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EFFECTS OF CHOLINERGIC MANIPULATIONS IN THE MEDIAL PREFRONTAL CORTEX ON CARDIAC CHRONOTROPIC REACTIVITY
DISSERTATION
Presented in Partial Fulfillment o f the Requirements for
the Degree Doctor of Philosophy in the Graduate School
o f The Ohio State University
By
Sheri Lyn Hart, B.S.
*****
The Ohio State University 1998
Dissertation Committee: Approved by Gary G. Bemtson, Ph.D., Advisor
Martin F. Sarter, Ph.D. I AdviAdvisor Cheryl M. Heesch, Ph.D. Neuroscience Graduate Program UMI Number: 9900840
Copyright 1999 by Hart, Sheri Lyn
All rights reserved.
UMI Microform 9900840 Copyright 1998, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
Consistent with its putative anxiogenic effects, the benzodiazepine receptor partial
inverse agonist FG 7142 has previously been shown to enhance the defensive-like
cardioacceleratory response to an acoustic probe stimulus. This FG-induced response
potentiation appears to be mediated by a basal forebrain cholinergic mechanism. The
present studies addressed the relevant projection sites o f the basal forebrain underlying this effect, and tested the hypothesis that the medial prefrontal cortex may be a critical target site.
Results supported this hypothesis. Medial prefrontal, and not lateral prefrontal or basolateral amygdalar, carbachol infusions mimicked the response potentiating effects of
FG 7142 administration. This effect was blocked by the muscarinic antagonist atropine.
These findings suggest that cholinergic activation o f the medial prefrontal cortex is sufficient to enhance the cardioacceleratory defensive-like response. Additional studies further demonstrated that cholinergic blockade or cholinergic-specific lesions o f the medial prefrontal cortex can block the response potentiating effects o f FG. These findings indicate that a cholinergic mechanism o f the medial prefrontal cortex is necessary for the response potentiating effects o f FG.
11 ACKNOWLEDGMENTS
I wish to foremost thank God for giving me the opportunity to better m yself with this pursuit. I also want to express my immeasurable thanks to my advisor. Dr. Gary
Bemtson, who offered an exceptional academic role model, skillful tutelage and inexhaustible patience throughout my graduate studies. Drs. Martin F. Sarter and Cheryl
M. Heesch are gratefully acknowledged for their interest in and guidance throughout this project. I would like to thank my colleague Janita Turchi for many productive conversations. To my parents, William and Evelyn Faber, I offer my most sincere thanks for their life-long unwavering confidence and loving support. I would like to express my gratitude to my brother, Gary, and his family, as well as to the rest o f my extended family, for their loving support. Finally, to my husband, Jeffery Hart, I would like to express my most sincere appreciation for his love, strength, humor, confidence and support, without which I would have been lost.
Ill VITA
July 4, 1969 ...... Bora- Binghamton, NY
1991 ...... B.S. The Ohio State University
1991 - present...... Researcher, The Ohio State University
PUBLICATIONS
Research Publication
1. Hart, S., Sarter, M., & Bemtson, G.G. (1998). Cardiovascular and somatic startle and defense: Concordant and discordant actions o f benzodiazepine receptor agonist and inverse agonists. Behavioural Brain Research, 90: 175-186.
2. Bemtson, G.G., Hart, S., & Sarter, M. (1997). The cardiovascular startle response: Anxiety and the benzodiazepine receptor complex. Psychophysiology, 34: 348- 357.
3. Bemtson, G.G., Hart, S., Ruland, S., & Sarter, M. (1996). A central cholinergic link in the cardiovascular effects o f the benzodiazepine receptor partial inverse agonist FG 7142. Behavioural Brain Research, 74: 91-103.
4. Quigley, K.S., Sarter, M.F., Hart, S.L., & Bemtson, G.G. (1994). Cardiovascular effect of the benzodiazepine receptor partial inverse agonist FG 7142 in rats. Behavioural Brain Research, 62: 11-20.
FIELDS OF STUDY
Major Field: Neuroscience
IV TABLE OF CONTENTS
Page
Abstract ...... ii
Acknowledgments ...... iii
V ita ...... iv
List of T a b les ...... vii
List of Figures ...... ix
Chapters:
1. Introduction
1.1. Cardiovascular reactivity and a n x iety ...... I
1.2. Benzodiazepine receptor (BZR) ligands ...... 4 1.2.1. GABA^/BZ receptor pharmacology ...... 4 1.2.2. BZR ligands and a n x iety ...... 6 1.2.3. Summary ...... 9
1.3. Basal forebrain - cortical cholinergic (BFCC) System ...... 9 1.3.1. BFCC anatomy ...... 10 1.3.2. Acetylcholine and a n x iety ...... 12 1.3.3. GABA/BZ - BFCC interactions ...... 13 1.3.4. Summary ...... 14
1.4. The medial prefrontal c o r te x ...... 15 1.4.1. General anatomy ...... 16 1.4.2. MPF and anxiety ...... 18 1.4.3. Modulation of MPF function by BZR ligands ...... 21 1.4.4. Summary ...... 22 1.5. The am ygdala ...... 22 1.5.1. General anatomy ...... 23 1.5.2. Amygdala and an x iety ...... 25 1.5.3. Modulation of amygdalar function by BZR ligands ...... 29 1.5.4. Summary ...... 30
1.6. General Summary ...... 30
2. General Methods
2.1. Surgical procedures ...... 32 2.2. Physiologic recording ...... 34 2.3. Testing procedures ...... 34 2.4. Pharmacologic agents ...... 35 2.5. Histological analysis ...... 35 2.6. General data reduction and analysis ...... 36
3. Effects o f infusions of carbachol into the medial prefrontal cortex on cardiovascular reactivity to an acoustic probe stimulus ...... 38
3.1. Methods 3.1.1. Subjects ...... 39 3.1.2. Testing procedures ...... 39 3.1.3. Data analysis ...... 39 3.2. Results 3.2.1. Baseline heart period ...... 40 3.2.2. Cardiovascular reactivity ...... 40 3.2.3. Histological analysis ...... 41 3.3 Discussion 49
4. Effects o f infusions of carbachol into the lateral prefrontal cortex or basolateral amygdala on cardiovascular reactivity to an acoustic probe stimulus ...... 51
4.1. Methods 4.1.1. Subjects ...... 52 4.1.2. Testing procedures ...... 52 4.1.3. Data analysis ...... 52 4.2. Results 4.2.1. Baseline heart period ...... 53 4.2.2. Cardiovascular reactivity ...... 53 4.2.3. Histological analysis ...... 53 4.3. Discussion ...... 61
VI 5. Effects o f infusions o f atropine into the medial prefrontal cortex, lateral prefrontal cortex, or basolateral amygdala on FG 7142 enhancement o f cardiovascular reactivity to an acoustic probe stimulus 63
5.1. Methods 5.1.1. Subjects ...... 64 5.1.2. Testing procedures ...... 64 5.1.3. Data analysis ...... 64 5.2. Results 5.2.1. Baseline heart period ...... 65 5.2.2. Cardiovascular reactivity ...... 66 5.2.3. Histological analysis ...... 67 5.3. Discussion ...... 75
6. Effects o f cholinergic specific medial prefrontal immimotoxic lesions on FG 7142 enhancement of cardiovascular reactivity to an acoustic probe stimulus ...... 77
6.1. Methods 6.1.1. Subjects ...... 78 6.1.2. Lesioning procedures ...... 78 6.1.3. Testing procedures ...... 78 6.1.4. Data analysis ...... 78 6.2. Results 6.2.1. Baseline heart period ...... 79 6.2.2. Cardiovascular reactivity ...... 79 6.2.3. Histological analysis ...... 80 6.3 Discussion ...... 88
7. General discussion ...... 90
Appendix
List of Abbreviations ...... 95
List o f References ...... 96
VII LIST OF TABLES
Table
3.1 Cardiovascular effects o f central carbachol/atropine infusions in the medial prefrontal cortex ...... 42
4.1 Cardiovascular effects of central carbachol/atropine infusions in the lateral prefrontal cortex or basolateral amygdala ...... 54
5.1 Cardiovascular effects of systemic FG 7142/central atropine infusions .. 68
6.1 Cardiovascular effects o f medial prefrontal cholinergic le sio n s ...... 81
V lll LIST OF FIGURES
Figure
3.1 Basal cardiovascular reactivity for subjects receiving central carbachol/atropine infusions in the medial prefrontal cortex ...... 44
3.2 Changes in cardiovascular reactivity following central infusions of cholinergically active drugs in the medial prefrontal c o r te x ...... 46
3.3 Location o f guide cannula tips within the medial prefrontal cortex for subjects receiving central carbachol/atropine infusions ...... 48
4.1 Basal cardiovascular reactivity for subjects receiving central carbachol/atropine infusions in the lateral prefrontal cortex or basolateral am ygdala ...... 56
4.2 Changes in cardiovascular reactivity following central infusions of cholinergically active drugs in the lateral prefrontal cortex or basolateral am ygdala ...... 58
4.3 Location of guide cannula tips within the lateral prefrontal cortex or basolateral amygdala for subjects receiving central carbachol/atropine in fu sion s ...... 60
5.1 Basal cardiovascular reactivity for subjects receiving systemic FG 7142 and central atropine infusions ...... 70
5.2 Changes in cardiovascular reactivity following systemic administration o f FG 7142 and central infusions o f atropine ...... 72
5.3 Location of guide cannula tips within the medial prefrontal cortex, lateral prefrontal cortex, or basolateral amygdala for subjects receiving systemic FG 7142 and central atropine infusions ...... 74
6.1 Basal cardiovascular reactivity for subjects with cholinergic specific immunotoxic or sham lesions o f the medial prefrontal cortex ...... 83
IX 6.2 Changes in cardiovascular reactivity following systemic administration of FG 7142 in animals with cholinergically specific immunotoxic or sham lesions o f the medial prefrontal cortex ...... 85
6.3 Location of 192 IgG-saporin and vehicle infusion sites within the medial prefrontal cortex ...... 87 CHAPTER 1
INTRODUCTION
1.1. Cardiovascular reactivity and anxiety
Historically, the autonomic nervous system (ANS) has been viewed as a system o f interoceptive reflexes designed to maintain homeostatic balance o f the internal milieux (Cannon, 1927). This autonomic nervous system is potently modulated by limbic and forebrain areas (Bemtson et al., 1998), however, and functional activity of the ANS has been shown also to reflect behavioral and cognitive processes such as perception, motivation, attention and learning (Quigley & Bemtson, 1990). Rostral neurobehavioral systems, such as those associated with the amygdala, prefrontal cortex, and hypothalamus, have relatively direct projections to brainstem autonomic mechanisms, and likely mediate links between behavioral and autonomic functions (Buchanan et al., 1994;
Danielsen et al., 1989; Luiten et al., 1985). Of particular interest in the present consideration is the fact that noxious or anxiogenic stimuli are often associated with changes in autonomic activity and that altered autonomic function is often seen in pathological anxiety states (Bemtson et al., 1998). In conditioned aversive paradigms, a neutral stimulus is temporally paired with an aversive event and comes to signal the impending onset o f that event. Further presentations o f this signal stimulus, even in the absence o f the aversive event, elicit a constellation o f fear related behavioral responses such as enhanced defecation, environmental scanning, freezing, and respiration as well as changes in autonomic outflow and associated alterations in heart rate and blood pressure. With the appropriate controls and caveats, components of this complex response, such as heart rate and blood pressure, may be used to index the central state of fear evoked by the signal stimulus
(Davis, 1990, 1992; LeDoux, 1995).
Like conditioned stimuli, novel stimuli with no explicit signal content also evoke changes in autonomic outflow. In a classical theory, Sokolov (1963) postulated the existence o f two opposing reflex systems - the orienting and defense reflexes. These responses, partially defined by differential effects on cephalic arteriole constrictor tone, were suggested to modulate stimulus processing. The orienting reflex, typically evoked by novel stimuli o f moderate intensity, is considered to be an attentional reaction comprised o f a constellation o f behavioral and physiological responses associated with enhanced stimulus perception. Conversely, intense or noxious stimuli were said to evoke a defensive reflex, including behavioral and autonomic responses associated with avoidance or the attenuation o f stimulus processing. In 1966, Graham and Clifton attempted to integrate Sokolov’s theory with Laceys’ (Lacey et al., 1963) model of heart rate changes associated with stimulus intake/rejection. Based on an integrative review of the literature, Graham and Clifton (1966) proposed that the orienting response is associated with stimulus intake and cardiodeceleration, whereas the defensive response is associated with avoidance or rejection accompanied by cardioacceleration. This simple dichotomy belies considerable complexity in the literature, and a wide range o f determinants o f heart rate response have been identified (Barry & Maltzman, 1985;
Bemtson et al., 1992; Graham, 1979; Graham, 1984; Turpin, 1986; Reyes del Paso &
Vila, 1993; Turpin & Siddle, 1983). Despite complexities and caveats, however, when the context is appropriately defined and controlled, the associations between orienting and cardiodeceleration and defense and cardioacceleration represent one o f the more consistent generalizations from the literature (Bemtson et al., 1992; Campbell &
Ampirero, 1985; Graham, 1979; Graham, 1984; Turpin, 1986).
These basic pattems of cardiovascular response may provide important tools for studying the relations between behavioral processes and autonomic functions, which are often altered in anxiety states (Bemtson et al., 1998). In fact, altered autonomic function is one defining characteristic of certain types o f anxiety (American Psychiatric
Association, 1994). Anxious compared to non-anxious subjects, for example, demonstrated a bias toward acceleratory responses to moderate intensity, non-signal tones
(Hart, 1975). Additionally, exaggerated cardiac responses have been observed to fear- evoking stimuli, such as recounts of personal war experiences in combat veterans, traumatic imagery in post traumatic stress disorder patients (Orr et al., 1993), phobic content images in phobic patients (Hoehn-Saric & McLeod, 1993; Fredrikson, 1981), open area exposure in agoraphobics (Hoehn-Saric & McLeod, 1993), and public speaking in social phobics (Hoehn-Saric & McLeod, 1993; Hoffrnan et al., 1995). Enhanced cardiovascular reactivity in anxiety is not invariable, however, as some anxiety disorders
may be associated with diminished autonomic variability and reactivity (Hoffinan et al.,
1995; Kelly et al., 1970; Roth et al., 1992; Thayer et al., 1996 ). What remains apparent
is that central states o f fear and anxiety may be associated with altered autonomic
functions (Bemtson et al., 1998). The present studies examined the potential
neurobehavioral systems that underlie links between fear, anxiety and autonomic
response. The backgroimd for this work derives from anxiolytic and anxiogenic actions o f drugs that interact with the benzodiazepine receptor complex.
1.2. Benzodiazepine receptor fBZR) ligands
1.2.1. GABA^/BZ receptor pharmacologv
The classic, most widely prescribed anxiolytic compounds are the benzodiazepine receptor agonists such as chlordiazepoxide (librium) and diazepam
(valium). The action of these compoimds is mediated largely by the y-amino-butyric-acid
(GABA) receptor/chloride (Cl ) ion channel complex (Haefley et al., 1975; Rabow et al.,
1995). The physiology o f this receptor is complicated and understanding o f its complexities is confounded by the lack o f isolation o f intact, native GABA^ receptor molecules (Barnard, 1995; Rabow et al., 1995).
However, from radioligand binding, mRNA, and reconstructive patch clamp studies, many solid inferences can be made. The GABA^ receptor complex is a transmembrane Cl" channel composed o f 5 subunits (Barnard, 1995; Rabow et al., 1995) with 15 distinct subunit types (6a, 3p, 3y, lô, and 2p) being identified to date. Using co localization restrictions, it is estimated that natural variants of this receptor complex range from a minimum o f 17 to a maximum of 850 (Barnard, 1995), with the majority composition believed to be in the form of 2a^(3^2y^ (Barnard, 1995; Mohler et al., 1995).
Functionally, each receptor complex binds two GABA molecules (at the a(3 interface) and two benzodiazepine receptor ligands at the benzodiazepine receptors (BZR) on the a subunits (Rabow et al., 1995).
Ligands bound at the BZ receptor alter GABA induced Cl' flow by changing the frequency o f channel opening, possibly due to altered GABA receptor-ion channel coupling (Rabow et al., 1995). This modulation of GABA induced Cl' flow is bidirectional at fully functional BZ receptors. Positive modulators (BZR agonists), such as the classic benzodiazepines, increase channel opening frequency and thus enhance
GABA induced Cl' flow. Negative modulators (BZR inverse agonists) decrease opening frequency and thus GABA induced Cl' flow, while null modulators (BZR antagonists) do not alter opening frequency but block action o f other modulators o f this site (Rabow et al., 1995).
Subunit composition alters physiologic function (Mihic et al., 1995; Mohler et al., 1995; Rabow et al., 1995), and a composition of a,, P^, y-, is required for full BZ receptor function (Herb et al., 1992; Rabow et al., 1995). a or y substitutions can eliminate BZR modulation o f GABA Cl- currents (Rabow et al., 1995; Sigel et al; 1990) or even alter the modulatory direction o f a ligand (Puia et al., 1991; Rabow et al., 1995).
The ubiquitous distribution o f GABA^ receptors, coupled with this complexity of expression and function allows for differential modulation o f diverse neural systems via
BZR ligands (Barnard, 1995; Rabow et al., 1995).
1.2.2. BZR ligands and anxiety
In addition to the efficacy in alleviating pathological human anxiety, BZR agonists exhibit anxiolytic effects in animal models o f anxiety. The anti-punishment
effects o f BZR agonists have been demonstrated by antagonism o f conditioned operant
suppression (Toal et al., 1991), punished suppression (Griebel et al., 1997; Quintero et
al., 1985), conflict behaviors (Corbett & Dunn, 1993), and elimination of a cardiac
conditioned response (McLendon et al., 1976). In more ‘ethopharmacologically’ based
studies, BZR agonists increase time spent in and entries into the putatively more aversive
open arms o f the elevated plus maze (Criswell et al., 1994; Grahn et al., 1995; File &
Johnston, 1989; Yasumatsu et al., 1994; Griebel et al., 1997; Millan et al., 1997), increase
time spent in the light side o f a light/dark choice box (Belzung, 1987; Chaouloff et al.,
1997; Yasumatsu et al., 1994; Griebel et al., 1997, Griebel et al., 1996) and increase
exploration of a novel environment (Griebel et al., 1996; Widgiz & Beck, 1990).
Additionally, BZR agonists increase measures of social interaction (File & Johnston,
1989; Yasumatsu et al., 1994), social competition (Joly & Sanger, 1991), and decrease
defensive reactions o f mice to a rat stimulus (Griebel et al., 1997). Perhaps because of
the decreased defensive reactions, BZR agonists may also increase offensive aggression
in some circumstances (Millan et al., 1997; Borsini et al., 1993). Consistent with their opposing actions on GABA induced Cl' flow, negative
BZR modulators (inverse agonists) and positive BZR modulators (agonists) have generally opposite functional effects, although these may be contextually dependant
(Theibot et al., 1988). In contrast to BZR agonists, inverse agonists produce anxiogenic and proconvulsant effects (Sarter et al., 1995). Inverse agonists decrease time in and entries into open arms of the elevated plus maze (Grahn et al., 1995; Cole et al., 1995;
Yannielli et al., 1996), increase time in the dark box o f a light/dark choice (Belzung,
1987), decrease environmental exploration (Kalin et al., 1992), and increase freezing and tonic immobility (Moriarty, 1995; Kalin et al., 1992). Inverse agonists also minor agonist effects in exerting proconflict effects in social interaction tests, although some authors report a decrease in offensive aggression along with an increase in flight from aggression (Cutler & Aitken, 1991). Additionally, inverse agonists demonstrate physiologic effects similar to acute stress, including increases in heart rate (Ninan et al.,
1982), blood pressure (Ninan et al., 1982), plasma cortisol (Kalin et al., 1992; Ninan et al., 1982) and plasma catecholamines (Ninan et al., 1982), as well as a state similar to fear induced analgesia (Fanselow & Kim, 1992). However, inverse agonists may decrease basal heart rate in non-stressed contexts (DiMicco, 1987).
In contrast to full inverse agonists, the P-carboline FG 7142 (FG) is a partial inverse agonist, and has much lower potency for the proconvulsant actions o f full inverse agonists (Jensen et al., 1983). FG retains the apparent anxiogenic properties o f the full inverse agonists, however, as evidenced by a human study (Dorow et al., 1983).
Although this single study in humans had methodological limitations (Thiebot et al.. 1988), FG-induced anxiogenesis has been demonstrated also in several animal models o f anxiety. FG has been shown to decrease open arm entries in the elevated plus maze (Cole et al., 1995; Rodgers et al., 1995), increase suppression o f operant responding on withdrawal of a safety signal (Thiebot et al., 1991), and enhance measures of anxiety in social interaction tests (Beck & Cooper, 1986; File & Fellow, 1984; Rawleigh & Kemble,
1992; Kureta & Watanabe, 1996). Additionally, the state produced by FG 7142 generalizes to novelty and shock conditions in a drug discrimination paradigm
(Leidenheimer & Schechter, 1988), decreases open field exploration (Meng & Drugan,
1993), mimics the effects o f inescapable shock on social interactions (Short & Maier,
1993), and enhances novelty-induced increases in corticosterone levels in rats (Fellow &
File, 1985).
Although FG 7142 acts to decrease basal heart period (Wible et al, 1996) consistent with GABAergic modulation of parasympathetic output (DiMicco, 1987), evidence more consistent with FG-induced anxiogenesis is the finding o f an enhanced cardioacceleratory defensive-like response to moderately intense acoustic stimulus in rats
(Bemtson et al., 1996; Quigley et al., 1994). This effect of FG was similar to that seen with an increase in stimulus intensity (Quigley & Bemtson, 1990) and the presence o f a behavioral stressor (Hart et al., 1998). Based on these and other findings, the effects o f
FG 7142 have been considered a pharmacological model for anxiety (Sanger & Cohen,
1994). 1.2.3. Summary
Benzodiazepine receptor ligands allosterically modulate GABA induced chloride ion flow at the GABA^ receptor/ionopore complex. Ligands at the BZ receptor can enhance (agonists) or decrease (inverse agonists) GABA induced chloride influx.
Similar to their opposing effects on GABA^ chloride influx, BZR agonists and inverse agonists frequently exert opposing behavioral effects with agonists exerting anticonvulsant and anxiolytic effects and inverse agonist demonstrating proconvulsant and anxiogenic effects. In contrast to full modulators, partial modulators demonstrate differential potencies for specific functional properties. The P-carboline FG 7142 (FG) is a partial inverse agonist, and has much lower potency for the proconvulsant actions of full inverse agonists yet retains anxiogenic properties. Consistent with this, systemic administration o f FG 7142 enhances cardiovascular reactivity to probe acoustic stimuli.
The present studies utilize FG in the investigation of neuronal substrates which modulate cardiovascular responses.
3.1. Basal forebrain-cortical cholinergic fBFCCl svstem
Consistent with the complex GABA^ receptor physiology (Rabow et al, 1995) and regionally specific anatomic subunit distribution (Wisden et al., 1992) it has been suggested that BZR ligands may differentially affect multiple neurological systems mediating varied behaviors (Bemtson et al., 1997). One o f the systems that has been suggested to be active in contexts that entail cortical/cognitive processing and thus be amendable to BZR ligand action is the basal forebrain cholinergic system (Bemtson et al.,
1998; Sarter & Bruno, 1997).
1.3.1. BFCC Anatomv
The basal forebrain cholinergic system is a set of cholinergic projection neurons extending through the ventral forebrain grey matter. Neurons belonging to this population are found in scattered, variously named, and sometimes overlapping nuclei, such as the Nucleus Basalis (NB), Substantia Innominata (SI), Sublenticular Grey, Ansa
Lenticularis, Nucleus o f Ansa Peduncularis, Nucleus of Diagonal Band, and Medial
Septal Nucleus. (Mesulam, 1991; Koliatsos & Price, 1991). However, recent theories based on hodological and chemoarchitectural data have postulated that parts o f the
Amygdala, SI and Bed Nucleus of the Stria Terminalis (BNST) may be one anatomic, and possibly functional, entity (Heimer & Alheid, 1991). Based on these data, Golgi staining, and common connective and developmental features, Alheid et al. (1995) have put forth the concept o f an extended amygdala - a continuum of intermpted cell columns stretching dorsomedially from the amygdala proper to the BNST, forming a ring around the intemal capsule.
The cholinergic projection neurons follow a topographical projection pattem.
The medial septum and vertical limb of the diagonal band innervate the hippocampus, the horizontal limb o f the diagonal band and the magnocelluar preoptic nucleus project to the
10 olfactory bulb, piriform cortex and entorhinal cortex, while the neocortex and amygdala receive cholinergic projections from the ventral pallidum, substantia innominata, intemal capsule, and nucleus o f ansa lenticularis (Zaborszky et al., 1991). This latter set o f projections, termed by Mesulam (1991) as NB/C4, follows a roughly medio-lateral, anterior-posterior topography (Zaborszky et al., 1991). It is specifically the NB/C4 area that is the major source o f cortical acetylcholine (Woolf, 1991). Consistent with this, selective immunotoxin lesions of the cholinergic neurons in the SI result in relatively complete neocortical loss of cholinergic markers (Heckers et al., 1994). Additionally, electrical stimulation o f the nucleus basalis increases cortical cholinergic efflux as measured by cortical cup (Casamenti et al., 1986) and microdialysis (Rasmusson et al.,
1992).
Functionally, the BFCC has been implicated in a wide range of tasks including motor function, arousal, attention and mood (Mesulam, 1991). Some have speculated that the basal forebrain may act as a gating mechanism for sensory information flow into limbic structures (Mesulam, 1991; Zaborszky et al., 1991). Consistent with this, motivationally correlated changes in neuronal activity have been demonstrated in the basal forebrain (Zaborszky et al., 1991). Additionally, electrophysiological recordings o f nucleus basalis/substantia innominata/diagonal band neurons have demonstrated maximal responsiveness to novel (Wilson & Rolls, 1990) and aversive (Richardson & DeLong,
1991) stimuli.
11 1.3.2. Acetylcholine and anxiety
Tangential evidence for a role for acetylcholine in anxiety comes from the clinical literature o f humans exposed to cholinesterase binding organophosphates. Low level occupational exposure to organophosphates was found to be associated with elevated levels of anxiety when accompanied by decreased plasma cholinesterase (Levin et al., 1976). Additionally, multiple high level exposures resulted in elevated measures of anxiety on one year follow-up (Reidy et al, 1992). However, this latter study is not conclusive as experimenters were not blind to subject condition and were part of the litigation team (Meams et al, 1994).
More substantial supporting evidence is found in the animal literature. Rats receiving intracerebral injections o f the cholinergic agonist carbachol emit ultrasonic vocalizations similar to those induced by handling and foot shock (Brudyznski et al,
1991). Cats treated with intracerebroventricular (ICV) muscarine exhibit fear-like aggression with escape behaviors (Beleslin & Samardzic, 1977) whereas low doses of
ICV carbachol enhanced defensive fear reactions with no effect on basal behavior
(Johansson et al., 1979). Moreover, low dose ICV carbachol has been demonstrated to act as a peripheral adrenergically sensitive cardiovascular stimulant, while high doses elicited a striking fear reaction (Day & Roach, 1977). Furthermore, learned fear responses in conditioning paradigms are sensitive to systemic administration of the muscarinic antagonist scopolamine (Anagnostaras et al., 1995; Young et al, 1995).
12 1.3.3. GABA/BZ - BFCC interactions
Consistent with a postulated basal forebrain function in anxiety and the involvement o f BZR ligands in modulating anxiety, the NB/C4 complex has been implicated as a site o f action for BZR ligands (Sarter et al., 1992; Sarter & Bruno, 1994;
Barter & Bruno, 1997). Anatomically, GABAergic synaptic contact is a general feature of ventral pallidal, corticopetal, cholinergic neurons (Zaborszky et al., 1991). Functionally, this is evidenced by a decrease in cortical acetylcholine efflux with the GABA^ agonist muscimol (Casamenti et al., 1986) and the reversal o f scopolamine induced acetylcholine depletions by the inhibitory GAB A modulator DM-9384 (Abe, 1991). The SI shows a high density o f BZR binding sites (Sarter & Schneider, 1988), and specifically a high concentration o f a,P,Y 2 G ABA subunit mRNA (Wisden, 1992) consistent with full BZ receptor function.
Congruous with a NB/C4 cholinergic modulation of BZR ligand function, intrabasalis infusions of BZR ligands have demonstrated functional effects and BZR modulation is sensitive to pharmacologic cholinergic manipulations. Systemic FG 7142 enhances cortical acetylcholine efflux (Moore et al., 1995). Furthermore, and consistent with the modulation of cortical cholinergic transmission, the functions frequently affected by intrabasalis BZR ligand infusions include more cognitive processes such as learning and vigilance. The cholinesterase inhibitor physostigmine antagonized the negative effects of chlordiazepoxide on learning and memory and the passive avoidance response
(Nabeshima et al., 1990). Additionally, infusion of a weak BZR inverse agonist antagonized the attentional effects of systemic scopolamine (Duka et al., 1996). Intra
13 nucleus basalis infusions o f the partial inverse agonist FG 7142 resulted in enhancement of working memory (Smith et al., 1994). Similarly, infusions of the agonist chlordiazepoxide and the inverse agonist (3-CCE into the substantia innominata bidirectionally modulated vigilance performance (Holley et al., 1995). It has been suggested that cortical efflux of acetylcholine acts to allocate processing resources to the detection and association o f stimuli o f current behavioral significance (Sarter & Bruno,
1994; 1997) and it is this enhanced signal processing that may account for modulation of responses to fear relevant stimuli by BZR ligand administration.
In support of this, studies of the modulation of anxiety by BZR ligands have implicated cholinergic systems in these effects. The potentiating effects of FG on the cardioacceleratory defensive response were mimicked by intracerebroventricular (ICV) administration o f carbachol, and blocked by ICV atropine (Bemtson et al., 1996).
Furthermore, the effect o f FG on the defensive-like response was eliminated by selective lesions of basal forebrain cholinergic neurons with the cholinergic-specific immunotoxin
192 IgG-saporin (Bemtson et al., 1996).
1.3.4. Summarv
The basal forebrain cholinergic system is a set of cholinergic projection neurons extending through the ventral forebrain grey matter. The NB/C4 section of the basal forebrain, innervates the neocortex and amygdala and has been termed the basal forebrain cortical cholinergic (BFCC) system. Functionally, the BFCC has been implicated in a wide range o f tasks including motor function, arousal, attention and mood.
14 Consistent with a postulated basal forebrain function in anxiety, and the involvement of
BZR ligands in modulating anxiety, the NB/C4 complex has been implicated as a site of action for BZR ligands. Congruous with this, cholinergic specific lesions of the NB/C4 region of the basal forebrain block enhancement of cardiovascular reactivity by the BZR partial inverse agonist FG 7142. The present studies investigate the potential cholinergic target sites o f the NB/C4 projections that underlie effect o f FG on cardiovascular responding.
1.4. The medial prefrontal cortex
The functional role of the prefrontal cortex has been o f long standing interest to psychologists, physiologists and anatomists alike (Nauta, 1971; Neafsey, 1990). The prefrontal cortex is defined by the projections o f the medial dorsal nucleus of the thalamus. In primates this includes cortical areas with limbic and visceral ties such as the posterior orbital and anterior cingulate. In rodents the prelimbic and infralimbic areas are included (Reep, 1984). The history of links between the autonomic nervous system and the medial division o f the prefrontal cortex dates back to the 1920s (Neafsey, 1990). In fact, prefrontal lobotomies were widely used to treat such disorders as ‘anxiety neurosis’ and ‘conversion hysteria’ (Freeman & Watts, 1942). Many have postulated that the prefrontal cortex is a visceral cortex (Neafsey, 1990; Reep, 1984; Tyakagishi & Chiba,
1991) based on the affective changes in humans with frontal lobe dementia/degeneration
(Neary, 1995), as well as ictal anxiety and fear (Myers, 1972; Chauvel et al., 1995) and
15 frank autonomic manifestations (Chauvel et al., 1995) in cases of epileptic discharges originating in the prefrontal cortex. In fact, the prefrontal cortex has been included in a definition o f the visceral forebrain based on projections to the nucleus tractus solitarius
(NTS) (van der Kooy, 1984) and other anatomical connections suggesting that sensory input to the prefrontal cortex is highly integrated with input from cortical areas associated with emotional processes (Barbas, 1995). Indeed, in an influential paper Nauta (1971) proposed that the prefrontal cortex was a cortical-limbic interface, and functional deficits observed with prefrontal cortical injury were considered ‘visceral agnosia’.
This putative viscero-emotional function o f the medial prefrontal cortex is in contrast to the working memory function postulated for such lateral areas o f the prefrontal cortex in primates as the principle sulcus. Goldman-Rakic (1988), however, has emphasized the role o f all subdivisions o f the prefrontal cortex as components o f a network o f parallel circuits which operationalize working memory in the form o f response delay activation, with each aspect o f the circuit functioning in a different information-type domain (Goldman-Rakic, 1990; 1996).
1.4.1. General anatomv
The prefrontal cortex is defined by the projection field o f the medial dorsal
(MD) nucleus of the thalamus (Reep, 1984, Sarter & Markowitsch, 1984; Sesack et al.,
1989). In the rat, the central division o f MD projects to the ventral agranular insular cortex, the medial division innervates the dorsal agranular insular and prelimbic cortices, while the anterior cingulate cortex is innervated by the lateral MD division (Sarter &
16 Markowitsch, 1984). Also included in the MD projection field are the infi'alimbic cortex and frontal pole (Sarter & Markowitsch, 1984).
It is especially the afferent and efferent connections of the prelimbic and infiralimbic areas o f the prefrontal cortex that contain other afferent and efferent connections that implicate this structure in affective modulation. In the rat the prelimbic and infiralimbic cortical areas are located in the ventral portions of the medial prefrontal cortex. Amygdaloid nuclei, which have been implicated in affective processes, provide both target and source for connections to the prelimbic and infralimbic regions (Reep,
1984; Tyakagishi & Chiba, 1991). The posterior aspect o f the basolateral nucleus of the amygdala irmervates prelimbic and infralimbic regions (Sarter & Markowitsch, 1984). In turn, the prelimbic area projects to the basolateral and basomedial amygdalar nuclei
(Buchanan et al., 1994; Sesack et al., 1989), while the infralimbic cortex projects to the
central nucleus (Buchanan et al., 1994). The prelimbic cortex represents the medial zone
o f convergence o f medial dorsal and amygdalar projections, a structural relationship that
is the most consistent across species (Reep, 1984).
As with the rest of the cortical mantle, basal forebrain cholinergic projections
to the MPF are supplied by the NB/C4 region (Zaborszky et al., 1991). The GABA^ subunit mRNA detected in this area is (Wisden, 1982). GABA^ receptors with
this subunit configuration would thereby be expected to display full BZR ligand
modulation (Rabow et al., 1995).
Other connections support a role for the prelimbic and infralimbic cortices in
autonomic regulation. Prelimbic and infralimbic projections have been documented to
17 such structures as the lateral hypothalamus (Hurley et al., 1991; Sesack et al., 1989;
Tyakagishi & Chiba, 1991), nucleus tractus solitarius (Bacon & Smith, 1993; Buchanan et al., 1994; Hurley et al., 1991; Terreberry & Neafsey, 1983; Terreberry & Neafsey,
1987), specifically the medial/ventral parts (van der Kooy et al., 1984), the nucleus ambiguous (Buchanan et al., 1994; Hurley et al., 1991), dorsal motor nucleus of the vagus
(Buchanan et al., 1994; Hurley et al., 1991), ventrolateral medulla (Buchanan et al., 1994;
Hurley et al., 1991) and the intermediolateral cell columns (Hurley et al., 1991) and thoracic central autonomic region (Hurley et al., 1991).
1.4.2. MPF and anxietv
Changes in affect have been demonstrated in human subjects with frontal lobe lesions (Cummings, 1995), and although not universal (Divac et al., 1984), lesion studies with animals have supported a role for the MPF in emotional processing. Lesion studies of the MPF in anxiety paradigms have revealed differences along a dorsal-ventral dimension. Morgan & LeDoux (1995) demonstrated that whereas dorsal lesions enhanced contextual and conditioned stimulus (CS) induced freezing, ventral lesions only enhanced freezing to the CS. Additionally, Frysztak & Neafsey (1994) demonstrated that dorsal, but not ventral lesions enhanced the conditioned emotional response (CER), whereas subjects with ventral lesions demonstrated a CER less sensitive to autonomic blockade with either atropine or atenolol and a CS- response less sensitive to atropine
(Frysztak & Neafsey, 1994). Medial prefrontal cortical lesions have been shown also to enhance open field resistance to capture, urination, and defecation (Markowska &
18 Lukaszewska, 1980), increase the latency to eating in a novel environment (Bums et al.,
1996), increase time spent in a nest box with a decrease time spent in a novel box
(Holson, 1986), and increase time to enter a novel open field (Holson, 1986). On the
other hand, MPF lesions do not prolong e:ctinction to a conditioned context (Morgan et
al., 1993), increase latency to enter a dark box, and decrease latency to drink form a novel
water bottle (Holson & Walker, 1986). Thus, MPF manipulations appear to have a rather
complex effect on affect. Metrics associated with conditioned stimuli have proven to be
no more reliable. Medial prefrontal cortical lesions prolong extinction to a CS (Morgan
et al., 1993), yet have no differentiated effect on a CS+ and a CS- (Powell et al., 1994).
Moreover, in the same preparation, lesions of the MPF enhance respiration while
attenuating freezing and vocalization in response to a CS (Frysztak & Neafsey, 1991).
Together, these studies suggest that the medial prefrontal cortex may exert modulating
(both inhibitory and excitatory) effects on emotional processing. These effects may be
mediated by partially overlapping subsystems and/or may be dependent on specific
experimental context.
Studies o f the functions o f the intact MPF also support a modulatory role in
affective processes and anxiety. Increases in multiple unit activity in MPF, evoked by
novel stimuli and an aversive CS (proportional to the strength o f the aversive
unconditioned stimulus [US]) have been demonstrated (Powell et al., 1996). Moreover, controllable foot shock induced increases in MPF self stimulation (McGregor, 1991).
Measures o f 5-HT efflux and turnover in the MPF are sensitive to anxiety manipulations. Medial prefrontal cortical 5-HT metabolites and efflux levels are
19 increased by conditioned fear stress (Inoue et al., 1993; Inoue et al., 1994; Yoshoika et al..
1993), CS presentation (Goldstein et al., 1994; Goldstein et al., 1996), and the presentation o f a nonspecific being subjected to foot shock (Kawahara et al., 1993). Stress
induced enhancement o f MPF 5-HT measures has been blocked with either basolateral or central amygdaloid lesions (Goldstein et al., 1996).
In addition to 5-HT efflux, anxiogenic test conditions enhance MPF dopamine release. Increased dopamine turnover and efflux in the MPF have been demonstrated with foot shock (Davis et al., 1994; Dazzi et al., 1995; Rasmusson et al., 1994), conditioned fear stress (Inoue et al., 1994; Yoshioka et al., 1996) and presentation of a CS
(Goldstein et al., 1994; Goldstein et al., 1996). Additionally, novelty (Davis et al., 1994;
Feenstra et al., 1995), immobilization stress (Bertolucci-D’Angi et al., 1990; Hegarty &
Vogel, 1995), and handling (Feenstra et al., 1995) induce enhanced dopamine release.
Again these monoamine changes are ameliorated by lesions to the basolateral (Goldstein et al., 1996) or central (Davis et al., 1994; Goldstein et al., 1996) nuclei o f the amygdala.
There have been multiple other physiologic measures that implicate the MPF in anxiety. Restraint stress has been documented to increase MPF excitatory amino acid efflux (Moghaddam, 1993), cholecystokinin-like immuno-reactivity (Nevo et al., 1996), and acetylcholine efflux (Mark et al., 1996), and decrease cortical GABA^ binding
(Gruen et al., 1995). In addition, an aversive CS evokes norepinephrine efflux (Goldstein et al., 1996) and novel, but not habituated, stimuli evoke acetylcholine release (Acquas et al., 1996) from this area.
20 Consistent with its anatomical connections, MPF involvement in the modulation of autonomic functions has been confirmed by a variety of measures.
Decreases in gastric motility were demonstrated with MPF microstimulation in anesthetized rats (FIurley-Guis & Neafsey, 1986). Electrical stimulation o f the MPF has also been shown to decrease heart rate (Powell et al., 1994), and produce both depressor
(Bacon & Smith, 1993; Hardy & Holmes, 1988) as well as pressor (Neafsey, 1990) responses dependent on dorsal or ventral location respectively (Neafsey, 1990). These effects appear to be mediated, in part, by the posterior hypothalamus and the NTS (Hardy
& Mack, 1990). In other studies, excitotoxic MPF lesions were demonstrated to have no effect on mean arterial pressure or heart rate, but did reduce maximum and minimum heart rate plateaus and decrease baroreceptor reflex threshold to intravenous pressor and depressor agents (Verbeme et al., 1987). Additionally, MPF lesions increased plasma
ACTH and corticosterone in response to a 20 minute restraint stress, while crystalline cortisol implanted into the MPF has been demonstrated to decrease ACTH and corticosterone response to restraint stress (Diorio et al., 1993).
1.4.3. Modulation of MPF function bv BZR ligands
Although it is unclear if BZR ligand modulation o f the effects o f anxiety on the medial prefrontal cortex is mediated by a direct cortical action, by action in the amygdala, or through a projection system, potential links between the medial prefrontal cortex, anxiety and the actions of BZR ligands have been suggested by recent findings.
Complete neocortical aspiration in neonates, eliminated the ability of diazepam to
21 diminish ultrasonic distress calls in adult rats (Naito et al., 1995). Administration o f the full inverse agonist P-CCM and the partial inverse agonist FG 7142 increase MPF dopamine turnover (Bertolucci-D’Angio et al., 1990) and FG has been shown to enhance measures of tyrosine hydroxylation (Knorr et al., 1989). Moreover, BZR agonists attenuate the increases in MPF dopamine turnover induced by foot shock (Dazzi et al.,
1995), immobilization (Hegarty & Vogel, 1995), conditioned fear stress (Yoshioka et al., 1996) and novel environment (Feenstra et al., 1995). Finally, FG has been shown to potentiate decrements in open field exploration associated with MPF lesions (Jaskiw &
Weinberger, 1990).
1.4.4 Summarv
The medial prefrontal cortex has historically been implicated in anxiety and has direct anatomical ties to brainstem autonomic regulation centers. Additionally, the medial prefrontal cortex receives cholinergic projections from the basal forebrain. These factors suggest this area may be a relevant target site in the basal forebrain cholinergic modulation o f cardiovascular reactivity. The present studies investigate the effects o f cholinergic manipulations in this area on cardiovascular reactivity.
1.5. Amvgdala
The amygdala is a limbic structure that has classically been thought to be involved in emotional, especially fear, processing in both clinical and animal literatures.
In humans, the amygdala has been implicated in the processing o f emotionally significant
22 mnemonic events (Sarter & Markowitsch, 1985), while bilateral amygdaloid lesions impair recognition, recall, and intensity judgement specifically of fearful facial expressions (Adlophs et al., 1995). Other authors have postulated amygdalar function as one component in an emotional learning system ( Kapp et al., 1991 ; LeDoux, 1992;
LeDoux, 1995; McGaugh et al., 1996) or as a risk assessment system (Graeff, 1994).
Many animal models are centered around reactivity to a conditioned stimulus; the presentation of which produces a constellation of behaviors similar to those used to diagnose generalized anxiety in humans (Davis, 1992). With the combination of its many efferent projections (Davis, 1990) and the ability to elicit complex autonomic and behavioral fear-like patterns with electrical stimulation (Davis, 1992) it has been hypothesized that the amygdala mediates aversive conditioning. (LeDoux, 1992).
1.5.1. General anatomv
Anatomically, the amygdala is a complex o f nuclei that stretch through the ventrolateral aspects o f the brain. Caudally, amygdalar nuclei consist o f the basolateral nucleus (BLA) and the more ventromedial posterior cortical nucleus (CoAp). Moving rostrally, the basomedial nucleus is seen to be interposed between the BLA and CoAp, and the lateral nucleus is found in a dorsal position. Continuing forward, the discretely defined aspects o f the medial nucleus (MeA) and the more dorsal central nucleus (CeA) are found medial to the rest of the amygdalar complex (but see discussion o f the extended
23 amygdala above). More rostral still, the anterior cortical nucleus becomes evident in the ventral aspect o f the complex. Finally, the anterior amygdalar area is at the most rostral point of the complex.
The organizational framework of the amygdala is also very intricate. Within the amygdaloid complex there exist a core group made up o f the CeA and the MeA, a cortical like group including the superficial, basolateral, and basomedial nuclei, and olfactory and vomeronasal amygdalar groups defined by their reciprocal connections to the olfactory bulb and accessory olfactory system respectively. Extra dimensions of organization in the amygdala pertain to intemuclear and external connectivity.
Basomedial, posterior medial cortical, and amygdalo-hippocampal areas project to the core medial nucleus, whereas the basolateral nucleus demonstrates a preference for projection to the core central nucleus. In turn, it is the core nuclei that make the majority o f amygdalar efferent projections (Alheid et al., 1995).
In the context of fear processing, the cholinergic and autonomic connections o f the BLA/CeA nuclei stand out. It is the BLA that receives the most dense basal forebrain cholinergic innervation o f the amygdaloid complex (Gilmor et al., 1996; Price
& Cames, 1991; Ruggiero et al., 1990). Specifically, cholinergic projection neurons from the NB/C4 region o f the basal forebrain, supply the BLA (Heimer & Alheid, 1991).
Moreover, in relation to anxiety, the great majority o f neurons projecting to the MPF originate in the BLA, and the MPF in turn reciprocally projects to the BLA (McDonald,
1987; Sarter & Markowitz, 1984). Although the CeA receives little basal forebrain cholinergic innervation (Ruggiero et al., 1990), it does receive projections from the basal
24 and lateral nuclei, and provides connections to autonomically important areas including the lateral hypothalamus (Alheid et al., 1995; Cassell et al., 1986; Danielsen et al., 1989), nucleus tractus solitarius (Cassell et al., 1986; Danielsen et al., 1989; Rogers & Fryman,
1988; Schwaber et al., 1980), dorsal motor nucleus o f the vagus (Cassell et al., 1986;
Danielsen et al., 1989; Schwaber et al., 1980; Veening et al., 1982), nucleus ambiguous
(Danielsen et al., 1989) and rostral ventrolateral medullary Cl area (Cassell & Gray,
1989; Danielsen et al., 1989).
1.5.2. Amvgdala and anxietv
There is a great deal o f evidence which supports the hypothesized role o f the
amygdala as an integrative substrate through which aversive stimuli find behavioral and
autonomic expression. Support for this hypothesis is found in studies which measure
amygdalar activation in response to aversive events. Exposure to the Vogel conflict test,
in which subjects receive shocks simultaneously with water acquisition, (Moller et al.,
1994) and aversive air puff stimuli (Duncan et al., 1996) have been shown to enhance
amygdalar early gene activation as measured by c-fos detection. Additionally, amygdalar
manipulations have been shown to ameliorate central and behavioral outcomes of
aversive events. Intra-amygdalar application of morphine has been demonstrated to
impair fear acquisition and the development o f a hypoalgesic response to a hot plate
(Good & Westbrook, 1995) while amygdalar lesions reduce shock induced enhancement o f MPF dopamine turnover (Davis et al., 1994), and freezing (Roozendaal et al., 1991b).
25 In accord with a role in affective responding amygdalar manipulations also impact on learned aversive cues such as conditioned and contextual stimuli. Amygdalar lesions (Killcross et al., 1997; Kim et al., 1993; Maren et al., 1996) as well as intraamygdalar infusions o f glutamate antagonists (Maren et al., 1996) have been demonstrated to block contextually conditioned freezing. Furthermore, lesions o f the amygdala attenuate CS-induced suppression o f operant behaviors (Killcross et al., 1997), freezing (Goldstein et al., 1996; LeDoux et al., 1990; Lorenzini et al., 1991; Maren et al.,
1996; Phillips & LeDoux, 1992; Roozendaal et al., 1990), startle potentiation (Campeau
& Davis, 1995; Hitchcock & Davis, 1986; Hitchcock & Davis, 1987; Lee et al., 1996 ), avoidance (Killcross et al., 1997; Lorenzeni et al., 1991), and CS evoked ultrasonic vocalizations, defecation, and MPF dopamine turnover (Goldstein et al., 1996).
The amygdala has also been implicated in anxiety in paradigms consistent with a more general role in threat evaluation or risk assessment. Amygdalar c-fos expression is elevated with exposure to the elevated plus maze (Duncan et al., 1996), while time in the open arms is increased with blockade o f CRHl receptor formation by chronic intra-CeA antisense infusion (Liebsch et al., 1995). Modulation of social interaction has also been shown with amygdalar manipulations. Intra-amygdalar infusions of glutamate antagonists enhanced (Sajdyk & Shekhar, 1997) whereas a 5-HT agonist decreased (Gonzalez et al., 1996) time spent in active social interaction.
Additionally, infusions of glutamate antagonists into BLA blocked light-enhanced startle
(Davis et al., in press). Amygdalar activation has further been demonstrated with a variety of metrics and markers. Presentation of conspecific ultrasonic vocalization
26 (Beckett et al., 1997) enhanced amygdalar c-fos detection, whereas enhanced 5-HT efflux
was demonstrated with shock of a nearby conspecific ((Kawahara et al., 1993), and elevated CCK-4 levels were observed on the presentation o f a olfactory predator cue
(Pavlasevic et al., 1993).
The roles of the amygdala in the evaluation o f risk and conditioned aversive
behaviors are consistent with the hypothesis that the amygdala functions as a part o f an emotional learning system. This proposed function is supported by further evidence in
the literature. In vivo electrophysiological data reveal CS associated activity changes in
CeA and BLA that parallel development o f conditioned responding (Applegate et al.,
1982; Yejeya et al., 1991), as well as extinction (Yajeya et al., 1991). Similarly, c-fos activation in the amygdala is seen with CS presentation (Sotty et al., 1996). Furthermore, aversive conditioning has been demonstrated to be amendable to BLA excitability modulation (Davis et al., 1994). Intra-amygdalar administration of glutamate antagonists
block conditioned freezing (Fanselow & Kim, 1994; although this is not universal
Gerwirtz & Davis, 1997), as well as second order aversive conditioning (Gewirtz &
Davis, 1997). Additionally, amygdalar infusions of a norepinephrine agonist enhanced and atropine blocked retention o f an inhibitory avoidance response (Introini-Collison et al., 1996). Further support for a role in an emotional learning system is found in the human literature as subjects with amygdalar lesions demonstrate significantly less mnemonic enhancement by emotional arousal (McGaugh et al., 1996).
The amygdala has also been implicated in autonomic features o f fear and anxiety. Amygdalar lesions attenuate bradycardic response to immobilization
27 (Roozendaal et al., 1991), footshock delayed responding (Roozendaal et al., 1991b), CS presentation (Roozendaal et al., 1990), and defeat by a dominant conspecific
(Roozendaal et al., 1991), as well as tachycardie responses to CS presentation (Sananes &
Campbell, 1989, Gentile et al., 1986). These responses were specifically affected as heart rate responses to defensive burying (Roozendaal et al, 1991), US presentation
(Roozendaal et al., 1991b; Sananes & Campbell, 1989) and neutral stimuli (Sananes &
Campbell, 1989) were unaffected. Complementary to the effect on heart rate changes, amygdalar lesions attenuate pressor responses to CS presentation (LeDoux et al., 1990) and intracerebroventricular caibachol administration (Ozkutlu et al., 1995), as well as adrenocortical responses to US (Roozendaal et al, 1991b), CS (Goldstein et al., 1996), somatosensory and olfactory stimuli (Feldman & Conforti, 1981).
Other methods o f modulation o f amygdalar functions yield changes in somatic and autonomic components o f fear-like behaviors (Kapp et al., 1979). Electrical stimulation of the CeA enhanced acoustic (Rosen & Davis, 1988a,b) and evoked (Rosen
& Davis, 1990) startle, while partial BLA electrical kindling enhanced fear potentiated startle (Rosen et al., 1996). In addition, electrical stimulation o f the amygdala in anesthetized preparations evoked bradycardic responses (Iwata et al., 1987) while in conscious subjects, both electrical (Stock et al., 1978; Iwata et al., 1987) and glutamatergic (Iwata et al., 1987) amygdalar stimulation resulted in tachycardia.
Similarly, both pressor (Iwata et al., 1987; Gelsema et al., 1987) and depressor
(Mogenson & Calaresu, 1973; Gelsema et al., 1987) responses have been demonstrated
28 with CeA stimulation. Consistent with this, CeA stimulation yielded both inhibition and excitation o f barosensitive neurons in the rostral ventrolateral medulla (Gelsema et al.,
1989).
1.5.3. Modulation of amvgdalar function bv BZR ligands
Manipulation o f the GAB A^/BZ receptor system in the amygdala modulates measures o f fear in test subjects. Microinjection o f a GABA^ agonist into the BLA attenuated shock induced freezing (Helmstetter & Bellgowan, 1994), while similar infusions of BZR agonists impair elevated plus maze open arm avoidance (Resold &
Treit, 1995), and foot shock induced freezing (Helmstetter, 1993), avoidance (Harris &
Westbrook, 1995), and hypoalgesia (Helmstetter, 1993; Harris & Westbrook, 1995).
Infusions of a GABA^ agonist into the CeA enhanced social interaction (Sanders &
Shekhar, 1995b) and attenuated cold stress ulcer development (Sullivan et al., 1989).
Furthermore, shock probe avoidance was also impaired with CeA infusions o f an BZR agonist (Resold & Treit, 1995). In contrast, administration o f the GABA^ antagonist bicuculline or the BZR weak partial agonist flumazenil into the BLA blocked the anxiolytic effect of a peripherally administered BZR agonist on social interaction
(Sanders & Shekhar, 1995a). Consistent with this, administration o f bicuculline into the
BLA induces antisocial, proconflict, and sympathetically mediated tachycardie responses
(Sanders & Shekhar, 1995b) sensitive to previous subthreshold GABA^ blockade
(Sanders etal., 1995).
29 1.5.4. Summarv
Historically, the amygdala has been implicated in the mediation of anxiety.
This structure has direct anatomic ties to brainstem autonomic mechanisms.
Furthermore, the basolateral nucleus of the amygdala receives cholinergic innervation from the NB/C4 region o f the basal forebrain. Earlier results suggest that the basal forebrain cholinergic projection to the amygdala was not the critical pathway for the cardioreactive effects of FG, as this pathway is largely spared by immunotoxic lesions that abolished FG effects. Because o f its central role in anxiety and autonomic controls however, the present studies examined the effect of cholinergic manipulations in the basolateral amygdala on cardiovascular reactivity.
1.6. General summarv
Changes in autonomic outputs have traditionally been associated with responses to anxiogenic stimuli/environments (Bemtson et al., 1998), and the so-called defensive response to aversive stimuli is generally associated with cardiovascular acceleration and vascular pressor response (Graham, 1979; Graham, 1984; Turpin &
Siddle, 1983; Turpin, 1986). In fact such responses have been utilized to define fearful states in animal subjects as part of a constellation o f behavioral responses (Davis, 1990).
Moreover, fear and anxiety may lead to a shift in response to novel stimuli from the typical bradycardia o f the orienting response to tachycardia characteristic o f defense (Hart et al., 1998; Quigley & Bemtson, 1990). It is therefore not surprising that BZR ligands,
30 which have been implicated in modulating the anxious state, have been shown to also impact on cardiovascular reactivity. Previous studies have documented enhancement of cardiovascular reactivity to non-signal auditory stimuli by the BZR partial inverse agonist
FG 7142 (Quigley et al., 1994). Moreover, BZR agonists attenuate, and BZR partial inverse agonists augment, stressor-enhanced reactivity (Hart et al., 1998).
It has been postulated that one important locus o f action o f BZR ligands is the basal forebrain cholinergic projection system (Sarter et al., 1992; Sarter & Bruno, 1994;
Sarter & Bruno, 1997). Indeed, FG-poteniiation of cardiovascular reactivity has been mimicked by intracerebroventricular administration o f the cholinergic agonist carbachol, and blocked by intracerebroventricular infusion o f the muscarinic antagonist atropine, as well as by cholinergically specific lesions of the basal forebrain (Bemtson et al., 1996).
One likely rostral cholinergic target area in the BZR-basal forebrain cholinergic modulation o f cardiovascular reactivity is the medial prefrontal cortex, which has been implicated in animal models of anxiety and in modulation of cardiovascular function. Based on previous research as outlined above, it was speculated that the medial prefrontal cortex may be a critical target site o f the basal forebrain cholinergic system in effects of FG on the cardioacceleratory defensive response. Studies outlined below examined this hypothesis by: a) evaluation of the effects of cholinergic agonists and antagonists into the medial prefrontal cortex and other central sites; b) effects o f local cholinergic blockade in the medial prefrontal and other central sites on cardiovascular effects of FG; and c) effects of selective cholinergic immunotoxic lesions of the medial prefrontal cortex on the cardiovascular effects o f FG.
31 CHAPTER 2
GENERAL METHODS
2.1. Surgical procedures
Rats receiving intracranial drug infusions were cannulated under ketamine/xylazine anesthesia (90 mg/kg and 5 mg/kg, respectively, intraperitoneally).
Stainless steel guide cannulae (0.65 nun o.d.; Plastics One, Roanoke, VA) were lowered bilaterally through trephine holes to the medial prefrontal cortex, prelimbic area (AP +2.7
ML ±0.6 DV -3.6), the lateral prefrontal cortex (AP +2.7 ML ± DV ), or the basolateral amygdala (AP -2.4 ML ±4.9 DV -8.2), all coordinates from Paxinos & Watson (1987), relative to Bregma, midline, and durai surface with flat-skull orientation. Cannulae were affixed to the skull with stainless steel skull hooks and dental acrylic, subjects received ampicillin (0.01 ml/kg, s.c.) and were then allowed a minimum of seven days to recover prior to further instrumentation for cardiovascular recording.
Rats receiving central cholinergic lesions were anesthetized with ketamine/xylazine anesthesia (90 mg/kg and 5 mg/kg, respectively, intraperitoneally). A
10 pi, 26 gauge Hamilton Syringe was lowered through a trephine hole to the medial prefrontal cortex, prelimbic area (AP +2.7 from Bregma, ML ±0.6, DV -3.6 from dura;
32 Paxinos & Watson, 1987), with flat-skull orientation. One-half |il of DelBecco’s Saline
or 192 IgG-Saporin (0.01 pg/pl/hemisphere; Chemicon International Inc., Temecula, CA)
in DelBecco’s Saline was then injected. To allow for diffusion and minimize spread up
the needle track, the syringe was left in place for 3 minutes and then retracted at a rate of
0.6 mm per minute. The immunotoxin 192 IgG-saporin consists of a monoclonal
antibody to the low-affinity p75 nerve growth factor receptor coupled to saporin, a
ribosome inactivating toxin (Stirpe et al., 1992). The cholinotoxic selectivity of this
substance is due to the selective distribution o f p75 receptors on forebrain cholinergic
neurons and terminals (Koh et al., 1989; Wiley et al, 1991). Immimotoxin was kept
frozen in DelBecco’s saline until use. The concentration o f 192 IgG-saporin was based
on the findings of Holley et al. (1994) in which an estimated 40% reduction in cortical acetylcholinesterase was documented. Following completion o f bilateral injection, the
scalp was sutured closed, subjects received ampicillin (0.01 ml/kg, s.c.) and were allowed
10 days to recover prior to further instnunentation for cardiovascular recording.
Following recovery from cannulation or lesion surgery, subjects were
instrumented with a chronic, subcutaneous telemetry device (Data Sciences International,
Arden Hills, MN)- Electrode ends were tunneled subcutaneously and secured at two
separate ventral sites (1 cm caudal to the right clavicle, mid-clavicular line; 1 cm rostral to most caudal rib, mid-axillary line). The telemetry transmitter was then secured
33 interscapularly on the dorsum and all incisions were closed. Animals received ampicillin
(0.01 ml/kg, s.c.) immediately following surgery and were allowed 24 h to recover prior to testing.
2.2. Phvsiologic recording
Testing was conducted in a glass enclosure (51 cm x 30 cm x 25 cm), with a layer o f bedding in the bottom, located within a sound attenuated chamber (Industrial
Acoustics Company, Inc., New York, N ltl. EGG signals were transmitted to a PhysioTel receiver (Data Sciences International, Arden Hills, MN), coupled to a BIOPAC recording system for amplification, and the analog signal passed to an IBM compatible computer for A/D conversion (500 Hz/12 bit) and storage o f the digitized signals for offline processing.
2.3. Testing procedures
Animals were maintained ad libitum on food and water under a 12 h - light/dark cycle (06:30 on), with all testing during the light cycle. Subjects were handled five minutes/day for seven days and habituated to the testing apparatus 10 minutes per day over three days prior to formal testing. After recovery from surgery, each subject was placed in the testing chamber and the EGG signal was recorded throughout testing.
Background white noise (50 dB) was presented continuously. Following 10 minutes of habituation, a series o f 5 auditory stimuli were presented (1000 Hz, 70 dB, 20 s duration,
90 s variable intertrial interval) through a ffee-field speaker (Pre-drug tones). Upon
34 completion o f the stimulus series, subjects were removed from the test chamber and a drug administration protocol was carried out. Upon completion o f the drug protocol, subjects were returned to the testing environment and allowed to rehabituate for 10 minutes. Another series o f 5 stimuli was then presented (Test tones).
2.4. Pharmacologic agents
Intracranial infusions o f the cholinergic agonist carbachol (Sigma, St. Louis;
0.1 |ig/0.5|il/hemisphere in physiologic saline) and the muscarinic antagonist atropine sulfate (Sigma, St. Louis; 10 pg/0.5 p 1/hemisphere in physiologic saline) were performed over a one minute injection period by a MEDEX 2000 infusion pump (Medex Inc.,
Hilliard, OH) connected to chronic guide cannulae by special infusion tubing (Plastics
One, Roanoke, VA). The BZ receptor partial inverse agonist FG 7142 (RBI
Biochemicals, Natick, MA; 8 mg/kg) was suspended and sonicated in 10% cremophor EL
(BASF, Ludwigshafen ,Germany) and administered intraperitoneally. The dose o f the BZ receptor ligand was chosen based on a previous dose response study (Quigley et al., 1994) and is within the reported range for induction o f putative anxiogenesis (Petersen et al.,
1982; Petersen & Jensen, 1984) while minimizing the risk of proconvulsant activity
(Stutzmann et al., 1987).
2.5. Histological Analvsis
Upon completion of testing, all subjects were deeply anesthetized and transcardially perfused with saline followed by 10% formalin. The brains were removed
35 and cryo-protected in 30% sucrose/phosphate buffer. Sections were then Nissl stained and cannula/infusion sites were mapped onto atlas plates o f Paxinos & Watson (1987). In order to estimate parenchymal spread o f drugs, unilateral cannulation o f the medial
prefrontal cortex, lateral prefrontal cortex and basolateral amygdala were carried out on three separate subjects. These subjects received a 0.5 pi injection o f 1% fluorogold, as
outlined above. Medial prefrontal, lateral prefrontal and basolateral amygdalar flourogold
infusions demonstrated an infusion area of less than one millimeter in diameter, with
some dorsal extension along the cannulation tract.
2.6. General data reduction and analvsis
The transient, stimulus evoked cardiac responses were analyzed for 5 s before
(baseline) and 15 s after stimulus onset. The digitized ECG was submitted to an R wave peak detector to derive R-R intervals. Movement or recording artifacts were identified and corrected with assistance of an automated algorithm (Bemtson et al., 1990), and beat
by beat and second by second heart periods were derived by custom software. Trials were removed from analysis if the prestimulus baseline was unstable (^ 15 ms change in baseline heart period, or a mean baseline heart period > 2 standard deviations from the mean for that subject condition), or if excess movement artifacts were apparent (<2% of trials were removed). In order to capture potentially bidirectional responses, integral areas under the acceleratory and deceleratory response functions were derived separately.
An overall index o f reactivity, or lability, was based on the arithmetic difference o f the integral area under the deceleratory and acceleratory components o f the response (lability
36 score = integral acceleration - integral deceleration). Effects o f drugs on phasic cardiac responses to the acoustic stimulus were derived as the difference in integral response areas after drug treatment (test tones), relative to pre-drug trials for each animal in each test session. In order to adjust for possible baseline differences in pre-drug response levels, drug effect data were submitted to repeated measures analysis of covariance
(ANCOVA) in which the pre-infusion reactivity scores served as the covariate. Dunnett’s post hoc analyses were used to test specific predictions of a priori hypotheses, while, for data for which there were no specific a priori hypotheses, the more conservative Tukey’s test was used.
37 CHAPTERS
EFFECTS OF INFUSIONS OF CARBACHOL
INTO THE MEDIAL PREFRONTAL CORTEX
ON CARDIOVASCULAR REACTIVITY TO AN ACOUSTIC PROBE STIMULUS
Administration of the benzodiazepine receptor partial inverse agonist FG 7142 enhances defensive-like cardiovascular reactivity to acoustic probe stimuli (Quigley et al.,
1994). FG is know to potently activate basal forebrain cortical cholinergic projections
(Moore et al., 1995), and previous work has shown that the response potentiating effect of
FG can be mimicked with intracerebroventricular (ICV) administrations o f the cholinergic agonist carbachol, and blocked with ICV administrations of the cholinergic antagonist atropine (Bemtson et al., 1996). These findings raise the possibility that the cardiovascular effects of FG are mediated by the release of cortical acetylcholine by the basal forebrain cholinergic system. Further support for this hypothesis comes from the finding that cholinergic specific lesions of the basal forebrain also block the cardioreactive enhancing effects o f FG 7142 (Bemtson et al., 1996). O f cortical areas, the medial prefrontal cortex has historically been implicated in anxiety, is innervated by basal forebrain cholinergic projections and has direct anatomical ties to central autonomic
38 regulatory nuclei. It was therefore hypothesized that the medial prefrontal cortex may be an important target site for the anxiogenic effect of FG. This initial study examined this possibility by testing the ability o f local infusions o f a cholinergic agonist to mimic the effect of FG.
3.1. Methods
3.1.1. Subjects
Subjects were 12 adult, male Sprague-Dawley rats (Zivic Miller, Zelinople,
PA) weighing 395±5 g (Mean±SE).
3.1.2. Testing Procedures
Testing followed the general procedures outlined above. The drug
administration protocol consisted of an intracranial infusion o f atropine (10 pg/ 0.5 pi/
hemisphere) or its vehicle followed by an intracranial infusion o f carbachol (0.1 pg/ O.Spl/
hemisphere) or its vehicle. Drug combinations o f vehicle-vehicle (W ), carbachol-
vehicle (CV), and carbachol-atropine (CA) were conducted within subjects, across three
successive days, in counterbalanced order.
3.1.3. Data Analvsis
Effects o f drug infusions on cardiac reactivity to the acoustic stimulus were investigated using a repeated measures analysis of covariance (ANCOVA: 3 drug
39 conditions) in which the pre-infusion responses served as the covariate. Ancillary analyses were based on a simple repeated measures analysis o f variance (ANOVA: 3 drug conditions). Tukey’s tests were used for post hoc analyses.
3.2. Results
3.2.1. Baseline Heart Period
Administration o f the cholinergically active compounds did not significantly affect baseline heart period measures (Table 3.1). Analysis o f variance revealed no effect of drug on either the test session baseline heart periods, nor on the change in baseline heart period from the pre-drug to test session. (Base: F(2,22)=2.152, NS; Change:
F(2,22)= 1.407, NS).
3.2.2. Cardiovascular reactivitv
Administration o f the cholinergic agonist carbachol into the medial prefrontal cortex yielded significant enhancement o f the cardioacceleratory response to the acoustic probe stimulus. This effect was blocked by the co-administration o f the muscarinic antagonist atropine (Table 3.1; Figure 3.2) Analysis o f variance confirmed significant stimulus-evoked cardiovascular responding, as subjects demonstrated a significant overall increase in the post-stimulus lability score (Figure 3.1: F( 1,11)=32.166, p<0.001).
Analysis of covariance demonstrated a significant main effect of drug on the change in magnitude of cardiac reactivity (Figure 3.2: F(2,21)=4.10, p<0.05). Tukey’s post hoc
40 analysis revealed that the carbachol-vehicle treatment significantly increased the stimulus-evoked cardiovascular response compared to the vehicle-vehicle condition.
Furthermore, co-administration o f the cholinergic antagonist atropine significantly blocked this effect. In order to further characterize this medial prefirontal cholinergic effect, separate analyses o f covariance were performed on the acceleratory and deceleratory components of the stimulus evoked responses. These analyses revealed that
infusion of carbachol significantly enhanced the acceleratory component of the response, while not significantly affecting the deceleratory component (Accel: F(2,21)=3.80, p
<0.05; Decel: F(2,21)=1.51, NS).
3.2.3. Histological analysis
As illustrated in Figure 3.3, all placements were confirmed to reside in the medial prefirontal cortex.
41 Integral Area Pre Infusion HP A Integral Area HP Pre Infbsion HP Post Infusion HP Lability Lability Baseline (ms) Baseline (ms)
Vehicle-Vehicle 68.7±15.8 -6.9±13.0 145.0±3.3 154.8±4.5 Carbachol-Vehicle 34.0±12.7 40.5±16.4* 142.0±3.3 151.24=2.9 Carbachol-Atropine 57.9±18.3 -24.5±17.4 144.6±3.1 147.7±4.5
Table 3.1; Cardiovascular effects of central carbachol/atroplne infusions in the medial prefrontal cortex. * p<0.05 ANCOVA by groups; Tukey’s post hoc p<0.05
6 Figure 3.1 Basal cardiovascular reactivitv for subjects receiving central carbachol/atropine infusions in the medial prefrontal cortex. Second by second stimulus- evoked heart period responses for pre-drug and test presentations under the vehicle- vehicle inftision condition in the medial prefrontal cortex.
43 2 Pre-Drug Tones Test Tones
0
U) E 2 T3 0 L. 0) £L ■c 3 -4 1
6
8
-10 -4 -2 0 2 4 6 a 10 12 14 Seconds
Figure 3.1 Figure 3.2 Changes in cardiovascular reactivitv following central infusions of cholinergically active drugs in the medial prefrontal cortex. Effects o f medial prefrontal carbachol/atropine infusions on the change in mean integral area under cardiovascular response functions from pre-drug to test tone presentations.
45 0) c 40 (0
% 30
0 5 CL 4^ •c ON s 1 Ë O) I -20 2 a: -30 I 8 -40 CL -50
Vehicle-Vehicle Carbachol-Vehicle Carbachol-Atropine
Figure 3.2 Figure 3.3 Location of guide cannula tips within the medial prefrontal cortex for subjects receiving central carbachol/atropine infusions.
47 +2.70
-u .ST 00 +3.20
Medial Prefrontal Cortex
Figure 3.3 3.3 Discussion
Administration o f the cholinergic agonist carbachol into the medial prefrontal, prelimbic area (MPF) resulted in a significant enhancement of the defensive-like cardioacceleratory response to a moderate intensity acoustic stimulus. This enhancement o f the cardiac response appears to be mediated by muscarinic receptors as pretreatment with atropine eliminated the carbachol effect.
These data are consistent with previous literature which has linked the medial prefrontal cortex to the modulation o f autonomic outflow. The medial prefrontal cortex possesses direct connections to autonomic regulatory centers such as the nucleus ambiguous (Buchanan et al., 1994; Hurley et al., 1991), dorsal motor nucleus o f the vagus
(Buchanan et al., 1994; Hurley et al., 1991), ventrolateral medulla (Buchanan et al., 1994;
Hurley et al., 1991) and the intermediolateral cell columns (Hurley et al., 1991).
Furthermore, cardiovascular changes have been elicited with stimulation o f the MPF
(Hardy & Holmes, 1988; Hardy & Mack, 1990), and MPF lesions have been demonstrated to alter baroreflex sensitivity (Verbeme et al., 1987). Additionally, the
MPF has been conceptualized as a ‘visceromotor cortex’ (Neafsey, 1990).
Augmentation o f cardiovascular responding by medial prefrontal muscarinic activation is also consistent with previous studies investigating central cholinergic mechanisms of cardiovascular reactivity. Bemtson et al. (1996) demonstrated enhanced cardiovascular responding with intraventricular administration o f carbachol, an effect that was blocked by pre-treatment with atropine. The present results are consistent with the
49 hypothesis that the medial prefrontal cortex is an important target site o f the basal
forebrain cholinergic projection in the modulation o f autonomic activity in behavioral contexts.
50 CHAPTER 4
EFFECTS OF INFUSIONS OF CARBACHOL INTO
THE LATERAL PREFRONTAL CORTEX OR BASOLATERAL AMYGDALA
ON CARDIOVASCULAR REACTIVITY TO AN ACOUSTIC PROBE STIMULUS
Infusions o f the cholinergic agonist carbachol into the medial prefrontal cortex produced an enhanced cardioacceleratory response to the acoustic probe stimulus. As the basal forebrain cholinergic system projects to widespread areas o f the cortex, it is not clear whether this represents a specific cholinergic involvement o f the medial prefrontal cortex, or of the cortex more generally. To examine this issue, a similar regimen o f drug infusions was tested in the lateral prefrontal cortex. Earlier results suggest that the basal forebrain cholinergic projection to the amygdala is not the critical pathway for the cardioreactive effects o f FG, as this pathway is largely spared by immunotoxic lesions that abolished FG effects. Nevertheless, because it has been historically implicated in anxiety and autonomic regulation, effects of cholinergic manipulations of the amygdala were also examined.
51 4.1 ■ Methods
4.1.a. Subjects
Subjects were 24 (12 per group) adult, male Sprague-Dawley rats (Zivic
Miller, Zelinople, PA) weighing 383±4 g (Mean±SE).
4.1 .b. Testing Procedures
Testing followed the general procedures outlined above. The drug administration protocol consisted o f an intracranial infusion o f atropine (10 pg/ 0.5 pi/ hemisphere) or its vehicle followed by an intracranial infusion o f carbachol (0.1 pg/ O.Spl/ hemisphere) or its vehicle. Drug combinations of vehicle-vehicle (W ), carbachol- vehicle (CV), and carbachol-atropine (CA) were conducted within subjects, across three successive days, in counterbalanced order.
4 .1.e. DataAnalvsis
Effects o f drug infusions on cardiovascular reactivity were investigated using repeated measures ANCOVAs (3 drug conditions) within each infusion site, in which the pre-infusion responses served as the covariate. Ancillary analyses were based on repeated measures analysis of variance (ANOVA 3 drug conditions). Tukey’s test v/as used for post hoc analyses.
52 4.2. Results
4.2 .a. Baseline Heart Period
Administration of the cholinergically active compounds did not significantly affect baseline heart period measures in either the basolaterai amygdala or lateral prefirontal cortex (Table 4.1). Analysis of variance revealed no main effect of drug on either the test session baseline heart periods, nor on the change in baseline heart period from the pre-drug to test session. (All Fs < 2.52, NS).
4.2.b. Cardiovascular reactivitv
Administration of the cholinergic agonist carbachol had no significant effects on evoked cardiovascular responding when infused into either the basolaterai amygdala or the lateral prefrontal cortex (Figure 4.2). Analysis of variance confirmed significant tone evoked cardiovascular responding, as subjects demonstrated a significant increase in the post-stimulus lability score in both the lateral prefirontal and basolaterai amygdala
(Figure 4.1: Fs>25.298, ps<0.00l). Repeated measures ANCOVAs demonstrated lack of significant drug infusion effects in both the lateral prefrontal and basolaterai amygdalar groups (Fs<0.440, NS).
4.2.C. Histological analvsis
As illustrated in Figure 4.3, all placements were confirmed to reside in the lateral prefrontal cortex or around that basolaterai amygdalar nucleus.
53 Integral Area Pre Infusion HP A Integral Area HP Pre Inftision HP Post Infusion HP Lability Lability Baseline (ms) Baseline (ms)
Lateral Prefrontal Cortex Vehicle-Vehicle 50.7±15.9 -30.6±21.6 142.0±2.7 145.7±3.0 Carbachol-V ehicle 43.0±10.4 -25.8±18.3 138.3±2.0 146.1^=1.9 Carbachoi-Atropine 47.4±16.1 -7.1±26.2 138.3±2.1 147.8±2.8
Basolaterai Amygdala Vehicle-Vehicle 112.7^=27.1 -37.9=bl4.8 147.8=t3.5 162.4±3.4 Carbachol-V ehicle 69.2±18.4 -15.4±12.7 146.0±3.8 156.7±3.0 Carbachoi-Atropine 80.9±17.1 -27.3±17.0 146.0±4.3 157.3±4.3
4^LA Table 4.1 : Cardiovascular effects of central carbachol/atropine infusions in the lateral prefrontal cortex or basolaterai amygdala. Figure 4.1 Basal cardiovascular reactivity for subjects receiving central carbachol/atropine infusions in the lateral prefrontal cortex or basolaterai amygdala.
Second by second stimulus-evoked heart period responses for pre-drug and test presentations under the vehicle-vehicle infusion condition in the lateral prefrontal cortex and basolaterai amygdala.
55 Lateral Prefrontal Cortex
— — Pre-Drug Tones Test Tones
0-2
-10
Seconds
Basolaterai Amygdala
2
& 3 - :
1-6
•8
-10 -4 2 0 2 4 6 8 10 12 14 Seconds
Figure 4.1
56 Figure 4.2 Changes in cardiovascular reactivitv following central infusions o f cholinergically active drugs in the lateral prefrontal cortex or basolaterai amygdala.
Effects of carbachol/atropine infusions in the lateral prefrontal cortex and basolaterai amygdala on the change in mean integral area under cardiovascular response functions from pre-drug to test tone presentations.
57 6 0 ] Vehicle-Vehicle 50 / / / ] Carbachol-Vehicle 0) g V////v\ Carbachoi-Atropine (0 4 0 £ O 3 0 Si 20 T3 Oc 10
■c Ln m "7771 7----7- ’’-r r j 777 00 c -20 2 ûl -3 0 o -4 0 o_ -50 Lateral Prefrontal Basolaterai Amygdalar Figure 4.2 Figure 4.3 Location o f guide cannula tips within the lateral prefrontal cortex or basolaterai amygdala for subjects receiving central carbachol/atropine infusions. 59 +2.20 0\ 3.30 o +2.70 3.14 +3.20 Lateral Prefrontal Cortex Basolaterai Amygdala Figure 4.3 4.3. Discussion Administration o f cholinergically active agents into either the lateral prefrontal cortex or the basolaterai amygdala failed to significantly alter basal chronotropic state or reactive cardiac response to the acoustic stimulus. Although this does not rule out a potential cholinergic contribution to autonomic regulation in these areas, it does serve to highlight the potentially important role of the medial prefrontal cortex in mediating behavioral autonomic links. The lack o f effect o f cholinergic agents applied to the basolaterai amygdala may be somewhat unexpected based on the literature. In addition to being the primary amygdalar target o f basal forebrain cholinergic projection neurons (Heimer & Alheid, 1991), the basolaterai nucleus o f the amygdala can influence autonomic outflow through connections with the central nucleus, which projects directly to brainstem autonomic nuclei (Danielsen et al., 1989). Additionally, excitatory amino acid (Gelsema et al., 1987; Maskati & Zbrozyna, 1989; Soltis et al., 1997) and electrical (Gelsema et al., 1987; Gelsema et al., 1989; Iwata et al., 1987; Maskati & Zbrozyna, 1989) stimulation o f the basolaterai and central amygdalar nuclei have been shown to induce cardiovascular responses. However, in relation to cholinergic activation, it is the central (Aslan et al., 1997; Ohta et al., 1991; Ozkutlu et al., 1995; Tellioglu et al., 1997) and not the basolaterai (Aslan et al., 1997) nucleus in which activation has been associated with cardiovascular modulation. These results are consistent with the fact that immunotoxic lesions of the basal forebrain cholinergic neurons, which largely spare the amygdalar 61 projection (Heckers et al., 1994), are able to block FG-potentiation o f the cardiac response (Bemtson et al., 1996). The lateral prefrontal cortex is considered by some authors to be part o f the insular cortex. The insular cortex projects to such autonomically important structures as the lateral hypothalamus (Yasui et al., 1991) and the nucleus of the solitary tract (Van der Kooy et al., 1984), and also receives a large amount o f afferent visceral information via the nucleus o f the solitary tract, parabrachial nucleus and visceral sensory thalamic nuclei (Cechetto, 1987). Furthermore, stimulation o f the insular cortex can evoke functional cardiovascular changes (Hardy & Holmes, 1988; Hardy & Mack, 1990; Oppenheimer & Cechetto, 1990; Verbeme & Owens, 1998). Although many of the anatomic connections to brainstem autonomic nuclei demonstrated for the insular cortex are caudal to the lateral prefrontal cortical area, present infusions were located in the rostral depressor area previously mapped for the insular cortex (Hardy & Holmes, 1988; Hardy & Mack, 1990). Whereas the insular receives significant visceral sensory input, it makes few direct connections with the brainstem autonomic stmctures. Conversely, the MPF receives little general visceral information, but does receive copious projections from limbic stmctures, and has clear ties to brainstem autonomic nuclei. Functionally this may implicate the LPF in more of a visceral sensory role, and the MPF in the emotional modulation of cardiovascular function (Verbeme & Owens, 1998), a topic which shall be returned to later. The present results support a functional differentiation between the medial and lateral prefrontal cortex. 62 CHAPTERS EFFECTS OF INFUSIONS OF ATROPINE INTO THE MEDIAL PREFRONTAL CORTEX, LATERAL PREFRONTAL CORTEX, OR BASOLATERAL AMYGDALA ON FG 7142 ENHANCEMENT OF CARDIOVASCULAR REACTIVITY TO AN ACOUSTIC PROBE STIMULUS Enhancement of cardiovascular reactivity by the benzodiazepine receptor partial inverse agonist FG 7142 has been shown to be dependent on a basal forebrain cholinergic mechanism (Bemtson et al., 1996). Based on earlier findings, it was speculated that the medial prefrontal cortex may be a particularly important target site o f the basal forebrain cholinergic system in the cardiovascular effects o f FG. The prior studies supported this hypothesis: specifically infusion of a cholinergic agonist into the medial prefrontal cortex, but not other central sites, mimicked the response-potentiating effects of FG. Based on these findings, it was hypothesized that atropine infusions into the medial prefrontal cortex, but not the lateral prefrontal cortex or basolateral amygdala, would block the effects of FG on the cardioacceleratory response. The present study tested this hypothesis. 63 5.1. Methods 5.1.1. Subjects Subjects were 36 (12 per group) adult, male Sprague-Dawley rats (Zivic Miller, Zelinople, PA) weighing 357±5 g (Mean±SE). 5.1.2. Testing Procedures Testing followed the general procedures outlined above. The drug administration protocol consisted o f an intraperitoneal injection o f FG 7142 (8 mg/kg) or its vehicle. To allow the drug to come to full effect, subjects were returned to their home cage for 20 minutes. After this, atropine (10 pg/0.5 pl/hemisphere) or its vehicle was administered intracranially. Completion of the drug combinations o f vehicle-vehicle (W ); FG 7142-vehicle (FV); vehicle-atropine (VA) and FG 7142-atropine (FA) were conducted within subjects, counterbalanced across four successive days. 5.1.3. Data Analvsis Data analysis was based on the a priori hypothesis that administration o f the cholinergic antagonist atropine into the medial prefrontal cortex would attenuate FG induced enhancement of cardiovascular reactivity, relative to the central control sites (lateral prefrontal cortex and basolateral amygdala). The response-potentiating effects o f FG administration on phasic cardiovascular responses to the acoustic stimulus were analyzed by an overall mixed between/within analysis of covariance (ANCOVA: 3 64 anatomie sites x 2 drug conditions) on the vehicle-vehicle and FG-vehicle drug conditions. The a priori specific hypothesis that medial prefrontal infusion o f atropine would attenuate the FG induced enhancement of cardioreactivity was tested by repeated measures ANCOVAs on the FG-vehicle and FG-atropine conditions for each infusion site. Baseline data were analyzed using mixed between/within ANOVAs (3 anatomic sites X 2 FG X 2 atropine). Tukey’s test was used for post hoc investigation. 5.2. Results 5.2.1. Baseline Heart Period Consistent with previous studies, administration o f the benzodiazepine receptor partial inverse agonist FG 7142 evoked a modest baseline bradycardia in all conditions. This effect was not altered by central administration of the cholinergic antagonist atropine at any of the anatomic infusion sites (Table 5.1). A significant, but moderate, bradycardic effect of FG administration on baseline heart period during the test sessions, and on the pre-drug to test session change in baseline heart period were demonstrated (Base: F(l,33)=26.560, p<0.001; Change: F(l,33)=26.006, p<0.001). However, there were no significant main effects of atropine administration or anatomic infusion site, nor did these factors have significant interactions on baseline heart period (all Fs<1.258, NS). 65 5.2.2. Cardiovascular Reactivity The benzodiazepine receptor partial inverse agonist FG 7142 significantly enhanced the cardioaccelertory response to the acoustic stimulus, and this effect was significantly attenuated by administration o f the muscarinic antagonist atropine specifically into the medial prefrontal cortex (Figure 5.2). Analysis of variance confirmed significant tone evoked cardiovascular responding as subjects demonstrated a significant increase in the post-stimulus lability score (Figure 5.1 : F(l,33)=53.711, p<0.001). The ability of FG to enhance cardioacceleratory responses was confirmed by a mixed between/within analysis of covariance across anatomic group on vehicle-vehicle and FG-vehicle conditions (Figure 5.2: F(l,32)=22.870, p<0.001). This effect on reactivity did not differ across anatomic group, nor did group and drug factors show significant interactions (Fs<0.74, NS). Tukey’s post hoc analysis confirmed significantly greater changes in the evoked lability scores in the FG-vehicle condition when compared to the vehicle-vehicle condition in all anatomic groups. Separate ANCOVAs on acceleratory and deceleratory changes in evoked cardiovascular responses revealed significant FG mediated enhancement in cardioacceleration, as well a significant decrement in cardiodeceleration (Accel: F(l,32)= 16.71, p<0.001; Decel: F(l,32)=5.04, p<0.05). The a priori hypothesis that medial prefrontal atropine would attenuate the cardioreactive effects o f FG was confirmed with repeated measures analyses o f covariance within each anatomic group (Figure 5.2: MPF: F(l,10)=4.87, p=0.05; LPF: F(l,10)=0.08, NS; BLA: F(l,10)=1.71, NS). 6 6 5.2.3. Histological analvsis As illustrated in Figure 5.3, all placements were confirmed to reside in the medial prefrontal cortex, the lateral prefrontal cortex or in the vicinity o f the basolateral amygdalar nucleus. 67 Integral Area Pre Infusion HP A Integral Area HP Pre Infusion HP Post Infusion HP Lability Lability Baseline (ms) Baseline (ms) Medial Prefrontal Cortex Vehicle-Vehicle 24.8±10.6 -5.1±13.7 149.4±3.5 152.2±4.4 Vehicle-Atropine 33.8±9.3 -23.9±14.6 144.0±3.8 150.2±3.9 FG 7142-Vehicle 22.2±9.3 28.4±13.6“ 146.9±5.2 173.2±6.0* FG 7142-Atropine 18.2±12.2 5.7±15.4 139.4±3.3 172.8±5.3’ Lateral Prefrontal Cortex Vehicle-Vehicle 45.4±8.2 -6.0±16.5 142.8±3.1 148.8±3.6 Vehicle-Atropine 42.3±18.8 -20.4±28.1 140.7±4.1 144.4±3.9 FG 7142-Vehicle 31.5±11.0 32.0±9.4* 143.5±3.7 169.2±4.5’ FG 7142-Atropine 20.8±8.6 40.2±14.2* 145.8±3.3 167.8±4.4’ 0\ 00 Basolateral Amygdala Vehicle-Vehicle 31.5±5.0 -10.8±12.2 148.5±4.3 154.9±3.7 V ehicle-Atropine 29.4±10.0 -9.9±17.8 148.0±3.2 152.6±3.3 FG 7142-Vehicle 27.6±13.2 42.2±15.r 147.5±3.3 173.0±6.4’ FG 7142-Atropine 29.6±14.3 20.5±26.5* 146.0±3.5 168.6±6.0* Table 5.1 : Cardiovascular effect of systemic FG 7142/central atropine infusions: * p<0.01 overall ANOVA; Tukey’s post hoc p<0.05. * p<0.001 ANCOVA of all groups; Tukey’s post hoc p<0.05. ** p<0.05 ANCOVA by group; FG- Vehicle vs FG-Atropine. Figure 5.1 Basal cardiovascular reactivity for subjects receiving systemic FG 7142 and central atropine infusions. Second by second stimulus-evoked heart period responses for pre-drug and test presentations under the vehicle-vehicle infusion condition in medial prefrontal cortex, lateral prefrontal cortex, and basolateral amygdala. 69 Medial Prefrontal Cortex Pre-Drug Tones Test Tones X -6 -10 Seconds Lateral Prefrontal Cortex 0 - 2 -10 Seconds Basolateral Amygdala X - 6 -10 •4 2 0 2 4 6 8 10 12 14 Seconds Figure 5.1 70 Figure 5.2 Changes in cardiovascular reactivity following systemic administration o f FG 7142 and central infusions of atropine. Effects o f systemic FG 7142 administration and medial prefrontal, lateral prefrontal, and basolateral amygdalar atropine infusions on the change in mean integral area under cardiovascular response functions from pre-drug to test tone presentations. * FG potentiation o f cardiovascular reactivity, p < 0.001; ** atropoine attenuation of FG potentiation, p < 0.05. 7 1 6 0 50 0> O) c CO 40 7 ^ JC Ü 30 TV (0 20 ** 1 10 ■ c (0 0) X I t ro m -10 c -20 1 I Vehicle, Vehicle 2 r T t ! I] Vehicle, Atropine o_ -30 I ] FG 7142, Vehicle (0 f -40 ] FG 7142, Atropine -50 Medial Prefrontal Lateral Prefrontal Basolateral Amygdalar Figure 5.2 Figure 5.3 Location o f guide cannula tips within the medial prefrontal cortex, lateral prefrontal cortex, or basolateral amygdala for subjects receiving systemic FG 7142 and central atropine infusions. 73 +2.20 +2.20 +2.70 +2.70 3.30 -1 +3.20 +3.20' 3.14 Medial Prefrontal Cortex Lateral Prefrontal Cortex Basolateral Amygdala Figure 5.3 5.3. Discussion The enhanced cardioacceleratory response after FG 7142 is consistent with the putative anxiogenic profile o f this BZR partial inverse agonist, as aversive contextual changes can impart acceleratory shifts in cardiovascular responses to moderate acoustic stimuli (Hart et al., 1998; Saiers et al., 1990). This finding is consistent with previous reports on the effects o f FG on cardiovascular responses (Bemtson et al., 1996; Hart et al., 1998; Quigley et al., 1994) The new aspect o f the present results is that FG-induced potentiation o f the cardiac response can be attenuated by medial prefrontal atropine infusions. It is unlikely that the modest effects of FG on baseline heart period could account for the enhanced cardioreactivity seen with this agent. Previous studies have shown that the response potentiating effects of FG are specific to the cardiac defensive response, while the cardioacceleratory response to a startle stimulus is unaffected (Hart et al., 1998) or diminished (Bemtson et al., 1997) by FG administration. Moreover, these differential effects were observed concurrently with FG associated baseline bradycardia. Consequently, potentiation o f the cardioacceleratory response is not a simple consequence o f baseline effects. Moreover, the response potentiating effects o f FG in the present study were blocked by atropine, which did not alter the baseline bradycardia. This finding suggests that effects o f FG on basal and reactive heart period were mediated by separate mechanisms. One potential mechanism for the FG effects on baseline heart period is suggested by the report o f GABAergic attenuation o f vagal tone at the level of 75 the hypothalamus (DiMicco, 1987). Likely related to actions at GAB A receptors, BZR agonists have been shown to decrease vagal tone and vagal control o f the heart (Adinoff et al., 1992; Conahan & Vogel, 1986). The finding that the baseline effects o f FG were not altered by MPF atropine is consistent with the possibility o f a hypothalamic or other site of action in this effect. The administration o f the muscarinic antagonist, atropine, into the medial prefrontal cortex, but not the basolateral amygdala or the lateral prefrontal cortex, attenuated the cardioacceleratory enhancing effects o f FG. This data is consistent with previous studies in which intracerebroventricular atropine and cholinergic specific lesions o f the basal forebrain attenuated the effects of FG (Bemtson et al., 1996). Furthermore, the lack of effect o f atropine administration into the basolateral amygdala is consistent with the fact that immimotoxic lesions o f the basal forebrain cholinergic neurons, which block FG-potentiation, largely spare the amygdalar projection (Heckers et al., 1994). These data offer further support o f the hypothesis that the medial prefrontal cortex is an important target site o f the basal forebrain cholinergic projection in the modulation of autonomic activity in anxiogenic contexts. 76 CHAPTER 6 EFFECTS OF CHOLINERGIC SPECIFIC MEDIAL PREFRONTAL IMMUNOTOXIC LESIONS ON FG 7142 ENHANCEMENT OF CARDIOVASCULAR REACTIVITY TO AN ACOUSTIC PROBE STIMULUS Previous studies demonstrated that cholinergic activation in the medial prefrontal cortex, but not other sites, enhanced the cardiac defensive response. Moreover, muscarinic blockade in the medial prefrontal cortex, but not other sites, attenuated the response-potentiating effects of FG 7142. These results, together with prior studies, suggest that the medial prefrontal cortex may be an important target site o f the basal forebrain cholinergic system in mediating the cardioreactive effects of FG. This was further tested by selective cholinergic lesions of the medial prefrontal cortex. It was hypothesized that immimotoxic lesions of cholinergic projections to the medial prefrontal cortex would block the FG-induced enhancement of cardiovascular reactivity. 77 6.1 ■ Methods 6.1.1. Subjects Subjects were 24 (12 per group) adult male Sprague-Dawley rats (Zivic Miller, Zelinople, PA) weighing 361±3 g (Mean±SE). 6.1.2. Lesioning procedure MPF Infusions of the cholinergic specific immunotoxin 192 IgG-saporin (0.01 pg/pl/hemisphere) or its vehicle were made 11 days prior to testing (n=12 per group). 6.1.3. Testing Procedures Testing followed the general procedures outlined above. The drug administration protocol consisted of administration o f FG 7142 (8 mg/kg, i.p.) or its vehicle to both sham and lesioned animals. Following drug administration, subjects were returned to their home cage for 20 minutes prior to being returned to the test chamber. Administration of FG 7142 (FG) or vehicle (V) was conducted within both sham and lesioned groups, counterbalanced across two successive days. 6.1.4. Data Analvsis Effects of lesions on FG-potentiation o f cardiovascular reactivity were investigated using a mixed between/within analysis of covariance (ANCOVA; 2 group [lesion vs sham] x 2 drug [FG vs vehicle]) in which the pre-infusion stimulus-evoked 78 lability scores served as the covariate. A Duiinett’s post hoc was conducted on drug effect data (Sham FG responding > all other lesion/drug conditions). Baseline data were analyzed using mixed between/within ANOVAs (2 group [lesion vs sham] x 2 drug [FG vs vehicle]). Tukey’s test was used for post hoc investigation. 6.2. Results 6.2.1. Baseline Heart Period Cholinergic specific immunotoxic lesions of the medial prefrontal cortex produced a slight, but non-significant tachycardie effect on baseline heart period, and a weak attenuation o f FG induced bradycardia (Table 6.1 ). Pre-drug heart period baselines did not significantly differ between lesion groups (F( 1,22)=2.236, NS). Consistent with previous findings, analysis of variance showed a significant main effect of FG administration on baseline heart period in the test session, as well as pre- to post-drug change in heart period (Base: F(l,22)=28.030, p<0.001; Change: F(l,22)=36.827, p<0.001). This bradycardic effect of FG was weakly attenuated in the lesioned subjects, as evidenced by smaller pre- to post-drug change scores (Base: F(l,22)=2.502, NS; Change: F(l,22)=6.769, p<0.05). 6.2.2. Cardiovascular reactivitv Cholinergic specific immimotoxic lesions of the medial prefrontal cortex blocked enhancement o f cardiovascular reactivity by the BZ receptor partial inverse agonist FG 7142 (Figure 6.2). Analysis of variance confirmed significant stimulus-evoked 79 cardiovascular responding, as subjects demonstrated a significant increase in the post stimulus lability score (Figure 6.1: F(l,22)=57.398, p<0.001). Analysis of covariance revealed a significant group by drug interaction reflecting the selective attenuation of the effects o f FG in lesioned animals (Table 6.1, Figure 6.2: F(l,21)=6.03, p<0.05). No significant main effects of FG or of lesion were found (Fs<2.94, NS) and there was no significant main effect o f lesion condition were apparent in pre-drug lability scores (F(l,22)=0.028, NS). Dunnett’s post hoc analysis confirmed a significant enhancement of stimulus induced cardiovascular responding by FG in sham, but not cholinergically lesioned animals. Separate analyses of covariance on acceleratory and deceleratory components o f the cardiovascular responses revealed that the cholinergic lesions significantly diminished the cardioacceleratory effect of FG (F(l,21)=5.82, p<0.05), without significantly altering the deceleratory components (F(l,21)=3.33, NS). 6.2.3. Histological analvsis As illustrated in Figure 6.3, all infusion sites were confirmed to reside in the medial prefrontal cortex. 80 Integral Area Pre Infusion HP A Integral Area HP Pre Infusion HP Post Infusion HP Lability Lability Baseline (ms) Baseline (ms) Sham Vehicle 78.2±10.5 -27.8±13.0 151.1±2.8 156.6±3.1 FG7142 60.0±11.7 26.3±10.7* 146.5±2.2 183.0±4.6» Lesion Vehicle 61.9±16.3 -1.7±17.2 141.7±3.1 154.5±2.4 FG7142 84.3±29.1 -22.7±19.8 143.5±4.3 168.8±5.3^ Table 6.1 : Cardiovascular effects of medial prefrontal cholinergic lesions. * p<0.05 ANOVA of all groups, FG effect. 00 * p<0.05 ANCOVA of all groups; Dunnett’s post hoc; Sham-FG > Lesion-FG Figure 6.1 Basal cardiovascular reactivity for subjects with cholinergic specific immunotoxic or sham lesions of the medial prefrontal cortex. Second by second stimulus-evoked heart period responses for pre-drug and test presentations under the vehicle condition in animals with 192 IgG-saporin and sham lesions o f the medial prefrontal cortex. 82 Sham Lesion ■ Pre-Drug Tones / \ 0 • \\l \ - i •a \l o "C \\ 0) a. - •c V 00 3 w X V ^ < - -6 - < -8 -10 -10 : 1 I l l ' l l 1:1 -4 -2 0 2 4 6 8 10 12 14 0 4 6 8 10 12 14 Seconds Seconds Figure 6.1 Figure 6.2 Changes in cardiovascular reactivitv following systemic administration o f FG 7142 in animals with cholinergically specific immunotoxic or sham lesions o f the medial prefrontal cortex. Effects of systemic FG 7142 administration on the change in mean integral area under cardiovascular response functions from pre-drug to test tone presentations in animals with 192 IgG-saporin and sham lesions of the medial prefrontal cortex. 8 4 S8 Post - Pre Integral Heart Period Lability Change cn • k CO N3 rsj CO 05 o o o o o o o Ê g O —r CO 3" Q) 3 (Q C (D 05 N5 $ O 3 X' X ■n < G) ® ^ Ô ^ CD N5 Figure 6.3 Location o f 192 IgG-saporin and vehicle infusion sites within the medial prefrontal cortex. 86 +2.20 +2.20 •• +2.70 +2.70 +3.20 +3.20 Sham 192 IgG-Saporin Figure 6.3 6.3. Discussion Injection o f the cholinergic specific immunotoxin 192 IgG-saporin into the medial prefrontal cortex eliminated the heightened cardiovascular reactivity seen with administration o f the benzodiazepine receptor p artial inverse agonist FG 7142. Similar infusion of this toxin have been shown to specifically reduce cortical acetylcholinesterase-positive fibers by approximately 40% (Holley et al., 1994). The cardiovascular effects o f lesioning appeared to be specific to drug antagonism, as baseline heart period and basal measures o f cardiovascular reactivity were not significantly affected. Althogh lesions did also weakly attenuate the FG effects on baseline heart period, it is unlikely that this accounts for the changes seen in cardiovascular reactivity. Medial prefi-ontal administration of the cholinergic agonist carbachol had no impact on baseline heart period and similiar administration of the cholinergic antagonist atropine did not alter FG’s mild bradycardic effects. The blockade o f FG enhanced cardiovascular reactivity by medial prefrontal cholinergic lesions is consistent with similar atropine induced attenuation in this area. Furthermore, as 192 IgG-saporin is a specific toxin for basal forebrain cholinergic projection neurons (Wiley et al., 1991), this data is also consistent with previous studies which demonstrated lesions o f the basal forebrain cortical cholinergic projection system blocked FG cardioreactive effects (Bemtson et al., 1996). This data is also in keeping with the demonstrated stimulation o f cortical acetylcholine efflux by FG administration (Moore et al., 1995) and, consistent with the putative anxiogenic nature o f FG 88 administration, by stressors such as novelty, conditioned fear, and restraint (Acquas et al., 1996; Mark et al., 1996). These data offer further support of the hypothesis that the medial prefrontal cortex is an important target site of the basal forebrain cholinergic projection in the modulation o f autonomic activity in behavioral contexts, and specifically in anxiety and benzodiazepine receptor ligand interactions. 8 9 CHAPTER? GENERAL DISCUSSION Consistent with its putative anxiogenic effects, the benzodiazepine receptor partial inverse agonist FG 7142 has been shown previously to heighten defensive-like cardiovascular reactivity to moderate-intensity, non-signal auditory stimuli (Quigley et al., 1994). Previous studies have implicated a basal forebrain cholinergic mechanism in this FG-induced response potentiation as this effect is: 1) mimicked by intracerebroventricular carbachol; 2) blocked by ICV atropine; and 3) eliminated by cholinergic specific lesions of the basal forebrain-cortical cholinergic system (Bemtson et al., 1996). The present studies indicate that the medial prefrontal cortex is a critical basal forebrain-cortical cholinergic target site in the mediation of FG-enhanced cardiovascular reactivity. Local micro infusions o f the cholinergic agonist carbachol in the medial prefrontal cortex, but not in the lateral prefrontal cortex or basolateral amygdala, mimicked the cardioacceleratory enhancement seen with systemic FG administration. This effect was blocked by the local co-administration of the muscarinic antagonist atropine. Similarly, micro infusions of atropine into the medial prefrontal, but not lateral prefrontal cortex or basolateral amygdala, significantly attenuated the heightened cardiovascular reactivity induced by systemic administration of FG. Additionally, 90 cholinergic specific immunotoxic lesions of the medial prefrontal cortex prevented FG- induced cardiovascular response potentiation. These data are consistent with the postulated role o f the medial prefrontal cortex in emotional modulation of the cardiovascular system (Verbeme & Owens, 1998). Medial prefrontal lesions have been demonstrated to alter behavioral measures o f anxiety (Bums et al., 1996; Frysztak & Neafsey, 1991; Morgan et al., 1993; Morgan & LeDoux, 1995) and anxiogenic contexts have been shown to enhance activity in the MPF (Acquas et al., 1996; Mark et al., 1996; Feenstra et al., 1995; Nevo et al., 1996). The MPF has direct anatomie projections to such brainstem autonomie output structures as the nucleus ambiguous (Buchanan et al., 1994; Hurley et al., 1991), dorsal motor nucleus of the vagus (Buchanan et al., 1994; Hurley et al., 1991), and ventrolateral medulla (Buchanan et al., 1994; Hurley et al., 1991). Moreover, previous studies have demonstrated a functional regulation o f cardiovascular measures by MPF manipulations (Bacon & Smith, 1993; Hardy & Holmes, 1988; Hardy & Mack, 1990; Hurley-Guis & Neafsey, 1986; Neafsey, 1990; Powell et al., 1994; Verbeme et al., 1987). This convergence of evidence for cardiovascular regulation and anxiety modulation by the medial prefrontal cortex makes this stmcture a logical target site of basal forebrain cholinergic system in mediating links between anxiety and autonomic regulation. This hypothesis supported by the present studies. Negative results with cholinergic manipulations o f the LPF support a relatively specific role for the MPF. In contrast to the medial prefrontal cortex, there is little evidence to implicate the lateral prefrontal cortex in emotional modulation o f 91 cardiovascular function. Although stimulation studies have evoked cardiovascular responses from the LPF, this cortical area does not have substantial projections to brainstem autonomic mechanisms. Furthermore, the LPF has mostly been implicated in gustatory responses and has been conceptualized as a visceral sensory cortex (Neafsey, 1990; Verbeme & Owens, 1998). Although the negative findings in the present studies can not be taken as evidence o f complete lack o f involvement o f the LPF in cardiovascular reactivity, one can infer that FG-induced response potentiation is not due to general cortical activation by the basal forebrain-cortical cholinergic system. As outlined in the introduction, considerable data implicate the amygdala in fear, anxiety, and autonomic regulation. The lack o f significant effect of micro infusions of cholinergically active agents into the basolateral amygdala in the present studies does not challenge the previous findings. The current findings do suggest that basal forebrain cholinergic projections to the amygdala are not critical for FG-potentiation o f the cardiac defensive response. This is consistent with previous findings that cholinergic specific lesions o f the basal forebrain, which largely spare the amygdalar projection, are effective in eliminating the response-potentiating effects of FG (Bemtson et al., 1996; Heckers et al., 1994). Cholinergic neurotransmission in the basolateral amygdala has been especially implicated in the enhancement o f measures of memory (Mallet et al., 1995; Ohno et al., 1992; Oiino et al., 1993) and learning (Dumery & Blozovski, 1987), and emotional modulation o f memory storage (McGaugh et al., 1996; Sarter & Markowitsch, 1985). It remains possible that the amygdala may serve as an important descending way-station in the actions o f the MPF. 92 The present findings have important implications for the nature o f neural systems and processes underlying anxiety and its links with autonomic function. It has been postulated that cortical acetylcholine functions to facilitate stimulus detection and processing (Sarter & Bruno, 1997). Congruous with this, activation o f the basal forebrain-cortical cholinergic system enhances attentional performance (Sarter & Bruno, 1997). Furthermore, benzodiazepine receptor agonists and inverse agonist have been shown to bidirectionally modulate attentional performance by their effects on cortical acetylcholine release (Holley et al., 1997). Based on these and other findings, Bemtson et al. (1998) suggest that FG may exert its anxiogenic effects and potentiate cardiovascular responding by augmenting basal forebrain cholinergic activity and thus the cortical processing of fear related stimuli. This model is consistent with cognitive theories of anxiety (Eysenck, 1991) and may represent an important substrate for “top-down” cognitive activation o f circuits related to anxiety and autonomic regulation. This top- down processing is particularly salient in anxiety disorders, and the BZR agonists may exert their anxiolytic effects by attenuating basal forebrain-cortical cholinergic activity and diminishing associated cortical processing. This cognitive/attentional model is consistent with the report of enhanced cardiovascular reactivity to an intense auditory stimulus during externally focussed attentional tasks (Vila et al., 1997) and with reports of enhanced attention to aversive stimuli in patients with pathological anxiety (Ohman, 1996; Rosen & Schulkin, 1998). According to this cognitive model, the effect o f the basal forebrain-cortical cholinergic system would be expected to enhance anxiety and autonomic reactivity to the 93 extent to which relevant stimuli foster cortical processing. In this regard, FG does not enhance cardioacceleratory responses to all stimuli generally. Neither the cardioacceleratory startle responses nor the potentiation o f startle by a conditioned fear stimulus are enhanced with FG (Hart et al., 1998). This is consistent with the lack o f cortical processing required for startle responses. Indeed, the basic startle circuit is organized at a brainstem level (Davis et al., 1982), and even fear-potentiated startle appears to be mediated at the level o f the amygdala (Davis et al., 1993). In contrast, the novel stimulus employed in the present studies does not have inherent conditioned signal value, but rather would be expected to elicit attentional/evaluative processing for its potential adaptive significance. These considerations may account for the fact the F G potentiates the defensive-like response to the acoustic probe, but not to the startle stimuli. Although the amygdala may play a critical role in the processing of some types o f fear stimuli that do not require extensive cortical processing, such as conditioned aversive stimuli, the present findings suggest that the basal forebrain-medial prefrontal cholinergic system may play a more pivotal role in anxiety and autonomic reactivity for those stimuli and contexts that entail cortical/cognitive processing. 94 APPENDIX LIST OF ABBREVIATIONS 5-HT 5 -Hy droxytryptamine ACTH Adrenocorticotropic Hormone ANS Autonomic Nervous System AP Anterior-Posterior BFCC Basal Forebrain-Gortical Gholinergic System BLA Basolateral Nucleus of the Amygdala BNST Bed Nucleus o f the Stria Terminalis BZ Benzodiazepine BZR Benzodiazepine Receptor CeA Gentral Nucleus of the Amygdala GI Ghloride ion GS Conditioned Stimulus dB Decibel DV Dorsal-Ventral EGG Electrocardiogram FG FG 7142 GABA Y-amino-butyric-acid GABAa y-amino-butyric-acid receptor type A IGV Intracerebroventricular LPF Lateral Prefrontal Cortex MD Medial Dorsal Nucleus of the Thalamus MeA Medial Nucleus of the Amygdala ML Medial-Lateral MPF Medial Prefrontal Cortex NB/G4 Nucleus Basalis Gholinergic Projection System NB Nucleus Basalis NTS Nucleus Tractus Solitarius SI Substantia Innominata US Unconditioned Stimulus 95 LIST OF REFERENCES Abe. 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