aou et al., 1983), and more recently, have been found to be associated with active engagement in attentional processing (Himmelheber et al., 2000).

Prior to the development of the specific cholinotoxin 192 IgG-saporin (Wiley,

1992, Book et al., 1994), the behavioral effects of excitotoxic damage to the BFCN had been noted (Vanicky et al., 1998; Gutierrez et al., 1994; Andrews et al., 1994;

Abdulla et al., 1994; Weiss et al., 1994; Page et al., 1993; Muir et al., 1992).

Excitotoxic lesions in this region were later discussed in comparison with specific

cholinergic lesions, with respect to the resultant impairments of memory formation,

visual discrimination performance, sustained and divided attentional processes, as well

as other markers, which had been demonstrated (Wrenn et al., 1999; Gutierrez, et al.,

1999; Fine et al., 1997; Voytko et al., 1994; McGaughy et al., 1996; Turchi and Sarter,

1997). The advent o f the specific cholinotoxin provided useful tool for modeling of

attentional dysfunction associated with the neurodegenerative disorder of Alzheimer’s

disease.

The loss o fcholinergic function documented in Alzheimer’s disease has been

considered a major factor underlymg the cognitive deficits exhibited by individuals

with DAT (Perry et al., 1992; Bartus et al., 1982). Additionally, the potential role of

3 glutamatergic dysfünctîoa in Alzheimer's disease has been investigated (Ulas et al.,

1992; Greenamyre and Young, 1989). Distinct changes m NMDAR function have been documented, including decreases in NMDAR GLU and GLY binding sites, decreases in glycinergic affinity and diminished glycinergic regulation o f the NMDAR, differentiating individuals with DAT from normal, aged controls (Carlson et al., 1993;

Piggott et al., 1992; Del Bel and Slater 1991; Procter et al., 1989; Steele et ai., 1989).

The experûnents described herein examined the influence of glutamate upon the BFCN with respect to sustained attentional processes.

GLU/ACh interactions in BF

The NMDAR is a voltage dependent ionotropic glutamate receptor, requiring membrane depolarization for removal of the Mg^* block which is present within the ton charmel at resting membrane potentials (Nowak et al., 1984). The NMDAR requires glycine (GLY) binding to the NMDAR/GS for receptor activation (Cuiras and Pallotta,

1996; Benveniste et al., 1990; Kleckner and Dingledine, 1988) and is also regulated by polyamines and zinc at their respective binding sites (Rock and Macdonald, 1995;

Reynolds and Rothermund, 1995; Legendre and Westbrook, 1990). The NMDAR is

comprised of multiple subunits (NMDA-Rl, NMDA-R2[A-D]); while functional NRl

homomeric channels can be formed, subunits NR2A-D cannot form functional receptor

charmels without the presence of the NRl subunit (Laurie and Seeburg, 1994a;

Sprengel and Seeburg, 1993; Monyeret al., 1992). Furthermore, the NRl subimit

formed in a given NMDA receptor may reflect the expression of one o f several distinct

mRNA splice variants (Laurie and Seeburg, 1994b; Tingley et al., 1993). These NRl

4 subunits, including splice variant products, and NR2A-D subunits are differentially distributed iu mammaliau brain (Laurie et aL, 1995; Bresink et al., 1995; Wenzel et al.,1995; Monyer et al., 1994; Monyer et al., 1992) and different combinations of these subunits result in NMDA receptors with varied afSnities for agonists and antagonists

(Laurie and Seeburg, 1994a; Tingley et aL, 1993; Monyer et al., 1992 ). Native NMDA receptors tend to be present as hetero-oligomeric channels (Monyer et al., 1992) and the NR2A-D subunits lend specific characteristic properties to the receptor; fijr instance, the presence o f the NR2B subunit has been described to produce an agonist preferring configuration (Laurie and Seeburg, 1994a), and the sensitivity of the

NMDAR to the magnesium block has been shown to vary with the presence of the

NR2A versus NR2C subunit (Monyer et al., 1992). Moreover, the composition of individual subunits can alter the receptor's channel dynamics (e.g., specific substitution of an asparagine residue on NMDA-Rl alters Ca’* permeability, whereas this substitution on NMDA-R2 subunit affects the Mg^^ channel blockade: Sprengel and

Seeburg, 1993; Bumashev et al., 1992; see also Wafford et al., 1995; Laube et al.,

1993). This receptor’s characteristics are further complicated by the ability of various ligands to alter the nature of the interactions of other ligands with their binding sites

(e.g. agonists for the GLU recognition site can affect the afGnity of agonists and antagonists for the GLY site and vice versa: Priestley and Kemp, 1994; Grimwood et al., 1993; Lester et al., 1993; Kemp and Priestley, 1991).

In the rat, ultrastructural evidence o f glutamatergic synapses on cholinergic cells o f the basal forebrain has not been readily demonstrable. Anterogradely transported HRP 6om the basolateral amygdala has been noted in terminals apposed to choline-acetyltransferase (ChAT) immunoreactive (-IR) cells of the ventral pallidmn

(Zaborszky et al., 1984); however, intracortical (frontal, parietal and occipital regions) iontophoretic application of the anterograde tacec Phaseolus vulgaris leucoagglutinin

(PHA-L) failed to demonstrate glutamatergic axons terminating on BFCN ChAT-IR dendrites. Moreover, even the prefrontal corticofiigal terminals were not observed to make defrnitive synapse formations with cholinergic neurons, although the possibility of insufficient ChAT immunoreactivity within the distal dendritic zone, where corticofiigal terminations were anticipated, was noted (Zaborszky et al., 1997). Such findmgs parallel those discussed by Martin et al. (1993) with respect to differential localization of AMPA receptors in rats as compared to monkeys; while GIuRl nmnunoreactivity is present in nucleus basalis of Meynert for both rat and monkey, the

GIuRI-immunoreactive neurons in rat basal forebrain do not express ChAT, but rather express GABA and parvalbumin immunoreactivity. This is in contrast to the data in

monkeys, which demonstrated colocalization of GIuRl immunoreactivity with ChAT

(Martin et al., 1993).

Electrophysiology, microdialysis and behavioral data, however, stongly support

the notion o f positive glutamatergic mteraction with ACh neurons of the basal

forebrain, even though that positive modulation appears to be through an as yet

unidentified path in the raL Application of L-GLU to cultures of and slices containing

NB neurons, as well as SI/NB neurons in vivo, has resulted in «(citation of the ACh

neurons (Khateb et al., 1997; Lamour et al., 1986; Nakzgima et al., 1985). Likewise,

6 electrical stnnulation of NB neurons, as well as mtrabasalis micromjection of L-GLU have resulted in increased cortical ACh. efflux ^urosaw a et al., 1989; Casamenti et al.,

1992). Importantly, modulation of cortical ACh via glutamatergic stimulation of

NMDAR in the basal forebram has been demonstrated; mtrabasalis infusion of NMDA

(200 pmol) produced increases o f cortical ACh efflux in animals trained to associate darkness with the presentation of a palatable food (Fadeletal., 1998). Additionally, mtrabasalis administration of NMDAR antagonists (CPP, kynurenate) resulted in

decreases of evoked ACh release (Rasmusson et al., 1996; Rasmusson et al., 1994) as

well as non-evoked ACh release (Giovarmmi et al., 1997).

Effects of NMDAR and NMDAR/GS antagonism

Several studies have mvestigated the effects o f systemically administered

NMDAR antagonists and NMDAR/GS antagonists upon various cognitive processes.

In rats it was found that while NBQX, an AMPAR antagonist, disrupted response rates

in a task involving differential reinforcement of low response rates (DRL) as well as a

DMTP task, it failed to influence response accuracy; whereas rats receiving MK-801,

an NMDAR antagonist, exhibited an increase of burst responding and decreased

response accuracy for both paradigms (Stephens and Cole, 1996). Likewise, only

treatment with MK-801, not with the AMPAR antagonist YM90K, potentiated

scopolamine mduced deficits in animals performing a spatial learning and memory task

(Li et al., 1996). Moreover, transgenic modification of the NMDAR has an impact

upon long term potentiation (LTP), as mice lacking the NR2A subunit have been shown to have reduced hippocampal LTP (Kiyama et al., 1998). Such fîndfngs provide support for hypotheses arguing the importance of NMDAR function in LTP induction as a physiologic substrate of leaming and memory (see also Cammarota et al., 2000; Collingridge and Bliss, 1995; Corey-Slechta et al., 1997; Matthies et al.,

1995).

MK-801 administration has also been shown to impair performance of a delayed-non-match-to sample task (Ogura and Aigner, 1993). Intrahippocampal mfiisions o f AP5 have been shown to cause delay-dependent mnemonic impairment in a delayed-matching-to-place paradigm (Steele and Morris, 1999); similarly, intra- accumbens (core) infusions of APV yielded impairments of spatial task acquisition and performance (Smith-Roe et al., 1999). In addition, APV administration into insular cortex was found to impair memory in a conditioned taste aversion task (Rosenblum et al., 1997). Furthermore, subanesthetic doses of ketamine in healthy human subjects were reported to result in decreased performance accuracy for free recall, recognition memory and a task measuring attention, although the paradigm used for assessing attentional function was not described (Malhotra et al., 1996).

Antagonism of the NMDAR/GS has also been noted to impede cognitive processes. Intramuscular administration of HA-966, a ligand with properties of an

NMDAR/GS antagonist (Kemp and Priestley, 1991 ; Kloog et al., 1990; but see also

Henderson et aL, 1990), impaired DNMS performance in rhesus monkeys in a manner

similar to MK-801 (Matsuoka and Aigner, 1996b). Additionally, intracranial injection

of 7-Cl-KYNA, a potent and selective NMDAR/GS antagonist poster et al., 1992; Rao

8 et aL, 1992; Kloog et al., 1990; Henderson et al., 1990), mpanred acquisition of passive avoidance training in chicks (Steele and Stewart, 1993). Taken together, the anatomical, electrophysiological, neurochemical and behavioral studies described above indicate a significant basis for the examination of glutamatergic influence upon the BFCN as it relates to sustained attentional processes.

Viability of using antisense oligodeoxynucleotides for the NMDA-Rl subunit

Complementary deoxyribonucleic acid (cDNA) sequences have been cloned for each of the five NMDA receptor subunits (NRl, NR2A-D; Ishii et al., 1993; Moriyoshi et al., 1991). Based upon this work, it has been possible to generate appropriate antisense oligodeoxynucleotides targeting each of these receptor subunit mRNAs.

Antisense oligodeoxynucleotides (ODNs) provide an efficacious and reversible means for inhibition of specific gene expression, and in vivo infusions of antisense oligodeoxynucleotides represents a viable approach for examining the role of glutamatergic innervation of the SI/NB as it relates to attentional functions.

Specifically, the effects of decreased NMDA-Rl subunit density, following intrabasalis administration of antisense oligodeoxy-nucleotides for the NMDA-Rl subunit, upon sustained attentional processing will be discussed below in this document.

Antisense oligodeoxynucleotides consist of short chains of nucleic acids (15-

25 nucleotides) which are targeted to an mRNA sequence of complementary bases.

Inhibition of the targeted mRNA translation is achieved when the nucleotide bases of the antisense oligodeoxynucleotide form hydrogen bonds with the complementary nucleotide bases of the targeted mRNA, thereby blocking the mechanisms of

9 translation (Stein, and Cohen^ 1988; Walder and Walder, 1988). Another possible mechanism, o f action of ODNs is functioning as a substrate for en^onatic degradation by RNaseH (Wahlestedt, 1994; Touhne, 1992; Walder and Walder, 1988). The primary means of oligodeoxynucleotide uptake has been proposed as pinocytosis (Stein et al., 1993), or receptor-mediated endocytosis (Krieg, 1993; Yakubov et al., 1989;

Loke et al., 1989) although other mechanisms may be involved.

Antisense oligodeoxynucleotides (ODNs) provide a highly specific, when accompanied by adequate controls, and reversible means for inhibition o f particular gene expression; the transience of the effects generated by this type of ligand has been paralleled by the use of monoclonal antibodies to achieve similar “knock down” effects

(Cattaneo et al., 1999; Molnar et al., 1998). Antisense ODN’s have been employed in numerous studies, including, but not limited to, alterations of specific receptor tesponsivity (Dorri et al., 1997; Zhao et al., 1998), manipulation of behavioral characteristics (Kalra et al., 1999; Morrow et al., 1999; Otano et al., 1999; Landgraf et al., 1995; Akabayashi et al., 1994; Zhou et al., 1994; Skutella et al., 1994; Weiss et al.,

1993; Mani et al., 1994), as well as alteration of cortical plasticity (Roberts et al.,

1998).

Antisense oligodeoxynucleotides targeting NMDAR subunits have been used with success both in vitro (Zhong et al., 1996) and in experiments employing in vivo infusions into the brain (Kammescheidt et al., 1997; Matthies et al., 1995; Roberts et al., 1998; Dean et al., 1998); for example, NMDA-RI antisense ODNs bave been demonstrated to attenuate the neurotoxic effects of in vitro NMDA administration

10 (Wahlestedt et al., 1993). While modifîcatîoii of phoshpodiester oligodeoxy­ nucleotides is possible to increase cellular uptake and efScacy (Krieg et ai., 1993), phosphorothioate analogues o f unmodified, antisense oligodeoxynucleotides, wherein sulphur is substituted fi)r one of the non-bridging intemucleotide oxygen molecules, have demonstrable resistance to degradation following intra-cerebroventricular administration and have been infused, without apparent toxic effects, at continuous micromolar levels for one week (Whitesell et al., 1993). Entra-cerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides for the NMDA-Rl subunit has been shown to diminish the extent of ischaemic infarct damage (in a middle cerebral arterial occlusion model in rat: Wahlestedt et al., 1993) and to decrease tissue damage, mortality and behavioral deficits associated with lateral fluid percussion trauma in rats

(Sun and Faden, 1995). Likewise, i.c.v. infusion of NMDA-Rl antisense oligodeoxynucleotides in mice increased the time the mice spent in open arms of an

elevated maze (as compared to mice receiving sense ODN infusions) and increased the

latencies for seizure-like activity following NMDA administration (Zapata et al., 1997).

Additionally, it has been reported that intrastriatal NMDA-Rl antisense oligodeoxy­

nucleotides infusions dose-dependently elicit behavioral effects, with the highest dose

used producing spontaneous ipsilateral rotational befiavior and lesser doses

demonstrating ipsilateral rotation following D-amphetamine administration (Standaert

et al., 1996). The above mentioned ©cperiments indicate that in vivo infusion of

11 antisense oHgodeoxynncleotides into the bram represents a viable means for investigating the e& cts of inhibition of NMDA-RI receptor snbtmit expression in the SI/NB upon sustained attentional processing,

NMDAR and NMDAR/GS: changes with aging and Alzheimer^s disease

Several studies have indicated changes in either the brain levels of NMDAR GLU and GLY binding sites, or the afBnties of these sites, associated with aging in rats and mice (Magnusson et aL, 1996; Cohen and Muller, 1992), and with aging and AD m human brain samples. Data demonstrating decreases in GLU, GLY, and MK-801 binding sites m rats (Miyoshi et aL, 1990; Tamaru et aL, 1991) has been countered by other studies mdicating either increases in GLY binding sites (Saransaari and Oja, 1993) or a lack of true decreases in NMDA and GLY bindmg site density (Bonhaus et aL, 1990). hi normal aged humans' brain samples, certain studies found either no changes, or decreases of MK-

801 binding sites but not of affiiity (Johnson et aL, 1996; Piggott et aL, 1992). While some studies have shown decreases m glycinergic regulation of the NMDAR m AD

(Palmer and Bums, 1994; Procter et aL, 1989; Steele et aL, 1989), other studies have not disclosed the same Aidmgs, and intersubject variability may be an issue (Ulas et aL, 1992;

CowbumetaL, 1990).

Kynurenic add (KYNA NMDAR antagonist) levels m brain have also been investigated in rats as these levels relate to development and agmg; increases m KYNA have been noted with age, especially in cortex and hippocampus (Gramsgerben et aL,

1992; Moroni et aL, 1988). Additionally, brain levels of L-phosphoserme, a competitive

NMDAR antagonist, have been fbtmd to be elevated in AD (Klunk et aL, 1991). These

12 fedmgs, m conjanctioa with data demonstrating losses of NMDAR density in AD, may indicate another level of glntamatergic dysfunction underlying the cognitive deficits

associated with DAT.

While it would have been difficult to model cortical NMDAR decreases and

NMDAR/GS deregulation m behaving rats, use of the specific cholinotoxin 192 IgG-

saporin (Waite et aL, 1994; Wiley, 1992), which targets neurons bearmg the low affinity

p75 NGF receptor and spares ACh projections from SI/NB to amygdala (Heckers and

Mesulam, 1994), allowed for an effective model of the BFCN degeneration and attentional

impairments produced by cholmergic dysfunction described by the cholinergic hypothesis

of DAT (Perry et aL, 1992; Whitehouse et aL, 1982; Bartus et aL, 1982). The sustained

attention paradigm which was employed m the below described experiments, has sound

construct validity. It effectively allowed for the demonstration of attentional impafrments

incurred by SI/NB cholmergic lesions (McGaughy et aL, 1996), and provided suitable

measures for assessing the impact of ghitamatergic modulation of extant SI/NB

cholinergic projection neurons, following 192 IgG-saporin induced lesions, upon sustained

attentional processes.

Specific Amis

AIM l .Hvpothesis: Modulation o f basal forebrain cholinergic neurons by intrabasalis infusion o f NMDA receptor antagonists will produce attentional impairments in intact animals. Additionally, intrabasalis administration o f an NMDA agonist may yield attentional impairments but with opposing characteristics.

13 Administratioa of the NMDA receptor antagonist R(-)-3-(2-carfaoxypiperazme-

4-yl)-propyl-l-phosphonic acid (CPP) into the basal forebrain has been shown to decrease cortical acetylcholine efQinc following stimulation of glntamatergic projections to the BFCN from the pedimculopontine tegmentum (Rasmusson et aL,

1996). Additionally, it has been found that intrabasalis infusion o f the NMDA receptor antagonist Itynurenic acid (KYNA) decreases cortical acetylcholine efQux. associated with the presentation of behaviorally relevant stimuli (Fadel et al., 1998). Glycine binding to the strychnine-insensitive glycine site of the NMDA receptor is required for

NMDA receptor activation (Kleckner and Dingledine, 1988; Johnson and Ascher,

1987). Although physiological levels o f glycine are substantive, the ability of an

NMDAR/GS agonist to augment the influence of glutamate has been demonstrated

(Wood et al., 1989). It was hypothesized that infusion of an NMDAR antagonist, as it might be expected to decrease cortical acetylcholine efflux, similar to cholinotoxic

lesions of the BFCN, would thereby impair animals' ability to identify correctly the

signal events in the sustained attention task. Conversely, infusion of an agonist to the

NMDAR, as it might increase cortical ACh efflux, would impair the animals' ability to

identity correctly non-signal trial events, as has been observed with another agent

which has been demonstrated to increase cortical ACh efflux (B21R inverse agonist: see

Holley et al., 1995). As NMDA receptor modulation of cortical ACh was hypothesized

to be critically dependent upon the cognitive-demand status of the animal, no such

impairments o f performance were anticipated for the cued visual discrimination task.

14 AIM 2.Hypothesïs: Decreases in the density o fNMDA-Rl subunits, and concomitant diminution offim ctional hetero-oligomeric NMDA receptors, following intrabasalis administration o fantisense oligodeoxynucleotidesfor the NMDA-Rl subm it will result in attentional impairments.

The BFCN has been proposed to perfbrm a substantive role in attentional functions (Muir et al., 1994; Richardson and DeLong, 1991). Corticopetal cholinergic basal &rebrain neurons receive glntamatergic input horn multiple sources (Zaborszky et al., 1997; Zaborszky et al., 1991). Furthermore, intrabasalis administration of

NMDAR antagonists (ΠP, kynurenate) has been demonstrated to decrease evoked

ACh release (Rasmusson et al., 1996; Rasmusson et al., 1994). Antisense oligodeoxynucleotides provide an efficacious and reversible means for inhibition of specific gene expression. The effects of decreased NMDA-Rl subunit density, following intrabasalis administration of antisense oligodeoxynucleotides for the

NMDA-Rl subunit, upon sustained attentional processing were examined. This experiment provides additional information for efforts to describe the role of

glntamatergic innervation of the SI/NB as it relates to attentional functions. It was

predicted that any attentional impairments associated with intrabasalis infusions of

NMDA-Rl antisense oligodeoxynucleotides would depend upon suppression of

functional hetero-oligomeric NMDA receptors consequent to the disruption of NMDA-

Rl mRNA translation, and presumed (though not measured within these experiments)

accompanying decreases in cortical ACh efflux. As NMDA receptor modulation of

15 cortical ACh was hypothesized to be critically dependent upon the cognitive-demand status of the animal, no such impairments of performance were anticipated for the cued visual discrimination task.

AIM S.Hypothesis: Corticopetal cholinergic cell loss produced by infusions o f192 IgG-saporin into the basalforebrain w ill result in attentional impairments which will be attentuated by intrabasalis administration o f an NMDAR/GS agonist.

Although the losses of NMDAR binding sites and consequent glutamatergic abnormalities associated with AD suggest the need to increase glutamatergic activity, use of direct NMDAR stimulation appears contraindicated due to the potential for excitotoxic consequences (McEntee and Crook, 1993; Greenamyre and Young, 1989).

Therefore, investigation of NMDAR/GS ligands, such as the NMDAR/GS partial agonist D-cycloserine (DCS: Henderson et al., 1990), may reveal useful pharmacotherapeutic strategies.

hnpairments o f sustained attention associated with diminished cortical acetylcholine consequent to specific cholmergic lesions of the basal forebrain have been effectively demonstrated (McGaughy et al., 1996). It was hypothesized both that intrabasalis administration of the NMDAR/GS partial agonist D-cycloserine (DCS), would serve to augment cortical acetylcholine by and thereby ameliorate the deficits in sustained attentional processing resulting from infusion of the cholinotoxin 192 IgG- saporin into the substantia innominata/ nucleus basalis of Meynert (SI/NB). For the same reasons discussed in Aim 1 above, infusion of D-cycloserine, to the extent that it might incur a hyperactivated cholinergic system, was anticipated possibly to impair sustained attention performance in the sham-Iesioned animals.

16 CHAPTER!

GENERAL METHODS

Subjects

Male BNNia/F344 rats weighing approximately 200 g at the beginning of the experiments served as subjects for all experiments described. These rats were housed individually in a temperature- and humidity-controlled vivarium with a 12 h. light/dark schedule (lights on: 1:00, off: 13:00). Animals were moderately water deprived

(maintained at 90% pre-deprivation weight) and food was available ad libitum.

Experunents were conducted m AAALAC-accredited focilities and in accordance with the "US Government Principles for the Utilization and Care of Vertebrate Animals

Used in Testing, Research, and Training".

Subjects were trained in an operant system (MedAssociates, East Fairfield, VT) which consists of 12 operant boxes; each was enclosed in a sound-attentuating chamber and equipped with 2 retractable levers, three panel lights (2.8 W each, located above

the levers), a houselight (2.8 W), and water dispenser. The houselight is positioned

high on the wall opposite the levers. The water reinforcement was available on the

17 same wall as the intelligence paneL Signal presentation lever operation and water dispension were controlled by an IBM clone computer using MED-PC software

(MedAssociates).

Behavioral training and measures

After training to bar press for water remforcement (40 |il), the rats designated for the sustained attention task were trained initially to discriminate between signals (central panel light illumination: L s) and nonsignals (no Olummation), without the houselight illumination (see Hg. 2.1 for a schematic depiction of the task). For a behavioral session, animals were placed in the operant chambers and once the three minute adaptation period had concluded, the two levers were extended into the chamber 2 sec followmg the commencement of a trial and remained active for 4 sec. Left lever depression following signal events was recorded as a hit and rewarded, whereas depression of the right lever was not reinforced and was counted as a miss. Nonsignal events which elicited right lever depression were rewarded and recorded as correct rejections. Conversely, left lever presses for nonsignal trials constituted false alarms and were not reinforced. For this

■shaping stage, misses and false alarms resulted in correction trials (repetition of the previous stnnulus type). Failure to respond correctly after three correction trials resulted in a "forced trial", during which the same stimulus (signal or nonsignal) was repeated and onty the correct response lever was made available. Should a response not have been made within 4 sec of the trial event (signal or non-signal), levers was retracted and the lack of response was

18 recorded as an omission. At this training stage, one session was comprised o f 162 trials, with 81 trials of each stnnulus type (signal or nonsignal) pseudo-randomly presented and intertrial intervals of 9 ± 3 sec.

Once animals are able to respond correctly to 70% of signal and nonsignal trials, they progressed to the final task, wherein the correction and forced trials were no longer present, and the signal duration was of variable length (500,50 or 25 msec).

Within each 162 trial session, 27 trials o f each o f the three signal lengths were presented and 81 of the trials were of the nonsignal type. These signal and nonsignal trials were pseudo-randomly presented. Performance measures included percent hits, percent correct rejections, errors of omission, and indices of vigilance and side bias.

Animals were trained to asymptotic performance at the following minimum criteria:

>65% hits for 500-msec signals, >65% correct rejections, and <25 errors of omission.

Upon attainment of this criterion performance, animals were placed in the final task, wherein the abovementioned parameters (total trial number, signal lenghts and event rate) were maintained as described but the houselight was illuminated throughout the session. Once animats regained performance criteria (>65% hits for 500-msec signals,

>65% correct rejections, and <25 errors of omission), they were subjected to surgical and/or pharmacological manipulations indicated in the description of the individual experiments.

Animals designated for the cued visual discrimination task, a task which did not explicitly tax attentional processing as only the correct lever was cued by the visual

19 stimulus placement ^inunelheber et al., 1997), were similarly shaped to bar press for water reinfbrcement (40 pi); these animals were then trained to press the lever under one o f two possible illuminated panel lights (left or right). Levers were extended throughout the session following task onset, and light stimuli were 3 seconds in duration. A response of left lever pressing for left light illumination resulted in water reinforcement; likewise, right lever presses were rewarded following right light illumination (see Fig. 22 .for task schematic). This represented the final version of the task, which was comprised of 48 minutes of such visual stimuli, pseudo-randomly presented over the left or right lever, with a variable intertrial interval o f 12.0 ± 3 sec.

The relative numbers of both correct responses and omissions were recorded and analyzed for these experiments.

Behavioral measures

Data from the cued visual discrimination task (relative number of correct responses and omissions) was recorded both in 4 blocks of equal duration (12 minutes) as well as collapsed across all trials in the session. Records of the number of hits, misses, correct rejections, false alarms and omissions were obtained for each behavioral session of the sustained attention task. Each session, composed of 162

trials, was further analyzed as three blocks of 54 trials (27 trials each of signal and non­

signal events) in order to examine any vigilance decrement (decrease in task

performance over time). Calculation of the relative number of hits (hits/hits+misses)

was made for each signal length per 54 trial block of the task. Likewise, calculation of

20 the relative number o f correct rejections (correct rejections/correct rejections + false alarms) per block was made. A vigilance index (VI) was computed by collapsing values for the relative number of hits (h) and false alarms (fa) associated with each signal length according to the following formula* modified firom an index of signal sensitivity (Frey and CoUiver, 1973): VD= (h-f)/[2(h+f)-(h+f)’]. Values for VI approaching 1.0 indicate optimal discrimination of signal versus non-signal events for the animal performing the sustained attention task, whereas VI values approaching 0.0 indicate substantive impairments in the ability of the animal to distinguish these two types of trial event. Additionally, a measure of side bias, indicating the proclivity of an animal to respond on a single side (SB; SB= [hits+false alarms]/[totaI responses]) was included for each block of 54 trials. An SB value of 0.5 represents the absence of a side bias; namely, neither lever is preferentially manipulated during the task session, while a value less than 0.5 indicates a bias for the right (correct rejection/miss) lever.

Conversely, an SB value of greater than 0.5 reveals a bias to the left (hit/false alarm) lever. Most animals attain a stable performance criterion of at least 70% correct responses to the 500 msec stimulus as well as correct rejection trial events; however, the relative number of correct responses for the 50 and 25 msec stimuli are often lower

(50-60% and 40-45%, respectively). Thus more responses tend to be generated on the

right lever, yielding SB values of 033-0.44 for animals performing the task correctly.

Surgical protocols

After attaining a stable baseline performance m the final level of either the

sustamed attention task, or the cued visual discrhnination task, the rats were surgically

21 implanted with, chronic guide cannula mto the SI/NB; for Aims I and 2, this represented the only surgical manipulation. For Aim 3, implantation of chronic guide cannula into the SI/NB was performed in conjunction with 192 IgG-saporin lesions of the SI/NB. For any case, rats were anesthetized with ketamme (90 mg/kg, i.p.) and xylazine (6 mg/kg, i.p.) and all surgical operations occurred under aseptic conditions.

Following placement in a stereotaxic instrument ÇDavid Kop( Tij'xmga, CA) guide cannula (26 gauge, o.d. 0.46mm: Plastics One, Roanoke, VA) were implanted into both hemispheres according the following coordinates relative to Bregma: AP -0.8 mm; ML

± 2.5 mm; DV -12. mm from dura ÇPaxinos and Watson, 1997). Posterior to the cannula, two stainless steel screws were drilled into the skull and the entire assembly was covered in dental acrylic. Dummy cannula were inserted in order to prevent clogging of the cannula shafts. After surgery, animals were returned to their home cages and allowed to recover for 7 days with free access to food and water. Animals were then be trained until stable, asymptotic performance levels were reestablished.

Animals for Aim 3 were anesthetized with a coadministration of ketamine (90 mg/kg, i.p.) and xylazine (6 mg/kg, i.p.). Partial depletion of cholinergic basal forebrain projections to cortex was produced via bilateral infusion of the immunotoxrn 192 IgG- saporin (0.17 pg/pl in Dulbecco's saline, pH 6.9-7.4;0.5 p.l/hemisphere; lot #4-1, ATS,

San Diego, CA) using the following coordinates relative to Bregma: AP -0.6 mm, ML

±2.5 mm, DV -7.0 mm from dura (Paxinos and Watson, 1997). Sham-lesioned animals were subjected to the identical procedures but with infusions of 0.5 pl/hemisphere of

22 Dulbecco's saline substituted for the iramunotoxm. Bolus injections were made stereotaxicaHy using a 1 [ll microsyringe (Hamilton, Reno, NV) and the syringe remained in place for an additional 3 minutes following each infusion. Following surgery, anfmak were treated with Kdocaine and amoxicillni and have a 7 day pre-retraining recovery period (food and water available ad libitum).

Intracranial infusions

Dummy cannula were removed and polyethylene tubing was attached several times throughout the post-surgery training period to habituate the animals to intracranial mfusion procedures. Once annuals regamed baseime performance, they were subject to regimens of bilateral infusions mto the SI/NB: NMDA (Lot # 99F5805: Sigma, S t Louis,

MO) and APV (Lot #TBH-11-071B: RBI, Natick, MA: Aim 1); antisense and missense

ODNs for the NMDA-Rl subunit (Oligos Etc., Inc., WOsonville, OR: Aim 2); D - cycloserine (Lot ^Q9H5005, Sigma, St. Louis, MO: Ann 3). For each Am, bilateral infusions of 0.5 (il/hemisphere into the SI/NB occurred over a 1 mmute period, using a

BAS infusion pump (Model MDlOOl, West Lafayette, IN ) and the injection cannula remamed in place for 2 additional minutes. Intracranial mfusion sessions were separated by at least two traming sessions or by as many as was requned for the anmal to regam baseime performance. A Hamilton microsyringe, attached to polyethylene tubing and connected to internal cannula, was used to make the infusions (33 gauge, id. 0.10mm:

Plastics One, Roanoke, VA). Doses and volumes were sefected as suggested by previous studies where possible (APV: Maren et aL,

23 1996; NMDA: Pawley et al., 1996, Thanos et al., 1992, Boegmaa et al., 1992; NMDA-

Rl antisense and missense ODNs: Standaert et al., 1996; Matthies et al., 1995; Sun and

Faden, 1995; D-cycIoserine: Ohno and Watanabe, 1996, Steele et al., 1996).

Histological assessments

Following completion of the NMDA receptor and NMDAR/GS ligand testing schedules (Aims 1 and 3, respectively), or the NMDA-Rl antisense oligodeoxy­ nucleotide testing schedule (Aim 2), animals were transcardially perfused with heparinized saline followed by 10% formalin. The brains were removed and post-Sxed in 10% formalin for one day (Aims 1 and 2), or 9 h (Aim 3), prior to placement in 30% sucrose-phosphate buffer. Sections (40 pm) were Nissl stained to verify cannula placement (Aim 1,2,3). Fluoro-Jade processing (Histo-Chem, Inc., Jefferson, AR; following the method of Schmued et al., 1997 ) was performed on sections from Aim 1 and Aim 2 animals to assess any neurotoxic effects induced by the infusion protocols.

For Aim 3, adjacent sections were processed to demarcate acetylcholinesterase-positive fibers as an indication of the extent of BFCN damage produced by the 192 IgG-saporin lesion (according to the methods of Tago et al., 1986). Twenfy-four hours after the final intrabasalis infusion of either NMDA-Rl antisense or missense oligodeoxynucleotides (Aim 2, histologic component), animals were transcardially perfused with heparinized saline followed by 10% formalin. The brains were removed, post-fixed in 10% formalin for 24 h, and then placed in 20% sucrose-phosphate buffer.

Sections (40 pm) were processed to mdicate the density ofNMDA-Rl subumts

24 following NMDA-Rl antisense and missense oligodeoxynucleotide infusions

[followmg modified protocols fiom Upstate Biotechnology, Lake Placid, NY],

Statistical analysis

Statistical analyses of the behavioral data collected were conducted separately

for each task type withm each experiment. Detailed descriptions of the analyses conducted are provided within the chapters discussing the specific experiments; where

appropriate, data collected were also subjected to the below described analyses. For

all experiments, percentage data were normalized using an arcsine transformation (Zar,

1974: X’ = 2 * arcsine[ x] ) and statistical analyses were performed only on these

transformed values. For the sustained attention task in Aim 1, measures of

performance were subjected to repeated-measures analyses of variance (ANOVA) with

VI having 3 factors [dose (4 levels), block (3 levels), signal length (3 levels)] and SB,

as well as omissions, having 2 factors [dose (4 levels), block(3 levels)]. The relative

number of hits (h) and of correct rejections (cr) were also subjected to repeated-

measures ANOVA in order to clarify any significant effects foimd for VI [h: 3 factors

(dose X block x signal length); cn 2 factors (dose x block)]. Data from the cued visual

discrimination task was analyzed separately for each drug (NMDA or APV) by

repeated-measures ANOVA for the relative number o f correct responses and relative

number o f omissions per block of 12 minutes [correct responses, having 2 factors: dose

(3 levels), block (4 levels); and omissions, with 2 factors: dose (3 levels), block (4

levels)].

2 5 For the behavioral component of Aim 2, sustained, attention performance measures were subjected to repeated-measures analyses of variance (ANOVA) of VI, having 3 factors [dose (4 levels, including baseline and antisense, or missense, ODN concentrations), block (3 levels), signal length (3 levels)], and SB, having 2 factors

[dose (4 levels) and block (3 levels)]. The relative number of hits (h) and of correct rejections (cr) were also subjected to repeated-measures ANOVA in order to clarify any significant effects found for VI [h: 3 factors (dose x block x signal length); cn 2 factors (dose x block)]. Similarly, data fiom the cued visual discrimination task was analyzed by repeated-measures ANOVA for the relative number o f correct responses and relative number of omissions per block of 12 minutes [correct responses, having 2 factors: dose (3 levels), block (4 levels); and omissions, with 2 factors: dose (3 levels), block (4 levels)]. Data fiom the histologic component of Aim 2, comparing the amounts o f NRl-Iike immunoreactivity following intrabasalis infusions of either

NMDA-Rl antisense or missense ODNs, was not conducted at this time.

Sustained attention data in Aim 3 was analyzed as follows: the initial effects of

SI/NB lesions upon VI and the relative number of hits were assessed by mixed-fkctors

ANOVA, with the between-subjects factor of lesion (Dulbecco’s saline versus 192

IgG-saporin infusion) and the within-subjects factors of surgery (2 levels: pre- versus post-surgery), block (3 levels) and signal length (3 levels). The relative number of

correct rejections and SB were also subjected to mixed-factors ANOVA with the

between-subjects factor of lesion and the within-subject factors o f surgery ^re- versus

post-surgery) and block. Data fiom all behavioral measures for each dose of the drug

26 tested m Aim 3 (DCS) was analyzed separately in a repeated-measures ANOVA for each group of animals (sham- or 192 IgG-saporm-Iesioned) for VI, having 3 factors

[dose (3 levels), block (3 levels), signal length (3 levels)], and SB, as well as omissions, having 2 factors [dose (3 levels), block(3 levels)]. The relative number of hits (h) and of correct rejections (cr) were also subjected to repeated-measures ANOVA in order to clarify any significant effects found for VI [h: 3 factors (dose x block x signal length); cn 2 factors (dose x block)].

Possible violations of the assumption of sphericity were countered by evaluating repeated measures ANOVA with greater than two levels for any factor analyzed using Hitynh-Feldt e -corrected degrees of freedom (Vasey and Thayer,

1987); Huynh-Feldt corrected degrees o f freedom, as well as p values, are documented.

Further analysis o f significant F values was obtained by conducting one way AVOVAs

of relevant factors and Tukey’s Honestly Significant Difference (HSD) post hoc tests

(a = 0.05). Statistical analyses were performed using the SPSS/PC+ 10.0.0 Version

(SPSS In t, Chicago, II.).

27 CHAPTERS

EXPERIMENT!

Basal forebrain glutamatergic modulation of sustained attention performance in

rats: effects of APV and NMDA

Introduction

The BFCN receives input 6om diverse sources including indirect glutamatergic modulation from prefrontal cortical areas, as well as direct glutamatergic projections from the basolateral amygdala, GABAergic projections from the nucleus accumbens, noradrenergic input from the locus coeruieus and serotonergic projections from the dorsal raphe (Zaborszky et al., 1997; 2Iaborszky et al., 1991; Games et al., 1990). The

BFCN has been suggested to play a prominent role in attentional processes (Muir et al.,

1994), likely serving to enhance the processing of behaviorally salient stimuli, as data derived from in vivo microdialysis studies has shown increases of cortical acetylcholine efrlux in response to presentation o f sensory and behaviorally salient stimuli (Acquas et

al., 1998, Butt et al. 1997, Inglis et al., 1994). Electrical stimulation of NB neurons, as well as intrabasalis microinjection o f L-GLÜ have resulted in increased cortical ACh

efflux (Kurosawa et al., 1989). Additionally, intrabasalis administration of NMDAR

28 antagonists ( CPP, ^nnurenate) have resulted in decreases of evoked ACh release

(Rasmusson et al., 1996; Rasmusson et al., 1994). Importantly, modulation of cortical

ACh. via glutamatergic stimulation of NMDAR in the basal fbrebrain has been demonstrated; intrabasalis infiision of NMDA (200 jxmol; via reverse dialysis) selectively produced increases o f cortical ACh. efflux in animals trained to associate darkness with the presentation of a palatable food, as compared with awake, but entrainment-narve animals (Fadel et al., 1998).

The above mentioned data, in conjunction with in vivo microdialysis data demonstrating task dependent differential responsivity of the BFCN projection system, whereby cortical acetylcholine release is augmented only for instances of explicit attentional processing (Himmelheberet al., 2000; Himmelheber et al., 1997), suggests the utility of investigating the effects of intrabasalis NMDAR modulation during sustained attentional processing in comparison to non-attentional processing. It was anticipated that administration of the NMDAR antagonist APV would result in deficits in animals’ performance of the sustained attention task as compared with their performance following sterile saline infusions. Infusions of NMDA were expected to produce attentional impairments with different characteristics than those derived from

APV. Additionally, it was predicted that as the cued visual discrimination task does not tax attentional processes, impairments of response accuracy would not be observed

for either ligand admmistered.

29 Specific methods

Thirteen, male BNNia/F344 rats were used to examine the effects of intrabasalis infusion o f an NMDAR antagonist, as well as agonist, upon sustained attention performance. An additional five male BNNia/F344 rats were used to examine the effects o f intrabasalis infusion o f an NMDAR antagonist, as well as agonist, upon performance of the non-attention demanding cued visual discrimination task.

Behavioral training and intracranial infusions

The rats for this experiment were trained for either the sustained attention task or the cued visual discrimmation task as described above in the Chapter 2. After attaining stable, criterion performance (see General methods), all animals were implanted with bilateral chronic guide caimula. Animals had a 7 day recovery period

(food and water available ad libitum) after surgery. Following the post-operative recovery period, they resumed a water deprivation schedule and will return to behavioral training. Once their performance had reached asymptotic levels, a pseudo­ randomized schedule of infusions of NMDAR ligands (antagonist and agonist) was executed.

AIT o f the animals performing the cued visual discrimination task received a

series of 5 bilateral infusions including APV ( lOnmol, ZOrnnol, 0.5 pl/hs; Maren et al.,

1996), NMDA (3 nmol, 6 nmol; 0.5 pl/hs; Pawley et al., 1996; Boegman et al..

30 1992), and vehicle infiision (sterile saline: 0.5 |xl/hs; pH 7.4). For the animals performing the sustained attention task, one half of the animals received a series of infusions composed of APV (3 mnol, 10 mnol, 20 nmol, 0.5 pl/hs) and vehicle (sterile saline: 0.5 ptl/hs; pH 7.4). The other half of the animals received a series of infusions composed o f NMDA (1 nmol, 3 nmol, 6 nmol, 0.5 pl/hs) and vehicle (sterile saline:

0.5 pl/hs; pH 7.4). These infusions were executed 10 minutes prior to placement in the operant chamber (approximately 13 minutes prior to task onset) at an infusion speed of

0.5 pi/ min; the internal cannula remained in place for 2 minutes following the infiision to assure adequate perfusion of the drug. Each animal received 5 intracranial mfiisions and the interval between infiision sessions was a minimum of two training sessions, or as many training sessions as was necessary to regain baseline performance.

Histological procedures

Upon completion of the infiision regimen, all animals were perfused (as described in the General methods) and processed for Nissl staming. An additional five male BNNia/F344 rats were used in a histological experiment to assess potential neurotoxicity associated with the higher doses of NMDA selected (3 and 6 nmol).

These animals underwent stereotaxic singery (following the protocols described in the

General methods) wherein the animals received unilateral stereotaxic admistrations of

the same infiision parameters as those received by the behaving animals. Two pairs of

animals each received 3 or 6 nmol NMDA, and one animal received infiision of 30

nmol as a positive control fi>r neurotoxic insult (dissolved in sterile saline, 0.5 pi per

31 infusion» right hemisphere) mhised at a rate of 0.5 pi /minute mto the SI/NB via

Hamilton LG pi syringe; for control measures, these animals received 0.5 pi ihfiisions

of the vehicle for NMDA (sterile saline) into the same site on the left hemisphere,

followmg the same parameters of infusion speed. The syringe remained in place for 2

minutes following each infiision. Three days after surgery, the animals were perfiised

transcardially with heparinized 0.9% saline followed by 10% formalin, and postfixed/

cryoprotected for 2 days with 20% sucrose in 10% formalin. Sections (40pm thick)

were then collected using a cryostat for Fluoro-Jade processing according to the

method outlined in Schmued et al. (1997), with positive Fluoro-Jade label indicating

neurodegeneration.

First the sections were mounted onto gelatin coated slides and allowed to air dry

for 12 h. They were then placed in 100% EtOH for a 3 min. incubation followed

successively by rinses of 1 minute each in 70% EtOH and in distilled water. Sections

were then maintained on constant agitation, via orbital shaker, immersed in 0.06%

KMnO^ for 15 minutes. Following a 1 min. rinse in distilled water, sections remained

in a solution comprised of 10% Fluoro-Jade and 0.09% glacial acetic acid for 30

minutes- Upon the conclusion of this final incubation, sections were placed in three

successive 1 min. rinses in distilled water and then air dried before dehydrating through

ethanol, defatting in xylenes and coverslipping. For all incubations and procedures

including and following the Fluoro-Jade step, the sections were maintained in the dark

until examination under the microscope.

32 Statistical analyses

Statistical assessments were made in addition to the analyses described in the

General methods. The data collected fiom the cued visual discrimination task was exammed to reveal any differences between pre-surgical versus pre-infusion values for the relative numbers of correct responses (pc) and of omissions (o) with a repeated measures ANOVA, having 2 factors for each o f those measures, respectively: [surgery

(2 levels: pre-surgery and pre-infusion) and block (4 levels)]. Any differences between pre-infhsion baselines and saline for the relative numbers of correct responses Opc) and

of omissions (o) were similarly analyzed with a repeated measures ANOVA, having 2

factors for each of those measures, respectively: [dose (4 levels) and block (4 levels)].

Likewise, analysis of either APV or NMDA effects on the relative number of correct

responses, or of omissions, were conducted via repeated measures ANOVA, having 2

factors for each of those measures respectively: [dose (3 levels) and block (4 levels)].

For the sustained attention task, a 3-way mixed-factors ANOVA was

conducted to assess any differences in pre-surgical versus pre-infhsion baselines with

respect to the vigilance index [VI: between subject factor of group designation (2

levels, APV or NMDA); within subject factors: surgery (2 levels, pre- vs. post-op) and

signal length (3 levels)]. Examination of any differences between pre-infusion

baselineVI values for all stimulus lengths among the two groups was conducted by

oneway ANOVA, with factor group designation (APV or NMDA). Additionally, pre-

infhsion baseline Vi’s were compared with those of the saline infhsion by repeated

measures ANOVA within each drug group, having 2 factors [dose (5 levels) and signal

33 length. (3 levels). AU drug effects were analyzed as repeated measures ANOVAs within the two drug groups. Analyses for the effects of APV or NMDA were conducted for all of the behavioral measures collected as follows. Analyses of VI and of the relative number o f bits bad two factors [dose (4 levels), and signal length (3

levels)]. Effects on the relative number o f correct rejections, as well as SB and omissions, involved only 1 factor: dose (4 levels). Post hoc analyses o f any significant

F values were conducted as described in the General methods.

Results

For the cued visual discrimmation task, there were no effects o f cannula

implantation surgery upon either the relative number of correct responses or the relative

number of omissions (collapsed criterion data for 3 days preceding surgery were

compared with pre-infusion baselines for doses tested: all p’s >0.137). There was an

effect o f block observed for the relative number of omissions, F (2.11,8.445) = 7.737,

p =0.012; this reflected an significantly greater relative number o f omission for the

fourth 12 minute block of the task as compared to each of the preceding 12 minute

blocks [Tukey’s HSD < 0.001 for blocks I and 2; Tukey’s HSD = 0.005 for block 3;

omissions: block 1:18 i 2%,block 2:18 ± 2%, block3:201 2%, block4:28 ± 2%].

Similarly, while there was no difference detected between the relative number of

correct responses for pre-infhsion baselines versus saline infusion across all blocks (all

p’s > 0.24), an overall effect of block on the relative number o f omissions was present

34 for this comparison, reflecting a tendency Gir the relative number of omissions to mcrease as time on task progressed [omissions: block 1:14 ± 2%, block 2:12 ± 1%, block 3:15 ± 1%, block 4:22 ± 2%].

As anticipated, there were no discernable effects of either compound (APV or

NMD A) infused upon the relative number of correct responses, across all blocks, in the cued visual discrimination task (all p*s > 0.199; see Figures 3.1 and 3.3 for APV and

NMD A data, respectively). A main effect of dose upon the relative number of omissions for APV [F (2,8) = 29.181, p < 0.001] as well as strong trend for an effect of dose upon the relative number of omissions following NMDA infusions were present

[F(l.l 14, 4.457) = 6.920, p = 0.051]. Post hoc analyses revealed the effect of APV infusions upon the relative number of omissions to be significant for both doses employed [10 nmol APV: Tukey*s HSD =0.010; 20 nmol APV; Tukey’s HSD <

0.001; omissions: saline: 14 ± 1%, 10 nmol: 32 ± 4%, 20 nmol: 63 ± 5%]. Interactions between the effects of dose and block were not found for either compound tested (all p’s > 0.56).

No effect of the caimulation surgery upon the sustained attention task animals’ ability to discriminate correctly between signal and non-signal events was revealed

[collapsed criterion data for 3 days preceding surgery were compared with pre-infusion baselines for all doses adminstered; VI: F(l,l 1) = 0.285, p = 0.604 (VI pre-surgery:

0.51 ± 0.02, post-surgery: 0.52 ± 0.03)] ; likewise, significant differences between the anrmats designated for APV, as compared to NMDA administrations, with regard to either their collapsed pre-surgery or pre-mfbsion baseline VI values, were not present:

35 F (l,ll) = 1.881, p = 0.198 (APV group: presurgery VI: 0.48 ± 0.02, pre-infusion: 0.48

± 0.03; NMDA group: presurgery VI: 0.53 ± 0.03, pre-infusion: 0.55 ± 0.03). No significant differences were revealed within either group designation for any behavioral measure collected (VI, relative number o f hits, relative number of correct rejections,

SB, nor omissions) when pre-infusion baselines were compared with saline infusions

(all p’s > 0.055). Within each group, an effect of stimulus length upon vigilance performance was prominent [ APV: F (1.261,6.307) = 187J239, p < 0.001; NMDA: F

(1.740,10.439) = 88.445, p < 0.001], whereby decreases in the animals’ ability to discriminate signal fiom non-signal events were found at the shorter stimulus lengths

[APV: VI500 msec: 0.64 ± 0.01, VI50 msec: 0.46 ±0.01, VI25 msec: 0.36 ± 0.02;

NMDA: VI500 msec: 0.69 ± 0.01, VI50 msec: 0.51 ± 0.02, VI25 msec: 0.44 ± 0.01].

Significant effects of dose for both compounds upon the behavioral measures collected in the sustained attention task were observed. A main effect of dose on VI was found for the animals receiving NMDA infusions; [F (1.793,10.760) = 13.588, p = 0.001; VI: saline: 0.56 ± 0.02,1 nmol: 0.45 ± 0.03,3 nmol: 0.38 ± 0.03,6 nmol:

0.24 ± 0.06]. Post hoc multiple comparisons revealed significant differences between saline, 3 nmol and 6 nmol NMDA, as well as differences between the effects of 1 nmol and 6 nmol (Tukey’s HSD = 0.039,0.000 and 0.02, respectively), hi addition, although

the characteristic effect of sthnulus length was present, F (1.341,8.047) = 27.481,

p < 0.001, there were no interactions o f dose and signal length, F (2.282,22.544) =

0-991, p = 0-429. Further analysis was conducted to specify the source of the effect on

VI; while no effect of dose on the relative number of hits was demonstrated, F (1.790,

36 10.740) = 1.104, p = 0359, a main effect o f dose upoa the relative number of correct rejections was revealed, F (2.535,15311) = 10338, p = 0.001. This effect of dose was significant for saline versus 3 nmol as well as 6 nmol [Tukey’s HSD = 0.046 and

0.006, respectively; CR: saline: 86 ± 1%, 3 nmol: 72 ± 2%, 6 nmol: 66 ± 6%; see Fig.

3.4]. Additionally, while there was no main effect of dose on SB, [F (2.832,16.994) =

2.341, p = 0.112], there was an effect upon the number of omissions [ F (2.418,14.505)

= 15.732, p < 0.001]. Post hoc testing revealed this omissions effect to be significant for the 6 mnol dose only [Tukey’s HSD = 0.001; OM: saline: 9.0 ± 23,6 nmol: 78.0 ±

9.53].

Conversely, for the APV administrations, an important lack of effect of dose upon the relative number of correct rejections was noted, F (1383,7.917) = 0.130, p =

0.835, in the presence of a main effect of dose upon the relative number of hits, F (23,

11302) = 5.044, p = 0.024 (see Fig. 33). Post hoc analyses indicated that while a

trend existed for a significant difference between the effects of the 3 nmol dose and the

20 nmol dose (Tukey’s HSD = 0.067), the effect was actually reflecting a significant

depression in the relative number of hits following the 20 nmol infusion as compared

with the saline infusion (Tukey’s HSD = 0.024; h: salme: 66.6 ± 4.6 %, 20 nmol: 41.6

± 8.0% ). While there was also an effect of signal length [ F (2,10) = 16.146, p =

0.001], there was no interaction between the effects of dose and signal length [ F

(5.441,27304) = 1.787, p = 0.145]. There was no effect found of dose on SB [F (235,

11.761) = 3.46, p = 0.60]. Similar to the cued visual discrimination task, an effect of

37 APV adminstratioii upon omissions was recorded [F (2.72,11359) = 7306, p = 0.008]; this efifect was present for the 3 nmol as well as the 20 nmol doses (Tukey’s HSD =

0.003 and 0.004, respectively; OM: saline: 8.33 ± 3.0,3 nmol: 6.66 i 1.66,20 nmol:

70.1 ± 18.8).

Examination of the Fluoro-Jade processed sections revealed a lack of neurotoxic effects associated with mfiision of either of the higher doses of NMDA (3 and 6 nmol) employed in the behavioral component of this experiment, in contrast to the robust Fluoro-Jade staining observed for the 30 nmol NMDA infusion processed as a positive control for neurotoxic insult, (see Fig. 3.5 for photographic depiction of this data).

Discussion

Although both NMD AR and non-NMD AR are generally present on the same cell, each contributes differently to the response o f the cell to GLU (Mcoll et al., 1990); while AMPAR activation may allow for subsequent NMDAR activity (in response to

GLU), via removal of the Mg2+ block due to membrane depolarization, its rapid response and response decay may not subserve cognitive processes as the greater duration o f the NMDAR response to GLU has been implicated (NicoU et al., 1990).

That testing only NMDAR ligands was appropriate for this experiment is supported by the data o f Fadel et al. (1998), which indicated a requirement of cognitive/behavioral activation status for intrabasalis NMDAR agonism to yield increased cortical ACh

efflux, in contrast to agonism of kainate receptors. With respect to méthodologie

38 concerns, the degree of ligand difEusion, given the volnme (0.5 (xl) used, has been shown to be reasonably circumscribed to the area of mfiision (Routtenberg, 1972); maintenance o f an intracranial mfiision schedule of less than 5 infusions was followed in order to avoid excessive tissue damage with repeated intracranial infusions which might otherwise have precluded interpretation the behavioral data. Given that previous study has indicated behavioral and pharmacological manipulation of cortical ACh was not afEected by repeated (4 sessions) microdialysis testing in firontoparietal cortex

(Moore et al., 1995), the infusion regimen employed appeared to assure interprétable data

The lack of significant effects of NMDAR modulation, either agonism or antagonism, upon the relative number of correct responses exhibited by animals performing the non-attention demanding cued visual discrimination task provides additional empirical support for the notion that basal fbrebrain NMDAR-mediated increases or decreases in cortical ACh are critically dependent upon the cognitive demand status of the animal; thus effects would only be associated with tasks which explicitly tax attentional processes; data derived fiom the effects of basal fbrebrain modulation upon the behavioral measures in the sustained attention task provide evidence o f a substantive role o f glutamatergic modulation of corticopetal cholinergic neurons in attentional processing.

The finding of decreases in the relative number of hits following NMDAR blockade by APV infusions, in the absence o f efEects upon the relative num ber of

39 correct rejections, corresponds with previous data indicating the ability o f an aufmal to detect signal events in the sustained attention paradigm is critically dependent upon fimctional cholinergic output fiom the BFCN (McGaughy et al., 1996). As specific cortical cholinergic deafferentation induced by 192 IgG-saporm mfiision can yield both selective decreases m the relative number of hits (MgGaughy et al., 1996) and decreased cortical acetylcholine efflux (Fadel et al., 1996), the depression o f the relative number of hits in the animals performing the sustained attention task following intrabasalis APV infusion may correspond diminished cortical ACh efflux, although data for this interpretation is outside the scope of this experiment series.

The effect of NMDAR agonism upon performance measures in the sustained attention paradigm, namely the selective decrease in the relative number of correct rejections, is evocative of previous behavioral data wherein possible increases of cortical ACh efflux (see Moore et al. 1995) consequent to intrabasalis BZR inverse agonist administration was associated with selective increases in the rate o f false alarms

(the inverse o f the correct rejection measure; Holley et al. 1995). While it was not directly measured within the confines of this experiment, it may be possible to interpret the increase o f false alarms, i.e., the incorrect identification of non-signal as signal trial events, associated with intrabasalis NMDA infusion as reflecting a hyperattentional

state, perhaps associated with NMDA-induced cortical cholinergic hyperactivation.

This finding could have implications for modeling an aspect of cognitive dysfunction

(hyperattention) that over time could yield breakdowns m cognitive processing, or even

pqrchosis (see Sarterand Bruno, 1999; Mar et al., 1996).

40 CHAPTER#

EXPERIMENT 2

Antisense oligodeoxynncleotide-induced suppression of basal forebrain NMDA-

NRl receptor subunits: sustained attentional consequences

Introduction

The BFCN has been proposed to perform a substantive role in attentional

functions (Muir et al., 1994; Richardson and DeLong, 1991) and corticopetal cholinergic basal forebrain neurons have been shown to receive glutamatergic (both direct and indirect; see above) modulation &om multiple sources (Zaborszky et al.,

1997; Zaborszky et al., 1991). Furthermore, intrabasalis administration o f NMDAR antagonists (CPF, Iqmurenate) has been demonstrated to decrease evoked ACh release

(Rasmusson et al., 1996; Rasmusson et al., 1994). Antisense oligodeoxynucleotides

have been shown to provide an efficacious and reversible means for inhibition of

specific gene expression. While members of the NR2 group have been identified in

striatum, cerebellum, thalamus, hippocampus and brain stem nuclei, and the variations

of their representation have been noted (e.g., NR2A and NR2B are present in the

striatum, whereas NR2C and NE12D are not: Wenzel et al.,I995), such identification

41 profiles have not been executed for the SI7NB. Given the necessity of the NMDA-Rl subunit presence for functional hetero-oligomeric NMDA receptors (Sprengel and

Seeburg, 1993), targeting the NMDA-Rl subunit for antisense oligodeoxynucleotide mduced disruption appeared appropriate.

While unmodified antisense oligodeoxynucleotides (containing phosphodiester

[PDE] backbone linkages) have been used in various in vitro and in vivo experiments, their mcreased susceptibility to degradation by nucleases and the requirement for higher doses (which raises the likelihood for non-specific consequences) in order to obtain effects rendered this type o f oligodeoxynucleotide less desirable.

Phosphorothioate analogues, wherein sulphur is substituted in lieu of one of the non­ bridging intemucleotide oxygen molecules, are both more resistant to degradation and may have enhanced uptake relative to PDE-oligodeoxynucleotides; the work upon which selection of both the current sequence for the antisense oligomer as well as dose information employed fiilly phosphorothioate-substituted ODNs (Standaert et al.,

1996). hi an effort to avoid potential neurotoxicity associated with such fiilly substituted oligomers, the missense and antisense sequences selected for this experiment were “Second generation chimeras”, having less total sulfur substitutions along the phosphate backbone, and included 2’-0-methyl substitutions.

The present experiment examined the efiects o f decreased NMDA-Rl subunit density, fbllowmg intrabasalis administration of antisense oligodeoxynucleotides for the NMDA-Rl subunit, upon sustained attentional processing. It was predicted that any attentional impairments associated with intrabasalis infusions of NMD A-Rl

42 antisense oligodeoxynucleotides would depend upon the extent to which the number of functional hetero-oligomeric NMDA receptors was diminished consequent to the disruption of NMDA-RI mRNA translation. If moderate amounts ofNMDA-RI subunit synthesis inhibition had been obtained by this manipulation, the resultant attentional unpairments might appear as decreases in the number of correct visual signal detections (hits), similar to the efkcts of either partial cholinergic lesions o f the

SI/NB, or as discussed above, intrabasalis infusion of the NMDAR antagonist, APV.

Specific M ethods

Eight male BNNia/F344 rats were used to examine the effects of intrabasalis infusion of different doses ofNMDA-Rl antisense oligodeoxynucleotides, as well as missense oligodeoxynucleotides, upon sustained attention performance; six male

BNNia/F344 rats were used to test the effects of intrabasalis infusion of the highest dose of NMDA-RI antisense oligodeoxynucleotides, as well as missense oligodeoxy­ nucleotides, in the cued visual discrimination task. The goal of this experiment was to assess the capacity of different doses for eliciting behavioral efiects in the sustained attention task, and test the hypothesis that NMDAR involvement is selective for tasks which tax attentional processes. This dose regimen also revealed the reversibility of

NMDA-RI antisense oligodeoxynucleotide effects, with respect to behavioral

measures, as baseline performance levels had to be regained prior to subsequent

infusions.

43 Antisense and missense oligodeoxynucleotides

“Optunized oligomers”, processed through Level 1 purification (HPLC), were purchased fi»m Oligos Etc., fiic. (Wilsonville, OR). These oligomers contained a limited number o f phosphorothioate linkages in order to enhance their stabili^ but reduce potential non-specific eSects. A 20-polymer oligodeoxynucleotide with the

following sequence, which is complementary to a region including the putative

translation initiation site for the NMDA-Rl subunit, was used as the NMDA-Rl

antisense ODN: S*-G-CAG-GTG-CAT-GGT-GCT-CAT-G- 3’. The underlined portion

represents the region of the oligodeoxynucleotide which was complementary to the

translation initiation codon. The sequence for the missense control was 5’ -G-CAG-

GTG-CCT-GGT-GAT-CTA-G- 3’. Once received, the oligos were dissolved in fresh

sterile saline, lacking alcohol based bacteriostatic agents, and mulitple aliquots of each

dose of missense and antisense were made in gas sterilized, certified RNase-, DNase-

free microcentrifuge tubes; these aliquots were then lyophilized and maintained in

pellet form in a -80“C freezer until resuspension prior to infusions, as the dissolved

oligos were only viable for one week.

Behavioral training and intracranial infusions

These rats were teamed, as described above, in either the sustained attention

task or the cued visual discrimination task. Upon attaining stable, criterion

performance (see General methods), all animals were implanted with bilateral chronic

guide cannula. After surgery, animals had a 7 day recovery period (food and water

44 available ad libitum). Followmg the post-operative recovery period, they resumed a water deprivatioa schedule and returned to behavioral traming. Once their performance reached asymptotic levels, animals commenced oligodeoxynucleotide infusion schedules (2 doses as listed below), with each animal receivmg 3 mfusions each of NMDA-RI antisense oligodeoxy-nucleotides (1.0 nmol and 8.0 nmol) as well as missense oligodeoxynucleotides (8.0 nmol), m counterbalanced fashion, all infusions administered

12 h apart; animals in the cued visual discrimination task received 3 infusions each of missense (8.0 nmol) and NMDA-NRl antisense (8.0 nmol) oligodeo}qmucleotides (see

Table 4.1 for design of experiment). The same rate of infusion (0.5 |ti/min) was employed

for all administrations, and mtemal cannula were allowed to remain in place for 2 minutes

followmg all mfusions to ensure adequate perfusion of the respective solution. Behavioral

testing occurred 24 h after the third infusion of each treatment, although data was collected for analysis from the behavioral sessions 12 h following the second mfiision, as

well as 48 h followmg the third infiision treatment for both task types. Treatments 1,2 and 3 were separated by at least 2 behavioral sessions followmg the test session.

Histological procedures

After completion of the antisense or missense oligodeoxynucleotide infusion schedules, animals were perfused and processed for NissI stammg to establish cannula

placement (see General methods). An additional expernnental component of Aim 2 employed immunohistochemical technûpies to assess the effects of mtrabasahs

45 in filmons of antisense oligodeoiQmucIeotides targeting the NMDA-RI subunit upon expression of the NMDA-Rl subunit It was anticipated that whereas infusions of missense oligodeoxynucleotides would not affect the expression o f NMDA-Rl subunits, antisense oligodeoxynucleotides for NMDA-Rl might quantiffably inhibit the expression of NMDA-Rl subunits. Two groups of animals were implanted (as described in the General methods) with chronic guide cannula into the SI/NB. After a post-surgery recovery period, one group of animals was subjected to three unilateral infusions, each spaced 12 h apart, o f antisense oligodeoxynucleotides for the NMDA-

Rl subunit (right hemisphere: 8.0 nmol, 0.5 pi per infusion; 0.5 pl/min), and the other group received three infusions o f missense oligodeoxynucleotides (right hemisphere:

8.0 nmol, 0.5 pi per infiision; 0.5 p/min) following the same schedule. All animals

received infiision of the vehicle (sterile saline, 0.5 pi) for the oligodeoxynucleotides in

the same site on the left hemisphere, Billowing the same infiision parameters with

regard to speed, and mtemal cannula were allowed to remain in place for 2 minutes

following all infusions to ensure adequate perfiision of the respective solution

administered.

46 Design of experiment: I 2 h I 2 h 24 h Group 1 I antisense ----- 1 antisense ------I antisense ------perfuse

(n = 5 )

I2h 12 h 24 h Group 2 1-m issense ----- 1 missense------I m issense ------perfuse

(n = 5)

All animals were perfused with 10% Armalin, as described in the General

methods, 24 h after the Snal oligodeoxynucleotide inhision; the brains were placed in

10% formalin postfix for 24 h and then transferred to 20% sucrose in 0.1 M phosphate

buffer. One set of sections per animal were collected and subjected to immuno­

histochemical processing, while adjacent sections were processed for Fluoro-Jade

staining as described above in Chapter 3. Sections from each brain were examined for

the relative amount ofNRl-Iike immunoreactive cells in SI as well as evidence of

neurotoxic effects provided by the Fluoro-Jade procedure.

Processing for NMDA-Rl immunohistochemistry utilized a polyclonal

antibody directed against an intracellular loop of the NMDA-Rl subunit (anti-NRl-CT,

Lot #17580; Upstate Biotechnolosr, Lake Placid, NY); the specificity of this antibody

for NRl splice variant forms A, B, C, and F has been confirmed by immunoblot and

immunoprécipitation analysis. The antibody arrived as a frozen solution on dry ice,

and aliquots were made and kept at -20 "C as recommended. Sections (38 pm thick)

were collected using an electric stage coupled freezing microtome (“MG”, model

47 #PS10AD, and serial #5812, Spencer Lens Co., Buffalo, NY, respectively) into 0.1 M phosphate buffer (pH 7.4). The sections were first rinsed extensively m phosphate buffered safiie (PBS: pH 7.4) and then mounted onto clean slides. These slides were then placed m a Coplin jar containing 10 mM citrate acid solution; this jar was then placed in a water filled Pyrex© chamber for successive high temperature antigen retrieval treatments

(4 successively repeated sessions of microwaving for 1.5 mm. on high in a 600 W oven, and coolmg for 3 mm). The sections were cooled m the Coplin jar for 20 mm., removed

form the slides, placed into staming nets and rinsed m distilled water. They were then

placed m an 0.5% HiO? solution for 10 min., followed by extensive rinses in PBS. The next incubation was for 60 minutes in 1% bovme serum albumin/PBS solutioiL

Subsequently, a final blocking incubation (3% normal serum in 1% BSA/PBS) took place

for 30 minutes. Primary antibody incubation (1.0 p.g/ml of anti-NMDA-Rl) was

conducted at room temperature for 12 hours and involved contmuous agitation of the

sections. After the primary antiserum incubation, sections were rinsed extensively in

0.1 M PBS. The subsequent incubation in secondary biotmylated goat anti-mouse IgG

(1:400, Vector Laboratories) was 90 minutes m duration. Followmg rinses m PBS,

sections were then placed in avidin-biotin horseradish peroxidase complex (ABC Elite Kh:

Vector Laboratories) for 90 minutes. Sections were rinsed extensively m 0.1 M PB, and

next processed for 6 minutes in 3-3’ diamino-benzidine tetrachloride (100 mg, in

100 ml o f 50 mM PB) with 125 |il of 3% C 0 CI2 and 12.5 pi of 30% H 2O2- Rnal rinses m

50 mM PB followed by 0.1 M

48 phosphate bu& r preceded mounting o f the sections on gelatin coated slides and subsequent dehydration and coverslipping. Additional sections were processed as described above, save exposure to the prhnary antibody, for control measures. NRI- positive cells were evaluated m the SI by microscope observation and photographs of relevant sections from each brain were obtained (see Fig. 4.7).

Statistical analyses

Statistical assessments were made in addition to the analyses described in the

General methods. The data collected Gtom the cued visual discrimination task was examined to reveal any differences between pre-surgical versus pre-infusion values for the relative numbers of correct responses (pc) and of omissions (o) with a repeated measures ANOVA, having 2 factors for each of those measures, respectively: [surgery

(2 levels: pre-smrgery and pre-infrision) and block (4 levels)]. Likewise, analysis of either missense (8.0 nmol) or antisense oligodeoxynucleotide (8.0 nmol) effects on the relative number of correct responses, or of omissions, were conducted via repeated measures ANOVA, having 2 factors for each o f those measures respectively: [dose (4 levels) and block (4 levels)]; these analyses were conducted separately for each of the three different times points following the infiision regimen (12 h after the second infiision, 24 h after the third mfiision, 48h after the third mfiision).

For the sustained attention task, a repeated measures ANOVA was conducted to assess any differences in pre-surgical versus pre-infiision baselines with respect to the vigilance index [VI: withm subject factors: surgery (2 levels, pre- vs. post-op) and

49 signal length (3 levels)]. Pre-infusion baseline Vi’s were compared with those of the oligodeoxynucleotide mfusions by repeated measures ANOVA, VI having 2 factors

[dose (4 levels, including baseline, missense, antisense low dose, antisense high dose) and signal length (3 levels)]; this same analysis was conducted three separate times for each o f the three time points at which the data had been collected (12 h after the second infusion, 24 h after the third inhision, 48h after the third infusion). Likewise, three separate analyses for the effects of missense, and both doses of antisense, were conducted for all of the behavioral measures collected as follows. Analyses of VI and of the relative number of hits had two factors [dose (4 levels), and signal length (3 levels)]. Effects on the relative number of correct rejections, as well as SB and omissions, involved only I factor dose (4 levels). Post hoc analyses of any significant

F values were conducted as described in the General methods.

Results

As found for the first experiment, animals in the cued visual discrimination task failed to exhibit any effect of the cannula implantation surgery upon either the relative number of correct responses or the relative number o f omissions; this comparison involved the collapsed means of 3 criterion perfomance days preceding surgery and the collapsed pre-infusion baselines for all oligodeoxynucleotide infusions [correct responses: F (2,10) = 0.514, p = 0.613; omissions: F (1.940,9.701) = 3.814, p = 0.061].

Also, as observed in the first experiment, there was an eSect of block upon the relative number o f omissions, F (3,15) = 8.203, p = 0.002. Further analysis o f this effect indicated that the relative number of omissions was significantly higher in block 4 than

50 each o f the other blocks [Tukey*s HSD = 0.002 for block I; HSD < O.OOl for block 2, and HSD = 0.032 for block 3; omissions: block 1:0.16 ± 0.01, block 2:0.15 ± 0.01, block 3:0.19 i 0.03, block 4:0.28 ± 0.03].

As was hypothesized, the lack of «cplicit taxation of attentional processes afforded by the cued visual discrimination task was evident in the lack o f effects of antisense oligodeoxynucleotide adminstrations on either the relative number of correct responses or the relative number of omissions (see Fig. 4.1). These negative findings were observed for all time points of data collection: for the behavioral session conducted 12 h following the second infusion [correct responses: F (1.436,7.182) =

1.974, p = 0.207; omissions: F (1.736,8.682) = 1.927, p = 0.204], for the session 24 h

following the third infusion [correct responses: F (2,10) = 0.861, p = 0.452; omissions:

F (1.271,6.357) = 0.069, p = 0.856], and for the session 48 h following the third

infusion [correct responses: F (1.996,9.980) = 0.122, p = 0.886; omissions: F (1.573,

7.864) = 2.007, p = 0.198]. An effect of block was only found on the relative number

of omissions at the time point 48 h following the third infusion [F (1.717,12.645) =

4.279, p = 0.031]; however, post hoc analyses failed to reveal the locus of the effect

(Tukey’s HSD for blocks 1-4, all p’s > 0.111).

Analysis of the potential effects of the cannula implantation upon the animals’

ability to discrimmate signal and non-signal events, as measured by VI, yielded

negative results; this test involved comparison of the collapsed VI values finm 3

criterion days preceding surgery and the VI values fiom the pre-infusion baseline

sessions prior to each oligodeoxynucleotide dose administered [F (2.552,15.311) =

51 1243, p = 0324]. An effect ofsignallength was observed [VI: F (1391,8.344) =

75.001, p < 0.001]; post hoc analysis revealed decreases of VI values correspondmg to decreases in signal length [V I500 msec vs. V I50 msec: t(41) = 8.795, p < 0.001; V I50 msec vs. V I25 msec: t (41) = 5.941, p < 0.001; V I500 msec: 0.60 ± 0.01, V I50 msec:

0.43 ± 0.02, V I25 msec: 0.33 ± 0.02].

No eSect o f either missense or antisense infusions was found for the sustained attention behavioral session conducted 12 h followmg the second infiision upon VI [F

(3,18) = 2344, p = 0.107; see Fig. 42). While an effect of signal length upon VI was present [F (2,12) = 43.052, p < 0.001; V I500 msec vs. VI50 msec: t(41) = 6383, p <

0.001, V I50 msec vs. VI25 msec: t (41) = 5.381, p < 0.001; V I500 msec: 0.57 ± 0.02,

V I50 msec: 0.43 ± 0.02, VI25 msec: 034 ± 0.02], there was no interaction between the effects of dose and signal length upon VI for this time point. Likewise, no effects of dose were found on the relative number of hits, the relative number of correct rejections, SB, nor omissions for this time point ( all p's > 0270).

hi contrast to the data collected for the 12 h time point, a main effect of dose on

VI was observed for the behavioral data collected 24 h following the third infusion p (2.076,12.453) = 5.144, p = 0.023]. Post hoc analysis of this effect revealed a strong trend solely for infusion of the high dose of the antisense oligodeoxynucleotide at this time point (8.0 nmol: Tukey’s HSD = 0.050; VI: baseline: 0.48 ± 0.03, missense: 0.49 ± 0.05,1.0 nmol antisense: 0.43 ± 0.03,8.0 nmol antisense: 033 i

0.03). Further examination of this data was conducted by analysis of the relative number of hits and correct rejections, hiterestingly, a main effect of dose was found on

52 the relative number of hits (P (3,18) = 10.898, p < 0.001] in the absence of any effect

upon the relative number o f correct rejections [F (1.645,9.871) = 0.504, p = 0.584; see

Fig. 4.3]. Additional analysis revealed the effect of dose upon the relative number of hits was exclusive to the 8.0 nmol antisense oligodeoxynucleotide infiision, which

mduced a decrease in the relative number o f hits (h) as compared to both baseline and

missense mfiision values [baseline comparison: Tukey’s HSD = 0.001; missense

comparison: HSD = 0.026; baseline (h): 65 ± 2%; missense: 62 ± 3%; antisense, I.O

nmol: 58 ± 3%; antisense, 8.0 nmol: 48 ± 3%]. An effect of signal length upon the

relative number of hits was also noted [F (1.876,11.255) = 27.757, p < 0.001],

reflecting decreases in the relative number of hits corresponding to decreases in signal

length [h 500msec vs. h 50msec: t (41) = 6.491, p < 0.001; h 50msec vs. h 25msec: t

(41) = 5.253, p < 0.001; h: 500msec: 72.5 ± 2.0%, 50 msec: 56.2 ± 3.7%, 25 msec: 47.0

± 32%]. An effect of dose on SB was found for this time point [F(3,18) = 9.791, p <

0.001], reflecting a increased tendency for animals to respond to the right lever only

following the 8.0 nmol antisense administration (Tukey’s HSD < 0.001; SB baseline:

0.42 ± 0.007,8.0 nmol antisense: 0.33 ± 0.01); in the absence of any significant dose

effect upon the relative number of correct rejections (CR: baseline: 811 1%, 8.0 nmol

antisense: 81.5 ± 2%), it would appear that the dose effect upon the relative number of

hits reflected a suppression o f the animals’ ability to discriminate the signal events

following infusion of the highest dose of antisense oligodeoxynucleotide. No

interaction between the effects of dose and signal length upon the relative number of

53 hits were observed for this tune point [F (5.047,30.285) = 1.494, p = 0.221]. Likewise, there was no efifectofdose upon the number o f omissions |TF(3,18) = 0.586, p = 0.632].

Analysis of the sustained attention performance data collected 48 h following the third infusion of oligodeoxynucleotides revealed no effect o f dose upon any of the behavioral measures recorded (VL relative numbers of hits and correct rqections, SB

and omissions: all p’s > 0.40; see Fig. 4.4). The characteristic e& ct of signal length

upon VI was again observed at this time point [F (1.450,8.701) = 37J232, p < O.OOl],

with VI values decreasing concomitantly with decreases in signal length [ V I500 msec:

0.59 ± 0.01, V I50 msec: 0.45 ± 0.02, VI25:0.34 ± 0.02]. The analysis conducted at

this time point provided illustration of the transient nature of the antisense

manipulation (see Fig. 4.5 for a time course of the dose effect on the relative number of

hits and of correct rejections for the high dose of the antisense oligodeoxynucleotide).

For the histological component of this experiment, assessments were made of

the sections derived from the animals unilaterally infused with the high dose of either

missense or antisense oligodeoxynucleotides. Fluoro-Jade staining revealed a lack of

difference between the missense or the antisense infusions with respect to neurotoxic

damage (see Fig. 4.6). While positive label was present, it was restricted to the region

immediately proximate to the guide cannula track for both oligodeoxynucleotide

treated and untreated hemispheres, and did not extend into the region of the infiision

area (SI: see Fig. 4.7); although the internal cannula were flush with the guide cannula

54 length, the presence of this positive label suggested modest degeneration was yet induced by apects of the infusion process itself (e.g., internal cannula movement, perfusion of the solution).

hnmunohistochemical analysis o f the eSects of missense as compared to antisense oligodeoxynucleotide mfiisions revealed substantive suppression of NMDA.-

NRl immunoreactivity exclusively in response to antisense infusions (see Figures 4.8 and 4.9). Loss of positive label was noted in the SI (particularly the lateral aspect); this

loss extended into the less densely labeled population of cells in the globus pallidus

lateral to the infusion and, less prominently, into the horizontal nucleus of the diagonal

band. The extent of loss of label along the anterior-posterior gradient was rather

circumscribed and the pattern of antisense-induced suppression of NRl-positive label

was akin to areas of maximal functional reactivity that have been described in Pecina

and Berridge (2000), a study addressing a different compound and infusion site, but

involving similar infusions volumes ( 0.5 pi) and the same rate of infusion (0.5 pi/

min).

Discussion

The time point selected for behavioral testing reflected observations by

Standaert et al. (1996) which indicated that maximal expression of behavioral changes

associated with NMDA-Rl antisense oligodeoxynucleotide infusions occur at 24 hours

post infusion. Likewise, the doses selected for testing were similar to those which had

been successfoUy employed in several other studies involving NMDA-Rl antisense

55 oligodeoxynucleotide infusions and represented an eSbrt to elicit behavioral effects while avoiding substantial cellular pathology (Standaert et al., 1996; Sun and

Faden,1995; Matthies et al., 1995). Although the turnover rate o f NMDA-Rl subunits has been characterized as relatively rapid (hippocampal CAI region: Soltesz et al.,

1994), the issue has been raised that in order to inhibit successfully synthesis of a receptor component, at least 2 days of antisense oligodeoxynucleotide treatment may be required (Wahlestedt, 1994). The time point selected for primary behavioral data collection reflected observations by Standaert et al. (1996) which indicated that maximal expression of behavioral changes associated with NMDA-Rl antisense oligodeoxynucleotide infiisions occur at 24 hours post infusion. While the total number of infiisions to which each behaving animal was subjected (9) exceeded limitations originally suggested by Routtenberg (5:1972), all doses (8.0 nmol missense, 1.0 nmol antisense, 8.0 nmol antisense) were adminstered in counterbalanced order, and baseline performance values had to be regained prior to any subsequent infiision, thus there were behavioral data which indicated a lack o f excessive trauma to the region over the course of all infiisions (extensive damage associated with the

infusions would have likely yielded substantive impairments o f performance in the sustained attention task); similarly, the infiision schedule for all animals tested provides

a behavioral means of assessing the reversibility of the NMDA-Rl antisense

oligodeoxynucleotide effects. The missense sequence selected was in accord with

suggestions to establish adequately stringent controls (Wagner, 1994; Wahlestedt,

56 1994); this sequence was a mismatched analogue of the antisense sequence which contained the same base pair proportions and a minimal number of mismatches in order to preclude specific hybridization to the target mRNA.

Antisense oligodeoxynucleotide mfusions for the immunohistochemical portion of the experment were of the same concentration and volume as those found to produce substantive behavioral effects in this experiment, and the time point selected for perfusion was the same as that selected for behavioral testing. While there have been studies which reported a lack of difference between NMDA-RI antisense ODN infused and control nnfmals with respect to NRl-like immunoreactivity (Kammesheidt et aL, 1997) and

Western blot analyses (Standaert et aL, 1996), in spite of dose regnnes that had produced robust electrophysiological and behavioral effects as well as significant reductions in amounts of NMDA-Rl mRNA, as measured by in situ hybridization (Kanunesheidt et aL,1997; Standaert et aL, 1996), the data derived firom the immunohistochemical portion of this experment provided evidence of the selectivity of antisense-induced suppression of

NMDA-NRl immunoreactivity. The presence of positive Fluoro-Jade staining in the region immediately proximate to the end of the cannula track is possibly not unexpected as blood-brain-barrier disruption associated with cannula insertion has been documented to persist for up to 28 days (Groothuis et al., 1998). The exact mechanism of Fluoro-Jade staming of degenerating neurons has remained poorly characterized, but has been posited to reflect binding of the acidic Fluoro-Jade dye with a yet undefined basic component associated with degenerating

57 neurons (Schmued et al., 1997). This stainmg technique has been used with success to map degeneration in response to specific parenchymal insult (Hopkins et al., 2000;

Cirelli et aL, 1999; Eisch et al. 1998).

The significant effect of antisense administration (8.0 nmol), as compared to missense infusion, solely upon the relative number of hits, provided additional evidence for the role of NMDAR modulation in the SI upon sustained attentional processes; suppression of the functional NMDAR in SI consequent to these antisense

infiisions yielded results which correspond to those discussed in Chapter 3, whereby

APV induced NMDAR blockade likewise was associated with impairments in the

relative number of hits, in the absence of impamnents for the relative number of correct

rejections. Both of these manipulations presumably serve to depress cortical ACh;

thus taken together, these results are in keeping with data derived fiom 192 IgG-saporin

lesioned animals, with measurable losses of AChE -positive fibers (McGaughy et al.,

1996), performing in this sustained attention paradigm and exhibiting impairments of

the ability to detect signal events in the absence of impairments in the ability to identify

correct rejections. Importantly, the lack of any significant effects of either

oligodeoxynucleotide (missense or antisense) upon the relative number of correct

responses in the cued visual discrimination task served to buttress the hypothesis that

basal forebrain NMDAR modulation only mediates cognitive fimctions associated with

tasks which explicitly tax attentional processes.

58 CHAPTERS

EXPERIMENTS

Partial atténuation of Impairments of sustained attentional processes following

192 IgG-saporin induced cholmergic lesions of the SI/NB:

Effects of intrabasalis infusions of the NMDAR/GS partial agonist D-Cycloserine

Introduction

Accumulating data has suggested a significant role of basal forebrain NMDA receptor modulation in tasks taxing attentional processes, and it was hypothesized that positive NMDA receptor modulation via the glycine site might attenuate the substantive impairments of sustained attentional processing observed following specific lesions of corticopetal cholinergic neurons. As intrabasalis administration of

NMDA involves direct modulation of NMDAR without regard for normal patterns of synaptic transmission (e.g., the multiple cortical GLU inputs to BFCN), infiision of an

NMDAR/GS agonist, which has potential to modulate allosterically NMDAR within context o f normal patterns of GLU release (Watson et al. 1990), was of significant

interest for study. Although the losses of NMDAR binding sites and consequent

glutamatergic abnormalities associated with AD suggest the need to mcrease

59 glutamatergic a c tivity, use o f direct NMDAR stimulation appears contraindicated due to the potential for excitotoxic consequences (McEntee and Crook, 1993; Greenamyre and Young, 1989). Therefore, investigation ofNMDAR/GS ligands, such as the

NMDAR/GS partial agonist D-cycIoserine (DCS: Henderson et al., 1990), may reveal useful pharmacotherapeutic strategies. DCS has been found only to potentiate

NMDAR responses to NMDA if levels o f GLY were low (0.1 jiM); if GLY levels were high (1 pM or more), DCS (10 pm to 100 pm) dose-dependently curtailed NMDA induced response magnitude (asymptotic to 50% maximal response: Watson et al.,

1990). A study assessing the impact of DCS upon tritiated MK-801 binding in inferior parietal cortex samples from AD brains revealed positive modulatory effects (Chessell et al., 1991).

DCS has been found to enhance animal learning rates for a simple conditioning paradigm, to reverse scopolamine-induced deficits in both acquisition of spatial learning and in working memory performance, and to enhance passive avoidance performance (Thompson et al., 1992; Fishkm et al., 1993; Quartermainet al., 1994;

Pitkanen et al., 1995; Ohno and Watanabe, 1996; Steele et al., 1996; Maurice et al.,

1996). Likewise, improvements in animal performance of place discrimination and visual recognition memory have been ascribed to the effects of DCS administration

(Matsuoka and Aigner, 1996a; Baxter et al., 1994). Although claims o f DCS as a cognition enhancer based upon data derived from tasks such as passive avoidance require cautious consideration (Sarter et al., 1992), the above discussed neurochemical

60 findings associated with AD and the phannacological characteristics o f DCS indicated the utility of finther study. It is important to note that the regulation o f GLY at

NMDAR synapses somewhat unclear; although it has been suggested that the

NMDAR/GS is not ordinarily saturated (Chessell et ai., 1991). Therefore microdialj^s measures of stimulated cortical ACh release m response to intrabasalis DCS in intacL as well as 192 IgG-saporin SI/NB lesioned rats, would further substantiate the interpretation of behavioral data generated in these experiments.

It was expected that 192 IgG-saporin administration into the basal fbrebrain would produce attentional unpairments as compared with animals receiving Dulbecco's saline infusions. Moreover, it was anticipated that whereas intrabasalis administration of DCS might yield attentional impairments in non-lesioned animals, infusion of this

ligand m lesioned animals might attenuate their lesion-induced attentional deficits.

Specific methods

In order to investigate the possibility that attentional deficits associated with

specific cholinergic lesions of the basal forebrain might be attenuated by intrabasalis

administration o f DCS, an additional group often animals were trained in the sustained

attention paradigm.

Behavioral training and intracracial infasions

Once animals had achieved criterion performance levels in the sustained

attention task, they received bilateral guide cannula implants; within this same surgical

session, one group o f these animals also received bilateral infusions of 192 IgG-saporin

61 (0.17 0.5 |il/hemisphere; bolus infusion with 1.0 ^1 Hamilton syringe remaining in place for 2 minutes following toxin infiision), while the other group received infusions (03 pi/ hemisphere following the above listed parameters) of the vehicle for the hnmunotoxin. Attainment of asymptotic performance in the sustained attention task upon conclusion of the post-surgery recovery period was followed by a schedule of

intrabasalis administrations o f the NMDAR/GS partial agonist DCS; this infiision

regime was comprised of the 3 treatments listed, pseudo-randomly scheduled: DCS

(0.5 pg/pl, 5 pg/pl; 0.5 pl/hs) and vehicle (sterile saline: 0.5 pl/hs). The hafiisions

were conducted 10 minutes prior to placement in the operant chamber (approximately

13 minutes prior to task onset) at an infiision speed of 0.5 pF min; the internal cannula

remained in place for 2 minutes following the infiision to assure adequate perfiision of

the drug. Each intracranial infiision session was separated by a minimum of two

training sessions.

Histological procédures

Upon completion o f the intracranial infiision schedule, the animals were

perfused and brain sections will be processed to determine cannula placement (Nissl

staining) as well as degree of BFCN damage (acetylcholinesterase staining).

After completion o f post-surgical behavioral training rats were anesthetized

and transcardially perfused with 0.9% buffered saline (pH 72-7.4), followed by 10%

formalin (v/v solution). Perfiised brains remained in formalin for 9 h and were then

placed in 30% sucrose phosphate buffer (w/v solution) for cryoprotection. Brain

62 sections (40 ^tm) were processed to define acetylcholinesterase (AChE)-positive fibers

(Tago et aL, 1986). The validity of AChE-positive fibers as a method for revealing cortical cholinergic axons in rats has been fiequently demonstrated. Exceptions include the medial prefiontah dngulate and retrosplenial areas in which this method stains for AChE not associated with cholinergic neurotransmission (Lysakowski et aL, 1989; see also Mesulam and Geula^ 1992 for the validity of this method in the human bram). Initially, sections were placed hi 0.1% H 2O2 for 30 mm then rinsed in 0.1 M maleate bufer (pH 6.0) prior to placement in the first mcubation medium. Sections were incubated for 60 min in a solution composed of 5 mg acetylthiocholine, 0.5 mL of 0.1 M sodium citrate, 1.0 mL of 30 mM copper sulfate, and 1.0 mL of 5 mM potassium ferricyanide in 200 mL of maleate bufifer

(pH 6.0). Rinses in 50 mM Tris bufièr (pH 7.6) preceded placement of the sections in a second incubation medium, comprised of 0.05 g diamino faenzidhie and 3.75 g nickel ammoninm sulfate in 125 ml of 50 mM Tris buffer (solution pH = 6.4). Following 10 mm of incubation, 12 drops of 0.1% H 2O2 were added to this solution and sections remained for 12 min prior to fiial rinsmg m 5 mM Tris bufer. Sections were subsequently mounted on gelatin coated, air dried, dehydrated in ethanol and placed hi xylene prior to coversfipping. Parallel sections from the area of the infusion of 192 IgG-saporm into the basal forebrahi were stained with Cresyl violet for inspection of non-specific damage.

Employing procedures described in previous work (Holley et aL, 1994; McGaughy et aL, 1996; TurcM and Sarter, 1997) the relative cortical cholinergic fiber loss produced by the 192 IgG-saporhi administration was quantifisd. Briefly, a

63 focusing magnifier in a VANOX Olympus Research Microscope ^ o d e l AHBT; 25x magnification) provided the superimposed image of four orthogonal double cross-lines over each area o f cortex to be quantified- AChE-positive fibers which crossed these four lines were then counted- Fiber counts were taken from the somatosensory cortex

(area 3b; layers n/m and V, respectively) and the motor cortex (area 4; layers n /m and

V), and 43 (layers n/m and V). Two counts per hemisphere and per area were taken from two different sections perbrain-

Statistical analyses

For this experiment, a 3-way mixed-factors ANOVA was conducted to assess the differences in pre-surgical versus post-operative performance levels with respect to the vigilance index [VI: between subject factor of group designation (2 levels, sham- or

192 IgG-saporin lesioned); witfiin subject factors: surgery (2 levels, pre- vs. post-op) and signal length (3 levels)]. Examination of any differences between pre-infusion baseline VI values for all stimulus lengths as compared to one week of post-operative asymptotic performance data was conducted via repeated measures ANOVA, with VT having 2 factors [dose (4 levels, including collapsed post-operative means and pre- infiision baselines ft>r all three infusions tested), signal length (3 levels)]. Additionally, pre-infusion baseline Vi’s were compared with those of the saline infusion by repeated measures ANOVA within each lesion group, having 2 factors [dose (4 levels) and signal length (3 levels)]. All drug effects were analyzed as repeated measures

ANOVAs within the two lesion groups. Analyses for the effects of DCS were conducted for all of the behavioral measures collected as follows. Analyses of VI and

64 of the relative number of hits had two 6ctors [dose (3 levels), and signal length (3 levels)]. Effects on the relative number of correct rejections, as well as SB and omissions, mvolved only I factor: dose (3 levels). Post hoc analyses of any significant

F values were conducted as described in the General methods.

Results

Effects o f the cannula implantation surgery, as well as those associated with the lesion procedure, on the animals^ ability to discriminate signal and non-signal trial events, VI, were evaluated by comparing collapsed criterion values for 3 days preceding surgery and 7 days of asymptotic post-operative performance values. Both a significant between subjects effect (sham-lesioned vs. lesioned) was observed [F (I, 8)

= 5.878, p = 0.042], and main effects of surgery (pre- vs. post-surgery), as well as signal length, on VI were found [surgery: F (6.759,54.068) = 5.019, p < 0.001; signal length: F (2, 16) = 106.615, p < 0.001]. The main effect of surgery [VI pre-surgery vs. VI post-surgery: t(9) = 4.495, p = 0.001] was examined further using a one way

ANOVA comparison by group, and revealed VI values to be significantly different between sham- and 192 IgG- saporin lesioned animals only for the post-operative data collection period, [pre-surgery: F (1,8) = 0.716, p = 0.422; post-surgery: F (1,8) =

6J48, p = 0.036; VI: post-surgery: shams: 0.38 ± 0.02; lesioned: 0.27 ± 0.02]. The effect of signal length on VI values reflected overall decreases in the animals' ability to discriminate signal and non-signal events with decreases in signal length [VI 500msec vs. VT 50msec: t(9): = 8.760, p < 0.001; VI 50msec vs. VI 25msec: t(9) = 6.180, p

<0.001; VI 500msec: 0.471 0.03, VI 50msec: 032 i 0.02, VI 25msec: 025 i 0.02].

65 The effects o f pre- vs. post-surgery, lesion and signal length on VI did not interact significantly |TF (14,112) = 1.448, p = 0.143]. Also, no effect o f time ^re- vs. post­ surgery) upon the number of omissions was present [ F (4.33,34.664) = 0.824, p =

0.527]; likewise there was no between subjects effect for this measure |F (1,8) = 0.510,

p = 0.496].

The significant effects described above for the measure of VI were reflected in

the analyses of the relative numbers of hits and correct rejections. A main effect of

surgery (pre-vs. post-) was observed on the relative number of hits [ F(7,56) = 4.449, p

= 0.001]; additionally, there was a significant between subjects effect of lesion [F (1,8)

= 8.211, p = 0.021; see Fig. 5.1]. Post hoc comparisons revealed an decrease in the

relative number of hits (collapsed across signal length) following surgery [ t(9) = 3.664,

p = 0.005; h: pre-surgery: 63.6 ± 3.0%, post-surgery: 52.8 ± 1.3%]. The main effect of

signal length upon the relative number of hits was also present [F(2,16) = 110.014, p <

0.001], reflecting decreases in the relative number of hits accompanymg decreases in

signal length [h 500msec vs. h 50msec: t (49) = 11.540, p < 0.001, h 50msec vs. h

25msec: t(49) = 5.291, p < 0.001; h 500 msec: 63 ± 1%, h 50msec: 49 ± 2%, h 25msec:

43 ± 2%]. Although there were significant interactions between the effects of signal

length and lesion upon the relative number of hits [F (2,16) = 4.545, p = 0.027], as

well as between the effects of time (pre- vs. post-surgery) and signal length upon this

same measure [ F(14, 112) = 1.833, p = 0.042], a significant 3 way interaction between

the effects o f time, lesion and signal length upon the relative number of hits was not

present [ F (I4 ,112) = 1.651, p = 0.076]. Post hoc analysis indicated that the

66 mteractîon between the effects o f signal length and lesion upon the relative number of hits were only significant for the 500 msec stnnulus, post-operatively [ hSOO msec: F

(1,8) = 35.521, p < 0.001; h 50msec: F (1,8) = 5286, p = 0.051; h 25msec: F (1,8) =

2.048, p = 0.190; shamsdt 500msec: 75 ± 1%, h 50msec: 56 ± 2%, h 25msec: 45 ± 1%; lesioned: h 500msec: 58 ± 1%, h 50msec: 44 ± 1%, h 25msec: 39 ± 2%].

Importantly, and similar to previous work addressing sustained attention performance impairments in rats consequent to intrabasalis infusion of 192 IgG-saporin

(McGaughy et al., 1996), these effects upon the relative number of hits in response to cortical cholinergic deafferentation were not associated with concomitant depression of the relative number of correct rejections; neither an eSect of surgery (pre- vs. post-), nor a between subjects effect of lesion, upon the relative number of correct rejections was observed [surgery: F (1,8) = 4.397, p = 0.069; lesion: F (1,8) = 0.798, p = 0.398; post-operative CR: shams: 77.8 ± 0.9%, lesioned: 77.9 ± 6%}. While there was an effect o f surgery (pre- vs. post-) upon the measure of SB [ F (1,8) = 24.008, p = 0.001], as well as a between subjects effect for this measure [F (1,8) = 11.150, p =0.010], the absence of any effect of the lesion manipulation upon the relative number of correct rejections indicated that the effect of the cholinergic depletion upon the relative number of hits was due to impairments o f the animals’ abilities to discriminate the signal events. Post hoc analysis indicated that while pre-surgical and post-operative SB values differed [ t(48) = 3292, p = 0.002; SB: pre-op: 0.41 ± 0.07, post-op: 0 3 4 ±

0.06], the between subject difference for this measure was limited to the post-operative

67 coUectîoa period (pre-surgery: F (1,8) = 0,125, p = 0.733; post-surgery: F (1,8) =

59.550, p < 0.001; pre-surgery SB: shams: 0.40 ± 0.01, lesioned: 0.41 ± 0.01; post- surgery SB: shams: 0.40 i 0.007, lesioned: 034± 0.06].

Analyses were conducted within each group (sham- and 192 IgG-saporin lesioned) to address any possible dîfièrences in task performance between the post­ operative data collection period and pre-infusion baselines; no significant dififerences were found for the behavioral measure analyzed (VI: all p’s > 0.081). Similarly, the investigation of any possible difference in VI values between the pre-infiision baselines and the saline infiision yielded negative results (all p’s > 0.093).

The effects of D - cycloserine infusions (03 p g , 5.0 pg, dissolved in sterile saline: 0.5 pl/hemisphere) as compared to vehicle infusion (saline: 03 pLhemisphere) upon the relative numbers of hits and correct rejections were analysed separately within each lesion group. While the effect of signal length upon the relative number of hits was present for sham-lesioned animals [ F(2,8) = 17.055, p = 0.001], reflecting decreases in the percent of hits as signal length decreased (h 500msec: 803 ± 2.3%, h

50msec: 62.0 ± 8.0%, h 25msec: 56.0 ± 6.6%), no effect of dose was observed among the sham-lesioned animals for this behavioral measure [h: F (1.733,6.932) = 0.674, p =

0320; see Fig. 53]. Likewise, no effect of dose was visible upon the relative number of correct rejections [ F(1.602,6.408) = 0.573, p = 0.555]. Additionally, there was no effect of dose observed on the measures of SB or omissions ( all p’s > 0399).

68 la contrast, while a mam e% ct of dose upon the relative number o f hits was not present for the lesioned animals, there was a signihcant interaction between the effects of dose and signal length upon this measure for this animal group [ F( 4,16) = 4.190, p = 0.016; see Fig. 5.3]. Post hoc comparisons revealed this effect to be significant solely for the longest signal length [ h 500msec: F (2,12) = 4.360, p = 0.038; h 50msec:

F (2,12) = 1.129, p = 0.355; h 25msec: F (2,12) = 1.685, p = 0J227] and the highest dose o f D — cycloserine administered [5.0 pg, h 500 msec: Tukey’s HSD = 0.030; h 500 msec: saline: 50.8 ± 43%, 5.0 jxg DCS: 68.6 ± 2.9%]; comparisons for all other doses and signal lengths yielded HSD’s > 0321. Similar to the results found for the sham-lesioned animals, there were no significant effects o f dose observed upon the relative number of correct rejections, the number of omissions, or the side bias index,

SB (allp’s >0.136).

Analysis of within group (sham-lesioned and lesioned) data for effects of block

upon the relative number of hits yielded mostly negative results. An interaction

between the efîècts of block and signal length for the sham-lesioned animals

represented the only positive data for these analyses: [ F(4,16) = 5.881, p = 0.004]; this

interaction reflected a block effect for the 500 msec signal length only [ F(2,42) =

4354, p = 0.019, all other signal lengths, p’s > 0395], wherein the relative number of

hits for the 500 msec signal were higher in block 2 than in block 1 : [ Tukey’s HSD =

0.023; h 500msec: block 1:76 ± 2%, block 2: 86 ± 2%]. No effects of block, nor

69 interactions between the eSècts of dose and block, or block and signal length, or dose, block and signal length, upon the relative number of hits were observed for the lesioned annuals ( all p^s > 0.136).

Histologic analysis demonstrated marked, decreases (ranging from 57-67%) in the density of AQiE—positive fibers in the cortex for the 192 IgG-saporin lesioned animals as compared to the sham-lesioned animals (see Table 5.1), with relatively m inor amounts of non-specific damage near the infusion site (see Fig. 5.4 for a photographic depiction of damage associated with intrabasalis 192 IgG-saporin infusion). The animals* ability to perform the sustained attention task, as measured by

VI, correlated with AChE - positive fiber density in all areas inspected ( all r*s = 1.00; all p’s = 0.01). hi order to examine possible correlations between the relative amount o f extant ACZhE - positive fibers following the cholinergic lesion and any beneficial effects that may have been associated with intrabasalis D - cycloserine infusion, the fiber coimts from individual animals and areas measured were ranked in order of least to most fiber loss incurred by the lesion manipulation (see Table 5.2). These rank values were then subjected to post hoc correlation analysis (Spearman’s rho) with the ranked data indicating the degree of positive effects of D - cycloserine (5.0 pg)

infusion upon the relative number of hits for the 500 msec signal; the potency of the highest dose o f d - cycloserine to attenuate the lesion induced impairments on the

relative number of hits appeared to correlate with the AChE - positive fiber density

[ p= 0.709, p = 0.022; see Fig. 5.5].

70 Discussion

The lack of apparent hnpairments among the sham-lesioned animals in response to intrabasalis D—cycloserine mfüsions was not an expected Snding given the above discussed hypotheses concerning cortical cholinergic hyperactivation and concomitant impairments in the ability to detect non-signal events within the sustained attention paradigm. As the blocked analysis provides the greatest level of detail for examination of behavioral effects consequent to ligand infiision, the lack of dose effect findings for the sham-lesioned animals even at this level points to a need for réévaluation of the original hypothesis concerning the putative mechanism of beneficial efiècts mediated by D — cycloserine. Whereas an assumption implicit in the first version of the hypothesis was uniformity of NMDAR response to d - cycloserine administration, it is possible that the nature of this ligand (partial agonist) interacted with the differential neural substrate provided by the sham-lesioned versus 192 IgG-saporin lesioned subjects. Furthermore, while D—cycloserine administration has been associated with improvements in task acquisition and reversal learning (Riekkinen et al., 1998a;

Riekkinen et al., 1998b) in intact animals, there were windows for improvements of performance among these sham lesioned animals for either the relative number of hits

(for 50 and 25 msec signal lengths) as well as the relative number of correct rejections, however, none were observed among these animals; if the actions of D - cycloserine relative to the enhancement of glutamatergic transmission were not at the level of cholinergic hyperactivation, it is conceivable that some of the above listed behavioral measures could have exhibited enhanced performance accuracy. The doses of D -

71 cycloserine employed by Etiekkenen et aL (1998a & b) were admmistered intraperitoneally and ranged from 3-10 mg/kg; while the 5.0 pg dose was of apparent benefit among the 192 IgG-saporin lesioned animals, it is possible, though unlikely, that this dose was insufficient for visible efiècts upon the intact animal, hi either case, expansion o f the dose-response curve for further investigation of this compound may have been o f use.

On the other hand, the partial attenuation of the lesion-induced impairments upon the relative number of hits to the 500 msec signal afforded by infusion of the highest dose of D - cycloserine (5.0 pg) provided support for the hypothesis that basal forebrain NMDA receptor modulation plays a substantive role in the performance of tasks which tax attentional processes. Additionally, as modulation of the NMDAR at the glycine site has been characterized in terms of an allosteric nature (Priestly and

Kemp, 1994), administration of this partial agonist may have allowed for maintenance of the information flow in this region (much in the manner that administration of BZR modulatory ligands has been proposed to function; see Sarter et al., 1990). This finding of improved ability to detect the longest signal among lesioned animals suggested that impairments of sustained attention processing incurred by damage to the basal forebrain cholinergic ^stem may be effectively, if only partly, ameliorated by positive

NMDA receptor modulation via partial agonism of the glycine site; as this ligand has been shown to enhance NMDAR activation in tissue fiom Alzhenner’s diseased brains

(Chessell et al. 1991), the results fiom this experiment suggest therapeutic benefits may

72 be derived 6om this compound, and finther investigation of D—cycloserine in the context of attenuation of cognitive unpairments in fimctionally compromised systems is warranted (see also Maurice et al., 1996).

73 CHAPTERS

GENERAL DISCUSSION

The set of «cperiments described herein yielded the following principal findings: I. Basal forebrain NMDAR modulation has performance ramifications only for tasks which explicitly tax attentional processes. 2. Negative modulation of

NMDAR, observed following intrabasalis APV infusions, as well as suppression of functional hetero-oligomeric NMDAR via antisense oligodeoxynucleotide infusions, serves to depress solely animals’ ability to detect accurately signal events. 3. Positive

NMDAR modulation by direct agonism, as visible after NMDA infusions into SI, produces a possible hyperattentional state wherein the nmnber of false claims for signal events rises, in the absence of any significant effect upon the relative number of correctly identified signal events. 4. The effects of positive modulation of the glycine- site of NMDAR in the SI is only discernable in the damaged system: here, the specifically immunolesioned cortical cholinergic system.

An alternative to the NMDAR based glutamatergic hypothesis of neurodegenerative disorders has been described, mvolving the GluR2 receptors, as these receptors can be configured, based on subunit composition, as Ca~* -permeable

74 (Pellegrini-Giampietro et aL, 1997); this may be of interest as rodent (murine) basal fbrebrain cholinergic neurons have been documented as preferentially vulnerable to

AMPAR-mediated insult as opposed to NMDAR associated neurotoxicity (Weiss et aL,

1994). Additionally, NMDA and AMPA have been shown to induce c-fos expression differentially within the basal fbrebrain of rats, with AMPA infusions inducmg the greatest amount of FOS (Page et aL, 1993); however, as discussed above in Chapter 1, this GluR2 hypothesis may only have relevance for the human system and non-human primate models, as AMPA receptors in the rat brain were demonstrated to be coIocaHzed with GABAergic neurons as opposed to ChAT positive neurons (Martin et aL, 1993).

While the merely agonist requirmg nature of AMPA/KA glutamate receptors by deffution would seem the less tenable candidate for the phasic glutamatergic modulation of BFCN derived cortical acetylcholine output likely necessary for higher cognitive processing, a more stringent set of controls for assessing sustained attention effects of the

NMDA and APV infusions could have included administrations of AMPA and DNQX

(for AMPA/KA receptor blockade). As the precise location of the NMDAR in the SI has yet to be mapped, additional anatomical data would be required to map definitively the circuit whereby mesocortical glutamatergic inputs modulate NMDAR mediated performance characteristics in tasks which tax attentional processes. While AMPA receptors were showu to be colocalized with ChAT mmunopositive neurons in monkey

(Martm et aL, 1993), peptidergic colocalÊation with cholmergic neurons has been observed m monkey as well (Walker et aL, 1989); maps of possible corticofiigal glutamatergic mteraction with peptidergic populations m BF of rat may provide

75 elucidation of the path fay which glutamatergic modulation, specifically of NMDAR, m rat

SI/NB yields diffbrential responses with respect to cortical ACh efQux.

The absence of any effects of negative NMDAR modulation (APV infusion) or positive NMDAR modulation (NMDA administration) upon the cued visual discrimination task supports the hypothesis that faasal forefarain NMDAR modulation has performance ramifications only for tasks which explicitly tax attentional processes. Thus efiects of the possible ‘hyperactivated' cholinergic system mduced fay NMDA administration were only visible within the sustained attention task; the selective effect of NMDA upon the relative nmnber of correct rejections, as evidenced fay increases in the relative number of false alarms, corrrelated with previous data involving disinhibition of the BFCN, and consequently over activated cholinergic system for the mtact anhnal, and increases of apparent ‘hyperattentional' misidentification of non-signal events as signal trials (false alarms: Holley et aL, 1995). In light of other work mdicating beneficial effects of NMDA administration in the mtact animat (see Mason et al, 1999), it is possible that converse effects may have been obtained (e.g., improvements in the relative number of hits and/or correct rejections) if substantially lower doses had been employed. Future work could address a more comprehensive dose response curve for this ligand as the doses selected herem were prmcipally designated for the potential for visible e&cts m the absence of nemotoxic damage.

76 The snppressioa solely of the relative nmnber of hits foUowmg intrabasalis APV mfhsions paralleled previous findings (see below) associated with compromised integrity of the

BFCN corticopetal projection system.

The substantive and reversible effects yielded by the antisense administrations, which targeted the NMDA-NRl subunit in an effort to diminish functional hetero- oligomeric NMDAR in the SI, were an effective demonstration, both of the utility of the antisense **knockdown” technique and of the soundness of the hypothesis that NMDAR modulation of basal forebram cholinergic neurons is selectively mvolved in the processes which tax attentional functions. Furthermore, the selectivity of the effects of antisense administration, namely depression of the relative number of hits in the absence of effects upon the relative number of correct rejections, suggests that the antisense-induced suppression of functional NMDAR did not alter the animals’ processing of the prepositional rules of the task; and the effect of antisense (8.0 nmol) on side bias for the

24 hour time point, whereby animals’ were shown to prefer the right (correct rejection/miss lever), was actually an artifact of the suppression of the relative number of frits, as the relative number of correct rejections did not increase in response to tfris dose, and as a wmdow for such improvement to be visible was presenL This experimental evidence correlates with previous data generated from animals with compromised corticopetal cholinergic systems ( McGaughy et ai, 1996; McGaughy and Sarter, 1995), wherein selective impairments in the animals’ ability to detect signals was documented, and also elaborates the effects reported in Chapter 3, where NMDAR blockade by APV administration smrilarly yielded decreases in the relative number of hits while responses to

77 the non-signal events remained unchanged.

The missense sequence selected was an appropriate control of reasonably high stringency; to test the strictest control measure, and perhaps shed light on the mechanism of antisense oligodeoxynucleotide function, a missense strand composed of less than the 4 mismatched bases used in tins experiment could have been assessed. In particular, if the mismatches occurred downstream of the AUG initiation codon, lack of suppression of

NRl-immimoreactivity in response to this missense version might resolve if the mechanism by which the antisense oligomer functioned was exclusively due to RNase H cleavage of the mRNA. Further investigation of the effects of the antisense infusion may have benefitted from a larger pool of animals. Inspection of the data suggested the possibility of a “rebound’Tike effect for the relative number of correct rejections 48 hours after the final infusion of both the low and high doses (1.0 and 8.0 nmol), with an apparent increase in the number of false alarms for each compared to the missense data for that time point; this data did not, however, attain significance as the variability of the data precluded such a finding.

Likewise, individual variability of performance characteristics for the third experment may have obfuscated a dose dependent effect of the D- cycloserine infusions upon the relative number of hits. More detailed investigation of the potential for benfidal effects of positive NMDA receptor modulation in animals with partially deafferented cholinergic system could have included mfusions of NMDA, for data comparison with a dhrect agonist o f the receptor, with the presupposition that as

78 modulatiott of the NMDAR with the direct agonist functionally uncouples the mfbrmation flow of the neurons of interest, the greater likelihood of functional outcome would be a yet unpaired lesioned anima], as the data in Chapter 3 already demonstrate impairments of mtact animals in response to intrabasalis NMDA administration.

A growing body of literature demonstrates the interest in NMDA/GS modulation as a potential point of pharmacotherapeutic intervention for Alzheimer's patients (Kawabe et aL, 1998; Quartermam et aL, 1994; McEntee and Crook, 1993; Ulas et aL, 1992;

Lawlor and Davis, 1992; Del Bel and Slater, 1991; Cowbum et aL, 1990; Palmer and

Gershon, 1990; Deutsch and Morflnsa, 1988). This literature to date remams largely ambivalent with respect to D- cycloserine, as studies documenting beneficial effects in humans exist alongside those with negative results (see comments of Roesler et aL, 1996; but see also Tsai et aL, 1998; Mohr et aL, 1995); likewise, the literature mvolving animal models describes simOarly non-uniform results (see Land and Riccio, 1999; Maurice et aL,

1996; Matsuoka and Aigner, 1996; Thompson et aL, 1992; but see also Magnusson,

1996). As considerable evidence exists of NMDAR changes in the aging and neurodegenerating system for both human and rodent model subjects (Jasek and Griffith,

1998; Magnusson, 1998; Michaelis, 1998; Magnusson, 1997; BiDard et al, 1997; Du and

Walshm 1997; Shnnadaet aL, 1997; Ulas and Cotman, 1997; Duepree et aL, 1993;

Hashunoto et aL, 1993; Gonzales et aL, 1991;TamaruetaL, 1991; Mqroshi et aL, 1990;

Procter et aL 1989; Steele et aL, 1989), as well as changes in nenroactive metabolite levels, active at glutamatergic receptors (Khmfc et aL, 1991), and AMPAR (Ikonomovic and Armstrong, 1996), the need for further study of glutamatergic modulation of neuronal

79 populations involved in the execution of cognitively demanding processes is evident.

Exploration of polyamine site regulation modulation might prove of interest in this effort, as part of a “cocktail” administration hi the partial cortical cholinergically deafferented system, as a polyamine site agonist has been demonstrated to enhance glycmergic effects at the NMDAR (Reynolds and Rothermund, 1995).

Collectively, these experimental findings, in conjunction with the studies discussed within this document, suggest a demonstrable role for NMDA receptor-mediated glutamatergic modulation of the corticopetal cholinergic neurons of the SI for sustained attentional processes; these findings also indicate the requirement for continued investigation of positive NMDAR modulation via the glycme site as a potential route for pharmacotherapeutic intervention in Alzheimer^s disease.

80 Tables and Figures

81 Snstained attention task:

12h 12h 24 h

'Dreatment 1: 1 anti (low) I anti (low) I anti (low) test session

12 h I2h 24 h

Treatment 2: I anti (high) 1 anti (high) I anti (high) test session

I2h 12 h 24 h

Treatment 3: I miss (high) L miss (high) I miss (high) test session

Cued visnal discrimination task:

12 h 12 h 24 h

Treatment 1: I miss (high) I miss (high) I miss (high) test session

12 h 12 h 24 h

Treatment 2: I anti (high) I anti (high) I anti (high) test session

(anti: antisense oligodeoxynucleotide infusion; miss: missense oligodeoxynucleotide

infusiondow: LO nmol/0.5 |il/hemisphere; high: 8.0 nmol/0.5 |jJ/hemisphere)

Table 4.1. Experimental design fo r oligodeoxynucleotide infusions.

82 3b 3b 4 43 43 Group n /m V V nmr V Control 384561.91 40.2761.69 36.563.06 39.656L79 39.8561.53 Lesion 16.1064.56 17.0265.70 11.876326 17.6764.60 17.765.39 A% -58.6 -57.7 -67.5 -55.4 -55.6

Table 5.1 Cortical AChE—positivefiber density: group data± (M SD) (Arabic numerals indicate the brain area; Roman numerals indicate layer)

3b 3b 4 43 43 Rank Average Rat n /m V V nmr V (rank] Controls 989 40.0[3] 41.7(1] 38.1(2] 40.7(2] 39.6(4] 2.4 (3] 786 40.1(2] 41.0(2] 39.8(1] 402(3] 40.5(1] 1.8 (1] 289 37.6(4] 382(5] 32.0(5] 38.7(4] 38.6(5] 4.6(5] 451 40.3(1] 40.8(3] 37.1(3] 41.1(1] 40.3(2] 2.0 (2] 187 36.6(5] 39.5(4] 35.3(4] 37.2(5] 40.1(3] 42 (4] Lesion 388 14.2(9] 17.7(9] 12.6(8] 18.3(9] 192(8] 8.6 (9] 488 17J(8] 18.5(8] 13.3(7] 19.0(8] 19.2(8] 7.8 (8] 833 9.1(10] 7.1(10] 72(10] 9.5(10] 7.7(10] 10 (10] 741 18.7(7] 19.8(7] 13.8(6] 21.0(6] 20.7(7] 6.6(6] 185 . 2,L0[6L 21.8(6] 12,5(91.._ ,.20,5(2] , 2.1,5(61 ..... 6.8 (71 .

Table 5.2 Cortical AChE—positive fiber density: indmdual data(M) and ranking (in brackets) (Arabic numerals indicate the brain area; Roman numerals indicate layer)

83 Figure 2.1 Sustained attention task schematic.

Rats were trained to discriminate between signals and non-signals for 162 trials per training session. Two seconds following either stimulus, the levers were extended and remained active for 4 seconds. Responses of left lever presses after signal trials were rewarded as hits and right lever responses after non-signal trials were rewarded as correct rejections. Converse responses (right bar press for signal and left bar press for non-signal) were not rewarded and constituted misses and false alarms respectively. In the final task, the length o f the signal was varied (500,50 and 25 msec), the intertrial interval was 9 ± 3 sec, and the houselight was illummated throughout the session.

84 miss

Signal: ♦ s false alarm correct rejection

Non-signal: I ♦

Figure 2,1 ♦ lever press; R, reinforcement Figure 22. Cued visual discrimination task schematic.

Animals training in this task were merely required to press the lever located immediately below the left or right panel light in order to receive water reward.

86 hit miss

Left Cue: ± R _ — A miss hit i Ç i Right Cue: m Jt _ _ R 1

Figure 2,2 y lever press; R, reinforcement Figure 23 Cannula target placements in basalforebrain.

SI/NB targets are designated as the arrow tips on hgure; this depiction is adapted Grom Paxinos and Watson, 1997.

88 V N

Substantia innommata

Figure 2 J

89 Figure 3.1 Effects ofintrabasalis infîtsion o f APVon accuracy in animals performing cued visual discrimination task.

No significant effects of APV infusion were found upon the relative number of hits among animals performing the cued visual discrimination task; there was, however, an effect of dose on the relative number of omissions, with the greatest number of omissions occurring for in réponse to the highest dose admmistered (20 nmol).

90 1 0 0

90 Saline: Correct Saline: Omissions £ 80 APV lOnmol: Correct APV lOnmol: Omissions 70 APV ZOnmol: Correct I APV 20nmol: Omissions 8 60 g I 50 iSO, 40

30 I 20 10

0

Block 1 Block 2 Block 3 Block 4

Figure 3,1 Figure 3 J2 Effects o fintrabasalis AP V injusions upon sustained attention performance.

The highest dose of APV (20 nmol) m anhnals performing the sustained attention task yielded significant depression of the relative number of hits without concomitant change in the relative number of correct rejections.

92 % vehicle hits/(hits + misses) cr/(cr+false alarms) 2.6 APV 3 nmol APV lOnmol APV 20 nmol 2.4 87

2.2 80

2.0 71

1.8 62

1 iS CO 1.6 52 +1

1.4 42

1.2 32

1.0

0.8 500.00 50.00 25,00 signal length (msec)

Figure 3.2 Figure 3 3 Effects o f intrabasalis infitsion ofNMDA on accuracy in animals performing cued visual discrimination task.

No significant efiëct ofNMDA infusion was observed for either dose among animals performing this non-attention demandmg task.

94 1 0 0 = 5

90 Saline: Correct Saline: Omissions 80 NMDA 3nmol: Correct NMDA 3nmol: Omissions I 70 NMDA Gnmol: Correct NMDA 6nmol: Omissions 8 60

I 60

40 § 30 (X 20

10

0

Block 1 Block 2 Block 3 Block 4 Figure 3.3 Figure 3.4 Effects o fintrabasalis NMDA infusions upon sustained attention perfarmance.

Intrabasalis administration ofNMDA selectively decrease the relative number of correct rejections in the absence of any significant effects upon the relative number of hits.

96 X' % vehicle or/ (cr+ false alarms) 2.6 NMDA 1 nmol NMDA 3 nmol NMDA 6 nmol 2.4 87

2.2 80

2.0 71

1.0 62

r 52 I" +1 S 1.4 42

1.2

1.0 500,00 signal length (m sec) 50.oo 25.oo Figure 3.4 Figure 3.5 Lack o fneurotoxicity associated with NMDA doses tested in behaving animals: Fluoro-Jade assessment,

Fluoro-Jade staining following unilateral infusion of two different doses ofNMDA into SI/NB (ventral SI, 50X magnification is displayed). Item A depicts the neurotoxic effects o f 30 nmol NMDA infusion (note the green fluorescence of positive Fluoro- Jade staining is here indicated by black), while item B is the control hemisphere for that same animal (saline infusion). In. contrast, infusion of 6 nmol NMDA (item C) yielded no positive Fluoro-Jade stain, mdicating a lack of neurotoxicity associated with that dose (item D is the control hemisphere).

98 rjgiirc 3,5 Figure 4.1Effects o f intrabasalis infusions o fantisense or missense oligodeoxynucleotides in animals performing cued visual discrimination task.

As anticipated^ animals performing the non-attention demanding cued visual discrimination task were unaSected by missense or antisense infusions (both compounds: 8.0 nmol) with respect to either the relative numbers o f hits or omissions.

ICO 1 0 0 I 1 90 Baseline; Correct 0) Baseline; Omissions S 12hr Post Missense; Correct 8 12hr Post Missense; Omissions 2 80 12hr Post Antisense; Correct y — 8!— 12hr Post Antisense; Omissions 24hr Post Missense; Correct o f y 24hr Post Missense; Omissions 24hr Post Antisense; Correct 24hr Post Antisense; Omissions 30 § S. 20

10

0 BLOCK 1 BLOCK 2 BLOCK 3 BLOCK 4 Figure 4.1 Figure 4.2 Comparison ofeffects o f intrabasalis infusions ofantisense or missense oligodeoxynucleotides upon sustained attention performance 12 hours (fier second infitsion.

For animals performing the sustained attention task, no significant effects of missense or either dose of antisense (1.0 or 8.0 nmoQ was found upon either the relative numbers of hits or correct rejections at this time point.

102 X' 2.6 r % hits/(hjts+misses) cr/(cr+false alarms) Baseline Missense: 8.0 nmol 2.4 87 Antisense: 1.0 nmol Antisense: 8.0 nmol

2.2 - 80

2.0 - 71

1.8 - 62 s 1.6 - 52

1,4 - 42

+1 ^ 1.2

1 .0 600.00 signal length (msec) 60.00 26.00 Figure 4.2 Figure 4.3 Comparison of^ ects o f intrabasalis infusions ofantisense or missense oligodeojQ/nucleotides upon sustained attention performance 24 hours afterfinal infitsion.

Selectively, the relative number o f hits was significantly depressed in response to administration o f the highest dose of antisense (8.0 nmol) at this time point. No significant effects o f this dose were observed upon the relative number of correct rejections, nor were any effects present consequent to missense or the low dose o f antisense.

104 X' 2.6 % Baseline cr/(cr+false alarms)

Missense: 8.0 nmol 2.4 87 Antisense; 1.0 nmol Antisense; 8.0 nmol 2.2 60

2.0 h 71

1.8 62

8 I +1 1.6 52 S

1.4 - 42

1.2 32

1.0 # A 500.00 signal length (msec) 50,00 26.00

Figure 4.3 Figure 4.4 Comparison ofeffects o fintrabasalis infusions o f antisense or missense oligodeoxynucleotides upon sustained attention performance 48 hours after final infusion.

No significant effects of missense or antisense administration were observed upon any sustained attention performance measure depicted for this time point.

106 X' 2,6 r % hits/(hlts+mlsses) cr/(cr+false alarms)

2.4 83 2.2 60 75 2.0 71 66 1.8 62 o 57 1.6 52

3 47 V) +1 1 .4 2 Baseline Missense: 8,0 nmol 1.2 Antisense: 1.0 nmol Antisense: 8.0 nmol

1 .0 500.00 signal length (msec) 50.00 25.00 Figure 4.4 Figure 4.5 Effects o f intrabasalis antisense oligodeoxyrtucleotide (8.0 nmol) administrations upon sustained attention performance: temporal gradient o f impairments.

This Sgure depicts the effects of the highest dose of antisense upon sustained attention performance measures* the relative nmnbers of hits and correct rejections* for the three time points following the oligodeoxynucleotide infusion (12 h post 2“* mfusion* 24h post 3"* infusion and 48 h post 3"^ infusion).

108 X' 2 .6 % hits/(hits+misses) ANTISENSE ODN 8nmol cr/(cr+false alarms)

2.4 83 2.2 80 75 2.0 71 66 1.8 62 57

1.6 52 § 47 ^ 1 .4 42 2 37 Baseline 12hr Post Secondary Infusion 1.2 24hr Post Final Infusion 48hr Post Final Infusion

1 .0 600.00 signal length (msec) 50.00 25.00

Figure 4.5 Figure 4.6 Fluoro-Jade assessment o fthe effects o fantisense and missense doses tested in behaving animals.

Fluoro-Jade staining is visible for all sections shown, indicating that each infusion (saline, 8.0 nmol missense, and 8.0 nmol antisense) produced modest damage, possibly reflecting mechanical damage, immediately proximate to the tip of the guide cannula, but importantly not extending into the infusion area (SI). Item A depicts the missense control (saline) infusion [25x mag.]; item B shows the missense infusion effect[25x mag.]; item C depicts the antisense control (saline) infusion [37.5x mag.] and item D indicates the antisense mfusion effect [37.5x mag.].

no Figure 4.6 Figure 4.7 Lack o fneurotoxicity in the SI following infusions o fantisense (8.0 nmol).

This figure depicts a lack of Fluoro-Jade positive staining in the infusion area (SI), thus providing evidence of the lack of neurotoxicity associated with even the high dose antisense infusion employed in the behavioral testmg. Item A depicts the control infusion (salme): 37.5x mag.; item B shows the antisense infusion: 37.5x magnification.

112 Fiîiiire-Ï-.'

It: Figure 4.8 Antisense oligodeoxynucleotide-inducedsuppression o fNMDA-NRI subunit immunoreactivity: comparison with control (saline) infusion.

This figure depicts the suppression of NMDA-NRI immunoreactivity selectively following 8.0 nmol antisense infusions. Item A: 5x mag.; item B, the control hemisphere (saline mfiisions): 25x mag.; item C, the 8.0 nmol antisense infusions: 25x magnification.

114 Fisure 4.S

115 Figure AS High magnification evidence o f antisense-induced suppression o fNRl- immunoreactrvity.

Comparison of the effects of control (saline), missense (8.0 nmol) and antisense (8.0 nmoQ infusions upon NMDA-NRI immtmoreactivity in the SI. Item A depicts a control hemisphere and item B mdicates the lack ofNRI-immunoreactivity suppression following missense infusions; item C clearly demonstrates antisense- induced suppression of NRl-nmnunoreactivity (aH magnifications: 37.5x).

116 Ftüure 4 »

ll~ Figure 5 .1 Effects o fsham- or 192 IgG-saporin lesions on the relative number o f hits and correct rejections.

Selectively, and similar to previous &idmgs, the relative number of hits is significantly diminished by the intrabasalis infiision of 192 IgG-saporin as compared with control animals.

118 X' 2.6 % hits/(hits+misses) sham: pre cr/(cr+false alarms) sham: post 192: pre 2.4 192: post 83 2.2 80 75 2.0 71 66 1.8 62 57 VO

^ 1.6 52 47 +1 1.4 42 37

1.2

1.0 500.00 signal length (msec) 50.00 25,00 ^ Figure 5.1 Figure 5J, Effects of intrabasalis infusions of D-cycloserine upon sustained attention performance in sham-lesioned animals.

No significant effect of D-cycIoserine infusions were observed for any performance measure (the relative number of hits is depicted) among the sham-lesioned animals.

120 sham: salfne sham: DCS 0.5 microgram 2.4 sham: DCS 5.0 microgram

2.3 - “

2.2 - 80

2.1 - 75

1.9 - ^

1.8 - 62

1.7 - 57

5 1.6 ' 52

S 1 .5

1.4

500*00 signal length (msec)

Figure 5.2

121 Figure 5.3 Effects o fintrabasalis infusions o fD-cycloserine upon sustained attention performance in 192 IgG-saporin lesioned animals.

A significant beneficial effect of the infusion of 5.0 g of D-cycloserine was observed among 192 I^-saporin lesioned animals solely for the 500 msec sthnulus length. No other effects o f this compound were revealed for other performance measures.

122 lesioned: saline lesioned: DCS 0.5 microgram 2.1 lesioned: DCS 5.0 microgram

hits/(hits+misses)

1.6 -5 2

1.5 -47

1.3 -37

CO 1.2 -3 2

1.1 -27

1.0

500.00 50.00 2 5 .0 0 signal length (msec) Figure 5.3

123 Figure 5.4 Comparison ofAChE — positive fiber density in sham-lesioned and lesioned rats: cortical areas.

The left panel depicts a sham-lesioned animal’s AChE - positive fiber density, the right panel clearly shows the depletion of cholinergic fibers following 192 IgG-saporin infusion (acetylcholinesterase stain). The lower center panel mdicates the lack of substantive non-specific damage associated with the cholinotoxin infiision (NissI stain).

124 M & él • t . X

«- I

Figure 5.4

125 Figure 5.5 Post hoc correlation analysis o frelationship between —AChE p o sitiv e fiber density and increases in the relative number o f hitsfollowing intrabasalis D-cycloserine irfitsions.

Calculations of the percentage increase in the relative number o f hits for the 500 msec signal length following the administration of the high dose of D-cycloserine form the basis for the ordinate rank values. The rank orders o f relative AChE — positive fiber density finm Table 5 2 were used for the abscissa. The potency o f D-cycloserine to enhance the relative number of hits correlated with the extent o f fiber loss.

126 S10 ■ sham # lesioned F 9

O)

1 2 3 4 5 6 7 8 9 10 low loss AChE-posItive fiber loss (ranks) high loss Figure 5.5 BIBLIOGRAPHY

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