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THE EFFECTS OF SPECIFIC OPJATE RECEPTOR ANTAGONISTS ON THE
HABITUATION OF NOVELTYdNDUCED ANALGESIA
EMMA S. SPREEKMEESTER
Department of Psychiatry,
McGill University, Montreal
March 1997
A thesis submitted to the Faculty of Graduate Studies and Research in partial
fulfilment of the requirements of the degree of Master of Science
O Emma S. Spreekmeester, 1997
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ABSTRACT
Animals exposed to nociceptive stimulation for the first time display a
novelty induced hypoalgesia (NIH) that habituates with repeated exposure to
the same stimuli. The non-selective opiate receptor antagonist naloxone, has
been shown to attenuate this habituation. Antagonists selective for the p, a and
K receptors were used in order to elucidate the opiate receptor subtype that is
involved in this effect. Animals were exposed to a hot-plate apparatus set at
48.5OC once per day, for 8 days. The latency to lick the hind paw was used as
an index of pain sensitivity. CTOP (p), naltrindole (a), nor-binaltorphimine (r )
(0.5, 1 .O or 2.0 nM), or vehicle were injected ICV 30 min. prior to each plate
exposure. Only CTOP (1.0; 2.0 nM) and naltrindole (2.0 nM) prevented the
habituation of NIH. These results suggest that the specicific receptor subtypes p
and 3, are involved in the habituation of NIH.
On observe chez les animaux qui sont exposés à des stimulations
nociceptives pour la premiere fois une hypoalgésie induite par la nouveauté
(HIN) qui s'estompe aprés plusieurs r6pétitions du même stimuli (habituation).
II a été rapporte que le naloxone, un antagoniste des récepteurs des opiacés,
atténue cette habituation. Des antagonistes sélectifs pour chaques types de
récepteur aux opiacés (p, S et k) ont été utilisés dans le but de déterminer
lequel serait implique dans cette atténuation. Les animaux ont été exposés à
une plaque chauffante (48,5OC) une fois par jour pendant 8 jours. Le temps
requis pour que I'animal se liche les pattes arrières a été utilisé comme un
indice de la sensibilité à la douleur. Le CTOP (p), le naltrindole (6)' le nor-
binaltorphimine (k) (0.5, 1 .O ou 2.0 nM) ou le véhicule a été injecté ICV 30 min.
avant l'exposition de l'animal à la plaque chauffante. Seulement le CTOP (1.0
et 2.0 nM) et le naltrindole (2.0 nM) ont eu un effet préventif sur l'habituation de
l'animal à I'HIN. Ces résultats suggèrent que les recepteurs aux opiacés de
type p et 6 sont impliques dans les processus d'habituation à I'HIN.
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincerest gratitude to my
supervisor, Dr. Joe Rochford. Despite a busy schedule, he has upheld his role
as teacher and advisor, and rnanaged to pull things through when the going got
tough.
1 would also like to thank my colleagues and friends, Isabelle Rousse,
Nicky Richardson, Beth Tannenbaum, Dan Auld, lan Gilron, Andrea Jakob and
Adam Mar to mention a few of the many at the Douglas Hospital Research
Center, al1 of whom make going to work not only stimulating but fun. Wayne
Rowe and François Pomerleau also desewe mentioning as they always seem
to be there with help when it's needed.
I would also like to thank my family for always being there, for supporting
me and for beleiving in me. They may not think that they contributed, but
without them I wouldn't have been able to begin.
Some special friends, Tanya, Jeanne and Genevieve deserve
mention ing for their support and encouragement.
I would also like to thank Chris for his cornfort, reassurance and
motivation.
This work is dedicated to Mrs. C.
This work funded by the Natural Sciences & Engineering Research Council of Canada.
TABLE OF CONTENTS
EUCE
.................................................................................................. ABSTRACT l
RÉSUMÉ ...................................................................................................... II
.......................................................................... ACKNOWLEDGEMENTS Ill
............................................................................. TABLE OF CONTENTS IV
LIST OF FIGURES ................................................................................... VI
Problems with Specificity and Pattern Theories ....................... 2
The Physiological Assumption ......................................... 2
The Anatom ical Assumption ............................................. 2
The Psychological Assumption ........................................ 5
The Gate-Control Theory ............................................................... 7
Endogenous Pain Cont rol System .............................................. 8
Biochemistry of Antinociception:
Opiate and nonopiate mechanisms ............................................ 9
Opiate Receptors ........................................................................ O
.............................................................................................. Opiates 11
Enkephalins ......................................................................... 11
Endorphins ........................................................................... 11
Dynorphins ........................................................................... 12
Non-opiate antinociceptive systems ........................................... 2
Activation of EPCS ......................................................................... 3
......................................................................... Stress-induced analgesia 1 3
........................ Further evidence for plasticity within the EPCS 17
METHOD ...................................................................................................... 21
Experiment 1 .................................................................................... 21
Experiment 2 .................................................................................... 24
Experiment 3 .................................................................................... 25
RESULTS ................................................................................................... 27
Experiment 1 .................................................................................... 27
Experiment 2 .................................................................................... 30
Experiment 3 .................................................................................... 30
DISCUSSION .............................................................................................. 36
REFERENCES ............................................................................................ 49
LIST OF FIGURES
PAGE
................................................................................................. FIGURE 1 28
Mean (& SEM) paw lick latencies (s) of animals
administered 0.5nM (n=8), 1 .OnM (n=7), 2.OnM (n=9)
CTOP or vehicle (n=8) throughout al1 eight days of hot-plate
exposure.
FIGURE 2 ................................................................................................. 29
Mean (* SEM) area under the curve (AUC) for anirnals
administered CTOP or vehicle, calculated over al1 eight
days of hot-plate exposu re.
FIGURE 3 ...................................................................................... 3 1
Mean (i SEM) paw lick latencies (s) for animals
administered 0.5nM (n=7), 1 .OnM (n=8), 2.0nM (n=7)
naltrindole or vehicle (n=8) throughout al1 eight days of
hot-plate exposure.
FIGURE 4 ............................................................................................. 32
Mean (k SEM) area under the curve (AUC) for animals
administered naltrindole or vehicle, calculated over al1 eight
days of hot-plate exposure.
FIGURE 5 .......................................... ......... ....... ..... ...... . .....*................... 33 Mean (k SEM) paw lick latencies (s) of animals
adrninistered 0.5nM (n=9), 1 .OnM (n=7), 2.0nM (n=8)
nor-binaltorphimine or vehicle (n=8) throughout al1
eight days of hot-plate exposure.
FIGURE 6 ......................................................... . ................... . ................ 35
Mean (& SEM) area under the cuwe (AUC) for animals
administered nor-binaltorphirnine or vehicle, calculated
over al1 eight days of hot-plate exposure.
The progress in neurology and neuroanatomy made in the 19th century
dispelled the Aristoteiian view of pain as an emotion localized in the heart
(Merskey, 1980; Jaros, 1991 ). Pain became associated with the senses, and
thus a physiological cause was assigned to pain perception (Gamsa, 1994). In
1826 Johannes Müller published the "Doctrine of Specific Nerve Energies"
which suggested that pain, like any other f o n of sensory input, is perceived
when specific sensory receptors are stimulated (Jaros, 1 991 ).
The presumption of specific receptors for pain led to two distinct
physiological theories of pain (Jaros, 1991 ). The specificity theory, proposed in
1895 by Von Frey, posited that specific pain pathways exist to carry information
from peripheral pain receptors to a pain centre in the brain (Bonica, 1991;
Jaros, 1991 ; Melzack & Wall, 1983). The intensive (pattern) theory developed in
1894 by Goldscheider (Bonica, 1991 ; Melzack & Wall, 1983) proposed that pain
is governed by the intensity of a stimulus and the summation of nociceptive
sensory input at the level of dorsal horn in the spinal cord. According to this
hypothesis, pain is the result of either excessive stimulation produced by non-
specific tactile or thermal stimuli, or a pathological condition that strengthens the
summation of inputs from nonnoxious stimuli (Bonica, 1991 & Melzack & Wall.
1983). Despite the obvious discord between these two theories, bath are
derived from the same core assumption; each assumes a one-to-one
relationship between the intensity of a noxious stimulus and the ensuing
perception of pain (Bonica, 1 991 ).
th S~ecificitv and Pattern Theories
Specificity theory rests on three assumptions: the physiological
assumption that receptors are specialized; the anatomical assumption that
specific receptor types (0.g. tactile, thermal, nociceptive) lie beneath each
sensory spot on the skin and that specific fibers carry nociceptive information;
and the psychological assumption that the perception of pain bears a one-to-
one relation to the intensity of the stimulus and to a specific pain receptor
(Melzack & Wall, 1983).
The ~vsiological assumptiog : The physiological assumption that each
skin receptor type has an optimal level of energy to which it best responds is
known as the 'adequate stimulus' hypothesis and is generally well accepted
within the scientific community (Melzack & Wall, 1983). The adequate stimulus
hypothesis states that each specific type of skin receptor can be activated by a
specific types of information possessing certain stimulus properties. For
example, one type of receptor may be activated by pressure on the skin, while
another may be activated by specific temperatures. The physiological
assumption works well because it makes no underlying supposition regarding
the perceptual experience of pain (Melzack & Wall, 1983).
The anatomical assumption : The anatomical assumption that specific
receptor types lie beneath each sensory modality spot on the skin was based
on logical deductions. Von Frey assumed there were designated areas on the
skin specific for each sensory modality. For example, cold spots would contain
only cold receptors, and would be insensitive to any other sense. As yet there is
no solid experimental evidence to support this assumption (Melzack & Wall,
1983). However, specificity theory also predicted the existence of specific pain
transmission pathways and this prediction has been supported.
Nociceptors are the free endings of primary sensory neurons (Aghabeigi,
1992; Kandel et al., 1991). Nociceptive afferent fibers have their cell bodies in
the dorsal root and trigeminal ganglion, and terminate primarily in the dorsal
horn of the spinal cord (Cross, 1994; Kandel et al., 1991). These fibers can be
divided into two main groups: mechanoreceptors with small myelinated AS
fibers that conduct at approximately 5-30 mls and polymodal nociceptors with
unmyelinated C fibers that conduct at approximately 0.5-2 m/s (Kandel et al.,
1991; Cross, 1994). The rnechanoreceptor of the A6 neuron responds to
intense pressure on it's receptive field of approximately one square centimeter
(Dubuisson & Wall, 1980). The polymodal nociceptors of the C fibers respond
to pressure, heat and other irritants and are excited by bradykinins, histamine,
serotonin and substance P (Dubuisson & Wall, 1980). The mechanoreceptors
are more sensitive and transmit a "first" or "fast" pain that gives a discriminative
value to the pain that terminates upon removal of the nociceptive stimulus
(Cross, 1994). The threshold for first pain is invariable across individuals
(Cross, 1 994). The polymodal nociceptors require a stronger stim ut us and
transmit a "second" or "slow" pain that is broad and sustained even after the
removal of the stimulus (Cross, 1994). Secondary pain carries an affective
component and, unlike primary pain, varies in intensity from person to person
(Cross, 1994). The A6 fibers terminate both deep in the dorsal horn at laminae
IV and V and in the superficial area of lamina I (Kandel et al., 1991). The C
a* fibers terminate mainly in the superficial area of the dorsal horn of laminae 1 (the
marginal zone) and II (the substantia gelatinosa) (Kandel et al., 1991). Primary
afferent f ibers use L-glutamate as a transrnitter, and contain various
neuropeptides, such as substance P, which act as mediators in their
transmission (Cross, 1 994).
Second order neurons cross the anterior white commissure of the spinal
cord and ascend in the anterolateral quadrant (Cross, 1994). The three major
ascending pathways are the spinothalamic (Sm, spinoreticular (SRT) and
spinomesencephalic tracts (Kandel, 1991).
The SlT projection neurons can be divided into two major tracts: The
lateral nuclear group has axons that originate in laminae I and V of the dorsal
horn and project to the ventroposterolateral nucleus and the posterior nuclear
group. The medial nuclear group has axons that originate in laminae 1, IV and
VI and project to the central lateral nucleus and the intralaminar complex of the
thalamus (Cross, 1994; Kandel et al., 1991). The lateral nuclear group has a
smaller receptive field in the periphery and appears to be involved in
discriminative aspects of pain (Cross, 1994). The medial nuclear group has a
larger receptive field and is thought to be involved in affective aspects of pain
(Cross, 1 994).
The SRT projection neurons terminate in the reticular formation of the pons
(Cross, 1 994; Kandel et al., 1991 ). Nuclei receiving input from the SRT pathway
include the nucleus gigantocellularis, the nuclei reticularis pontis caudalis and
oralis, and the nucleus subcoeruleus (Cross, 1994). Neurons of the reticular
formation then send axons to the thalamus (Kandel et al., 1991).
Axons of the spinomesencephalic tract terminate in the periaqueductal
gray (PAG), the superior colliculi and the nucleus cuneiformis (Cross, 1994).
The brainstern and thalamus project to other diencephalic and cortical
structures (Dubuisson & Wall, 1980). Neurons from the lateral nuclear group
project mainly to the primary somatosensory cortex, where discriminative values
of pain are processed, and the medial nuclear group projects mainly to the
anterior cingulate gyrus where the affective components of pain are processed
(Cross, 1994; Kandel et al., 1991).
The Psvcholo_oial Assum~tion: The psychological assumption of
specificity theory posits that the physiological dimension initiated by nociceptive
receptor stimulation is translated into a corresponding psychological dimension
once it has reached the pain center in the brain (Melzack & Wall, 1983). In
order to assign a specialized role to a receptor type, we rnay rneasure the
physiological dimensions that are necessary to induce a response from that
receptor. In this way a receptor may be characterized by the physiological
stimuli to which it best responds, such as intense heat or pressure. However, to
classify the receptor as a "pain receptor" confuses the psychological
dimensions that rnay or may not be experienced as the result of the receptor
stimulation and the physiological dimensions that are necessary to evoke a
receptor response (Melzack & Wall, 1 983).
A principal obstacle to the specificity theory lies within the idea of an
invariant relationship between the magnitude of a psychological perception
and the force of the physical stimulus. A critical aspect of the specificity theory is
that a noxious stimulus that exceeds the threshold at which the pain receptor is
activated will cause a straight through message to the brain where a
corresponding psychological value is assigned, causing the appropriate
behavioral response. The direct connection from receptor to pain perception
implies that when a receptor is activated by the same noxious stimulus, it will
always result in pain and only pain (Melzack & Wall, 1983). Thus, information
about the nature of the stimulus occurs at the level of the receptor (Melzack &
Wall, 1983).
It is now well known that the quantity and quality of pain perceived in any
given situation is dependent not only upon receptor stimulation, but on many
psychological variables as well. Several variables such as cultural
background, past experience, possible reward and the perception of control dl
affect the individual's perception of pain (Melzack & Wall, 1983; Schmidt, 1985;
Cabanac, 1986; Goldberg & Maciewicz, 1994). In addition, cognitive-
behavioral techniques such as hypnosis, operant-conditioning and biofeedback
can be used to gain control over the perception of pain (Melzack & Wall, 1983;
Baker & Kirsch, 1991).
The pattern theory of pain perception posits that excessive stimulation of
non-specific stimuli results in the summation of neural firing rates, causing pain
(Melzack & Wall, 1983). Although the pattern theory allows for summation at the
level of the spinal cord, explaining the differences in pain perception across
individuals and pathological States of pain, it ignores the physiological
specialization of cutaneous receptors. (Melzack & Wall, 1983). The pattern
theory therefore, does not rely on the one-to-one relationship between stimulus
intensity and psychological response, but between the level of activity in the
spinal cord and a psychological response. In this way both the specificity and
pattern theories fail to account for the modulatory effects of psychological
variables on the perception of pain.
The Gate-Control Theorv
In 1965, Melzack & Wall proposed the Gate-Control theory in an attempt to
rectify the problems inherent in the previous theories. The gate-control theory
acknowledges the specialization of receptors and CNS pathways for pain, the
role of temporal and spatial patterning in the transmission of noxious
information to the CNS, the influence of psychological processes on the
perception of pain, and various clinical cases such as phantom limb pain
(Melzack & Wall, 1983).
The Gate-Control theory proposes that pain perception is controlled by a
neural circuit in the substantia gelatinosa (SG) in the dorsal horn of the spinal
cord (Melzack & Wall, 1983). The SG receives peripheral sensory transmission
from large (A-B) and small diameter (A-6 and C) peripheral fibres as well as
descending inhibitory input from the brain. Both the large and small peripheral
fibres and the central descending fibers influence transmission (T) cells which
send the summed information to the brain. This circuit, therefore, acts like a
gate which mediates the circulation of neural transmission from the peripheral
fibres to the CNS (Melzack & Wall, 1983).
The Gate Control Theory has received much experimental attention since
its introduction in 1965. It is now generally conceded that many of the
proposais inherent in the theory (such as, for instance, the neuroanatomical
circuitry proposed) are not likely correct. Nonetheless, the Gate Control theory
was the first to propose the existence of endogenous antiniociceptive pathways
that can be modulated by psychological factors. As will be detailed below, this
prediction has been supported by a considerable body of experimental
evidence.
ndoaenous Pain Control Svstem
The first evidence for an endogenous pain control system (EPCS) came
from Reynolds (1969), who demonstrated that electrical brain stimulation could
reduce the perception of pain, without general behavioral depression, a
phenornenon that has been termed stimulation-produced analgesia (SPA).
Subsequent research has extended this knowledge to include details on the
neural pathways involved in pain control mechanisms. These systems appear
to constitute a centrifuga1 control mechanism, since spinally mediated
nociceptive reflexes are blocked by SPA and lesions of the dorsolateral
funiculus block the inhibitory effect of SPA on dorsal horn cells (Terman et al.,
1984; Bausbaum & Fields, 1984).
These descending pathways originate in the cortex, the thalamus and the
brain stem, and synapse on dorsal horn projection neurons and interneurons
(Cross, 1994). Stimulation at various sites within these pathways has been
found to modify the activity of dorsal horn neurons and to inhibit pain
transmission (Willis, 1988; Reichling et al., 1988; Wang 81 Nakai, 1994). In
addition, there are many indirect descending pathways that synapse in the
dorsal horn (Holstege, 1988). Basbaum & Fields (1978) proposed that this
circuitry functions as a negative feedback loop. Within this model, noxious input
from small-diameter, primary afferent neurons would activate ascending
pathways in the anterolateral quadrant of the spinal cord (Fields & Bausbaum,
1978 ). The ascending pathways synapse ont0 the nucleus raphe magnus
(NRM) and possibly the nucleus reticularis magnocellularis (RMC), which form
the major brainstem descending outflow through a pathway in the dorsolateral
funiculus. These descending neurons terminate primarily in laminae 1, II, and V,
where they would be able to exert their influence on neurons receiving input
from primary afferent fibers (Fields & Bausbaum. 1978).
Biochemistrv of antinocicedion: O~iate and nonopiate mechanisms
In 1973, highly specific opiate receptors were discovered within the
mammalian brain and were soon followed by the discovery of endogenous
opioid ligands that act as transmitters at these receptors (Snyder & Pert, 1973;
Hughes et al., 1975). These discoveries corresponded closely in time with the
discovery of SPA, and led to the suggestion that SPA may be mediated by
endogenous opiate substrates (Berger & Nemeroff, 1987; Watkins et al.,
1992a). The ability of the opiate receptor antagonist naloxone to reverse the
analgesic effects of SPA and morphine led some credence to this suggestion
(Casey, 1982; Watkins et al., 1992a ). However, it was soon shown that the
ability of naloxone to reverse SPA was site dependent; that is the analgesia
evoked by stimulation of some sites was naloxone-resistent. These latter
findings led to the hypothesis that the EPCS consists of distinct opiate and non-
opiate neurochernical systems (Watkins & Mayer, 1982). Opioid analgesia was
identified as antinociception that was reversed by the administration of opiate
antagonists such as naloxone and displayed cross-tolerance with morphine
induced analgesia, whereas nonopioid analgesia was characterized as the
general class of antinociceptive effects that were insensitive to naloxone and
morphine (Watkins & Mayer, 1982; Grisel et al., 1993).
Opiate Recegtors
Opiates have their effects on opiate receptors located on cell membranes.
The three main families of opiate receptors, designated mu (p), delta (5 ) and
kappa (K) are located in various regions of the brain, spinal cord and the
periphery (Stein et al., 1993; Tyler, 1994). Morphine and most narcotics have
the highest affinity for the p receptor (Tyler, 1994). Postsynaptic p receptor
agonists increase potassium conductance and hyperpolarize the cell, whereas
presynaptically, p and 6 agonists may reduce transmitter release (e.g.,
glutamate) from primary afferents by reducing Ca++ influx during the action
potential (Fields et al., 1988). Kappa receptor agonists inhibit the opening of
voltage-dependent Ca++ channels (Fields et al., 1988).
Three classes of endogenous opiate peptides have been discovered;
enkephalins, endorphins and dynorphins. Each of the endogenous opioid
peptides are derived from separate genes, and each have different affinities for
the various subclasses of opiate receptors (Kandel et al., 1991).
En kephalins
The first two endogenous opioid pentapeptides were discovered in 1 975
by Hughes and Kosterlitz, and named met-enkephalin and leu-enkephalin
(opioid substances "in the brain"). The initial pentapeptide sequence for al1
opioid peptides contains the structure of met- or leu-enkephalin (Reisine, 1995;
Berger & Nemeroff, 1987). All enkephalins are derived from the inactive
precursor pro-enkephalin A, have modest to short projections and have a
selective affinity for 6 receptors (Cooper et al., 1991; Bausbaum & Fields, 1984;
Kandel et al, 1 991 ). Enkephalin-containing neurons are located in the
periaqueductal gray matter (PAG) and rostroventral medulla of the thalamus, as
well as in the dorsal horn of the spinal cord (Kandel et al., 1991).
end or ph in^
In addition to the discovery of the enkephalins, Hughes & Kosterlitz noticed
that the structure of met-enkephalin existed within the B-lipotropin pituitary
peptide (Reisine, 1995; Berger & Nemeroff, 1987). It was soon shown that B-
endorphin, adrenocorticotrophic hormone (ACTH) and B-l ipot ropin, were made
from the same precursor molecule, proopiomelanocortin (POMC) (Reisine,
1995; Berger & Nemeroff, 1987). Each B-endorphin molecule contains about
30 arnino acids. Whereas B-endorphin has the greatest affinity for the epsilon
receptor which is no longer considered opiate, it is also highly selective for the p
and 6 receptor (Reisine, 1995; Bausbaum & Fields, 1984). Neurons that contain
B-endorphin constitute long projection systems, which belong to the endocrine-
oriented systems of the medial hypothalamus, diencephalon and pons and
synapse at the PAG and noradrenergic nuclei of the brain stem (Kandel et al.,
1991 ; Cooper et al., 1991 ). Both ACTH and Bendorphin are released in
enhanced concentrations in respanse to acute pain or stress (Kandel et al.,
1991).
Dvnorphins
The dynorphins are made from the precursor pro-enkephalin 6, contained
mostly within the posterior pituitary and hypothalamus (Reisine, 1995; Berger &
Nemeroff, 1987). The dynorphinergic neurons have short projections and
dynorphins bind preferentially to the K receptor, although they also interact with
the p and 6 receptors (Cooper et al., 1991 ; Kandel et al., 1991). Like the
enkephalins, dynorphins are also located in the PAG and rostroventral medulla
of the thalamus, and in the dorsal horn of the spinal cord (Kandel et al., 1991).
Non-o~iate antinocice~tive svsterns
Although a thorough description of the neurotransmitters involved in the
analgesic systems is beyond the scope of this thesis, it should be noted that
epinephrine, norepinephrine, 5-hydroxytryptamine (5-HT), y-aminobutyric acid
(GABA), and a variety of peptide transmitters have been found to be
synthesized within the various nuclei and pathways of the EPCS, and that drugs
that activate these systems, have for the most part, been shown to evoke
antinociception (Cross, 1994).
Activation of EPCS
The existence of the EPCS begs the question of what type of stimuli are
able to induce its activation. One feature of the Bausbaum and Fields model is
that exposure to nociceptive stimulation can activate the EPCS. Indeed, there is
a substantial amount of evidence to support this assum ption (Basbaum &
Fields, 1984). Moreover. as detailed below, there is considerable evidence to
indicate that, in addition to noxious stimulation, environmental and
psychological factors can also activate the EPCS.
Exposure to a wide variety of noxious and non-noxious stimuli have been
shown capable of evoking an antinociceptive response (Watkins et al., 1984;
Wiertelak et al., 1 994). Activation of the EPCS by a stimulus which causes a
high level of arousal is termed "stress-induced analgesia" (SIA) (Watkins el al.,
1 992).
Some environmental stressors appear to elicit an opioid-mediated
analgesia, while others appear to activate nonopiate antinociceptive substrates
(Terman et al., 1984; Kirchgessner et al., 1982; Watkins et al., 1992a; Grisel et
al., 1993). The endeavor to delineate the stimulus qualities responsible for
activating the opioid or non-opioid analgesic systerns has led to several
different theories. The couiometric hypothesis posits that the analgesic system
which is activated is dependent upon the product of the intensity and duration of
the stressful stimulus (Terman et al., 1984; Grau, 1987). A stimulus with a weak
coulometric product will activate the opioid analgesic system, whereas a
stimulus with a stronger coulometric product will activate the non-opioid
analgesic system.
The perceptual-defensive-recuperative (PDR) theory proposed by Bolles &
Fanselow (1 980) assumes that the analgesic systems are not activated by
stressful stimuli, but by the fear evoked by such stimuli. One prediction that
arises from this hypothesis is that endogenous antinociceptive substrates can
be activated not only by fear-inducing stimuli, but by stimuli which predict the
occurrence of fear-inducing stimuli as well. Indeed, there is now extensive
evidence demonstrating the existence of such "conditioned hypoalgesia"
(Fanselow & Bolles, 1979; Calcagnetti et al., 1987; Fanselow et al., 1988;
Helmstetter & Landeira-Fernandez, 1990; Maier & Keith, 1987). That is, an
originally neutral stimulus (the conditioned stimulus or CS) acquires the ability
to elicit analgesia simply as a consequence of its being paired in
spatiotemporal contiguity with a stressor (which serves as the unconditioned
stimulus or US). The PDR theory differs from the coulometric hypothesis in that it
assumes that affective processes are required to elicit hypoalgesia. However,
the factor that predicts which analgesic system will be activated is the severity of
the US (Fanselow, 1 984; Grau, 1 987). Like the coulometric hypothesis, the
PDR theory predicts that CSs predicting the arriva1 of a US with a srnall
coulometric product will activate the opioid analgesic system, whereas CSs
predicting with a stronger coulometric US will activate non-opioid analgesic
systems (Fanselow, 1 984).
Grau (1987) has proposed the use of a working memory hypothesis in
order to predict which analgesic system is activated by exposure to nociceptive
stimuli. The working memory hypothesis is used as an extension of the
standard operating procedures (SOP) model of memory systems. Briefly,
according to the SOP model, information is coded into nodes, and these nodes
are connected by associations. At any given time a node can be in any of the
three memory states; "Al " and "A2" states of working mernory or the inactive
state of memory. The A l state is the focal point of working memory, and has a
small capacity. The A2 state is the peripheral part of working memory and
although still limited, has a larger capacity than the A l state. Presentation of a
stimulus activates it's node into the A l state, however, it is the duration and
intensity of the stimulus that determines the degree to which it is activated. A
node can decay to the A2 state, and a node can also be activated into the A2
state from the inactive state by an associative link.
Grau's working memory hypothesis states that opioid and non-opioid
analgesic systems are activated when an aversive stimulus is present within
one of the working memory states (Grau, 1987). That is, a nociceptive stimulus
present in the Al state of working memory will activate a non-opioid analgesia,
and one present in the A2 state will activate an opioid analgesia. Therefore, a
painful stimulus that is activated to the A l state and, due to a limited capacity,
decays to the A2 state, would be represented by a brief non-opioid analgesia
followed by a prolonged opioid analgesia. The distinguishing factor between
these theories lies in the degree to which they depend upon higher neural
processes. It is possible that the coulometric hypothesis best predicts the type
of analgesia elicited by direct incoming nociceptive stimulation, whereas the
working memory hypothesis is predictive in the case of learned associations.
Kirchgessner et al. (1982), suggested that the opioid and non-opioid
analgesic systems may not only interact, but may be mutually exclusive. These
authors argue that parallel activation of both opiate and nonopiate
antinociceptive substrates would be inefficient. Thus, the "collateral inhibition"
model predicts that activation of one antinociceptive substrate not only evokes
analgesia, but, in addition, prevents the other antinociceptive substrate from
being activated. Thus, the magnitude of stress induced opioid analgesia can be
attenuated by the subsequent activation of non-opioid analgesic substrates
(Kirchgessner et al., 1982; Grisel et al., 1993). Similarly, non-opiate analgesia
can be disrupted by activation of endogenous opioid analgesic mechanisms
(Kirchgessner et al., 1982).
Although a distinction is customarily made between opioid and non-opioid
analgesic systems, the validity of this distinction has recently been called into
question (Watkins et al., 1992). According to this argument, a given stressor
may activate parallel opiate systems. Consequently, administration of either
naloxone or specific opiate receptor antagonists may not affect each of the
opiate systems that are activated. As such, a hypoalgesic response that
appears to be nonopiate in nature (because it is resistant to naloxone
administration) may in fact be opiate. In support of this hypothesis, Watkins et
al (1992) demonstrated that some forms of stress induced analgesia that are
resistant to p opiate receptor selective antagonists can be completely abolished
by coadministration of selective p. and K or p and 6 receptor antagonists.
Walker et al. (1991) have also reported evidence for an interaction between
different opiate systems by demonstrating that K receptor mediated analgesia
can be enhanced by administration of a p receptor selective antagonist. These
observations suggest that stress-induced analgesia can be mediated by
multiple opiate systems activated in parallel, and that resistance to receptor
blockade does not necessarily dernonstrate that the analgesic response under
investigation is nonopiate-mediated.
Further Evidence for Plasticitv within the EPCS
The evidence reviewed to this point indicates that the perception of pain is
considerably less invariant than has been anticipated by either specificity theory
or pattern theory. Not only is there compelling evidence indicating the
existence of an interaction between stress and pain perception, the substantial
literature demonstrating the phenornenon of conditioned hypoalgesia indicates
a remarkable degree of plasticity within those neuroanatomical substrates
responsible for pain transmission. It is worth considering, however, whether this
evidence underestimates the true degree of plasticity that may occur within the
EPCS. That is, by focusing attention on the two-way interaction between stress
and pain perception and learning and pain perception, most investigators have
failed to appreciate the possibility of the existence of a three-way interaction
between learning, stress and nociception. Further, each of the models
described above makes the assumption that endogenous opiate systems
mediate the analgesic response evoked by a stressor. However, there is now
evidence to suggest that, under some circurnstances, endogenous opiate
substrates comprise, at least in part, the mechanisms responsible for mediating
plasticity within the EPCS (i.e., endogenous opiates mediate learning-induced
changes in EPCS function).
It has been shown that animals exposed to the stress of a novel
environment show a heightened level of hypoalgesia, known as novelty - induced hypoalgesia (NIH)(Bardo & Hughes, 1979; Sherman, 1979; Abbott et
al., 1986; Rochford & Dawes, 1993; Rochford & Stewart, 1987) . With repeated
exposure to the same environment, this hypoalgesia will habituate, therefore
habituation can modify at least one form of SIA.
It has recently been shown that administration of the non-specific opiate
receptor antagonist naloxone can prevent the habituation of NIH in rats
(Rochford & Dawes, 1993; Rochford & Stewart, 1987). This suggests that
endogenous opiates play a part in the habituation of NIH. In these studies, one
group of rats was administered naloxone prior to exposure to a novel hot-plate
apparatus, once a day for 8 consecutive days. Control animals were exposed
to the apparatus following saline administration; for these animals naloxone
was administered 2 - 4 hours after the exposure. The paw lick latencies in
control animals progressively declined over repeated exposures, an effect that
suggests the habituation of NIH. More importantly, whereas the latencies in
animals exposed following naloxone administration also declined, the
magnitude of this reduction was attenuated relative to the decline observed in
controls. Consequently. over the latter hot plate assessments, animals exposed
following naloxone administration displayed significantly longer paw lick
latencies than controls.
The longer paw lick latencies observed in animals exposed to the plate
after naloxone administration over the latter hot plate assessments are not likely
attributable to a drug-induced alteration in the sensitivity to noxious thermal
stimulation. First, as alluded to previously, naloxone administration does not
influence paw lick latencies during the first hot plate assessment. More
importantly, this effect occurs if animals are repeatedly exposed to a
nonfunctional, ambient temperature plate and then tested once on the
functional plate (Rochford & Stewart, 1 987). Moreover, since control animals
received the same quantity of naloxone, the longer latencies observed in
naloxone-exposed animals cannot be attributed to repeated opiate receptor
blockade alone. Considered collect ively, these results suggest that the
development of the effect is dependent upon opiate receptor blockade at the
time when animals are exposed to the plate apparatus, and are most
parsirnoniously accounted for by the suggestion that naloxone maintains longer
paw lick latencies by attenuating the habituation of NIH.
These considerations suggest that endogenous opiate substrates can be
involved in learning-induced changes in pain reactivity. Since naloxone is a
relatively nonspecific opiate receptor antagonist, what remains unclear at the
present time is the opiate receptor subtype(s) that may be involved.
Consequently, the present experiments were conducted to delineate the
relative importance of specific opiate receptor subtypes in the mediation of the
effect. To achieve this end, we examined the effects of the p-receptor selective
antagonist Cys2-Typ-OrnS-Pen7-am ide (CTOP), the &receptor select ive
antagonist naltrindole, and the K-selective antagonist nor-binaltorphimine
(NOR-BNI) on the rate of habituation of NIH.
METHODS
EXPERIMENT 1
Subiects.
Thirty six male Wistar rats (Charles River Breeding Farms, St. Constant,
Quebec), weighing between 300 and 350 grams were used in each experiment.
The animals were individually housed in polypropylene cages (34 x 29 x 17 cm)
and maintained on a 12-h lightldark cycle (lights on 0800h. lights off 2000h) at a
room temperature of 20 + 2%. Food and water were available ad libitum
throughout the experirnent. All experiments were conducted within the
guidelines of the Canadian Council on Animal Care and approved by the McGill
University Animal Ethics Committee.
Suroerv:
Each rat was anesthetized with ketamine anesthesia (Ketalak; Abbott
Laboratories, 50 mg / kg, i.m.) and Rompun analgesic (xylazine; Miles Canada
inc., 5 mg I kg, i.m.), placed in a stereotaxic apparatus and implanted with a
unilateral stainless steel 22-gauge guide cannulae (Plastic Products CO.) aimed
at the left cerebral ventricle [AP: 0.5mm; ML: 1.5mm; DV:3.5mm below dura
(Pellegrino 8 Cushman, 1 Q6i)l. Blockers extending 1 mm beyond the cannula
were inserted irnmediately following surgery. The blockers were cleaned and
immediately replaced at least twice during the week of recovery, in order to
familiarize the animals with the injection procedure before experimentation.
A ~ ~ r a t u s and Dru=
Pain sensitivity was assessed using the hot-plate apparatus, made of
20.3 x 38.1 x 20.3 cm clear plexiglass chamber mounted on a 0.6-cm thick, 26.7
x 30.5 cm piece of sheet metal. A wooden lid with air holes was hinged to the
top of hot-plate to prevent animals from escaping. The plate temperature was
controlled by immersing the sheet metal into a water bath heated by a Haake
E2 lmmersion/Open Bath Circulator. The hot-plate was located in a test room
illurninated by two 25 watt red light bulbs and maintained at a constant 20 e ° C .
CTOP (Cys2-Typ-0rn5-Pen7-amide) (Pen insula) was dissolved in distilled
water, alliquotted into 100 pl units, and frozen at -20°C in 1.5 ml eppendorph
tubes until used. Each testing day, one alliquot of each dosage was slowly
thawed, kept on ice and used within 5 hours. CTOP was delivered
0 intracerebroventricularly at either 0.5, 1 and 2 nM and vehicle (distilled water)
was used as control. Drugs were administered in the testing room, thirty
minutes prior to behavioral testing once a day for a total of 8 days. All drugs
were delivered in a volume of 2.0 pl, through injector wires which extended
1 mm beyond the cannulae (Plastic Products CO.). lnjectors were attached to
polyethylene tubing, which in turn were attached to a 10 pl Hamilton syringe.
All injections were delivered at a rate of 2 pl / 60s, following which the injectors
were left in place for another 60s.
Procedure
Following recovery from surgery, animals were randomly divided into
4@ four groups (n=9), receiving 0.5, 1 .O or 2.0 nM CTOP or vehicle. Behavioral
testing occured during the animal's light cycle. All animals were transported to
the test room where they received their respective injections. After drug
administration, al1 animals were individually kept in transport cages measuring
34 x 29 x 17 cm for 30 minutes prior to hot-plate exposure. Each animal was
placed on the hot plate where the latency to lick their hind paw was recorded to
the nearest 0.1 s with a timer (Lafayette Instrument Co.). The temperature of the
water was maintained at 48.5 (k.2) O C , this temperature has been found to be
ideal for the expression of novelty-induced hypoalgesia (Rochford & Stewart,
1987). If an animal failed to lick its paw within 90 s, it was removed from the hot-
plate in order to prevent tissue damage, and a 90 s maximum score was
recorded. All animals were returned to their home cages upon completion of
testing. This procedure was repeated once a day for a total of 8 days.
H istoloay
One animal from both the vehicle and 0.5 pg CTOP groups, and 2 from
the 1 .O pg group were exluded due to damage of their cannulae. All remaining
anirnals were injected with 5pg of blue dye following euthanization by carbon
dioxide asphyxiation. The brains were then removed from the skull and
cannulae placements were verified by visually confirming staining around the
walls of the ventricle.
Stat istics
The data from Experiment 1 were analyzed by a 4 x 8 (group x day)
analysis of variance (ANOVA). Significant interactions were analyzed by F-tests
for simple main effects. Significant simple main effects were further analyzed by
Tukey's honestly significant difference test. To better characterize the CTOP
dose response curve, the area under the curve over the eight days of the
experiment was calculated for each group, using the trapezoidal rule. These
data were analyzed by a one way (group) ANOVA; significant differences
between groups were confirmed using Tukey's honestly significant difference
test.
EXPERIMENT 2
Subiects
Thirty six experimentally naive male Wistar rats, weighing between 300-
350 g upon arrival, were obtained and housed as described in Experiment 1.
Suroerv
AH animals underwent ICV cannula implantation as described in
Experiment 1. Animals were allowed a minimum one week recovery period
before experimentation.
Ag~aratus and Druas
The hot-plate apparatus used in Experiment 1 was set at the same
temperature and used in Experiment 2. Naltrindole (Research Biochemicals)
doses were determined using the equimolar concentration of 0.5, 1 .O and 2.0
nM (0.21, 0.42 & 0.84 pg). Drugs were disolved, stored and administered in the
same manner as described in Experiment 1.
Procedure
Al1 animals were randomly assigned to one of the four groups; vehicle,
0.5, 1.0 and 2.0 nM naltrindole. Transportation, injections and testing were
conducted in the same manner as in Experiment 1.
Histology
One animal from both the vehicle and 1 .O nM groups and two from each
of the 0.5 and 2.0 nM groups were exluded due to dammage or misplacement
of their cannulae.
Statistics
All data from Experiment 2 were analyzed as described previously for
Experiment 1.
EXPERIMENT 3
subiectç
The subjects were thirty six experimentally naive, male Wistar rats
weighing between 300-350 g at the start of the experiment. The animals were
obtained and housed as described in Experiment 1.
¶tus and D r u g
Nor-binaltorphimine dihydrochloride (Research Biochemicals) was
prepared in 0.5, 1 .O and 2.0 nM (0.35, 0.69, & 1.38 pg) concentrations as
described previously.
Procedure
One week following surgery, animals were randomly assigned to one of
four groups; vehicle, 0.5, 1 .O and 2.0 nM NOR-BNI. Transportation, injection
and testing was conducted in the same manner as described in Experiment 1.
Histology
One animal from each of the vehicle and 2 nM groups and two from the
1 .O nM groups were exluded due to damage or misplacement of their cannulae.
Stat istics
The data from Experiment 3 were analyzed as described previously.
RESULTS
EXPERIMENT 1: CTOP
The mean paw lick latencies for the 4 groups over the 8 days of testing are
displayed in Figure 1. Examination of Figure 1 reveals that al1 4 groups
displayed relatively long and equivalent paw lick latencies on the first test day.
On subsequent tests, the paw lick latencies for vehicle-treated animals
gradually declined, as did those in animals treated with 0.5 nM CTOP. Note,
however, that the reduction in paw lick latencies over days was attentuated in
the groups receiving 1 .O and 2.0 nM CTOP. These observations were
confirmed by the ANOVA performed on the data, which yielded a significant
Dose x Days interaction, F (21, 196) = 1 -95, p < ,025. Subsequent tests for
simple main effects, conducted between groups for each day, revealed
significant group differences from days 2 through 6, inclusive, Fs(3, 224) 24.01,
p s c .O25 Tukey's HSD tests revealed that the mean paw lick latencies for al1
three groups receiving CTOP were longer than vehicle-treated animals on day
2 (p s < .05). Over days 2-6, the mean latencies for animals treated with 1 .O and
2.0, but not 0.5, nM were greater than vehicle-treated animals. There were no
differences between animals treated with 1 .O and 2.0 nM of CTOP.
Figure 2 shows the mean area under the curve (AUC) calculated over the
entire 8 days of testing for al1 4 groups. A one way, between-groups ANOVA
revealed significant group differences, F(3, 28) = 3.88. p c .025. Tukey's
pairwise cornparisons revealed that the mean area under the curve for animals
receiving 1 .O and 2.0 CTOP was significantly greater than that for vehicle-
m r e 1. Mean (k SEM) paw lick latencies (s) of animals
administered 0.5nM (n=8), 1 .OnM (n=7), 2.0nM (n=9) CTOP
or vehicle (n=8) throughout al1 eight days of hot-plate exposure.
* p c 0.05 versus vehicle treated animals.
CTOP
0.5nM 1 .OnM DOSE
Fiaure 2. Mean (f SEM) area under the curve (AUC) for animals
administered CTOP or vehicle, calculated over al1 eight
days of hot-plate exposure. ** p É 0.01, * p c 0.05 versus vehicle
treated anirnals.
treated animals (ps c .05). There was no significiant difference between the
vehicle and 0.5 nM groups, or between the 1 .O and 2.0 nM groups.
EXPERIMENT 2: NALTRINDOE
Figure 3 portrays the mean paw lick latencies for al1 4 groups over the 8
days of testing. As in the previous experiment, latencies in the vehicle-treated
group were initially high, and declined over days. The rate of decline observed
in animals treated with 2.0 nM naltrindole was attenuated. ANOVA yielded
significant main effects for both dose, F (3,26) = 4.88, p < ,009, and days, F (7,
182) = 18.38, p < .001, but no dose x days interaction, F (21,182) = 1.09, p >
.05. The days main effect reflects the general decline in latencies over days in
al1 groups. Tukey's tests revealed that the dose main effect was due to the 2.0
nM naltrindole group displaying significantly longer paw lick latenies than the
vehicle-treated group. No other group differences were significant.
Figure 4 shows the mean AUC for al1 4 groups over the last 4 days of
testing. Analysis of the data with a one way (Group) ANOVA revealed a
significant difference between groups, F (3, 26) = 5.1 1, p c .01. Subsequent
analysis using Tukey's tests confirmed that the mean AUC for the 2.0 nM group
was significantly greater than the vehicle-treated group. No other group
differences were significant.
EXPERIMENT 3: NOR-BNI
Figure 5 displays the mean paw lick latencies for al1 4 groups over the 8
days of testing. A Dose x Days ANOVA revealed a significant main effect for
days, F (7, 196) = 12.75, p c ,001, indicating the decline in latencies over days.
NOR-BNI did not influence the rate of decline as both the main effects for dose,
F c 1 .O, and the dose x drug interaction, F (21, 196) = 1.25, were not significant,
p s > .05.
NALTRINDOLE 4 VEHICLE
0.5nM
l.OnM
I 2.OnM
. 8
1 P 4 5
DAYS
Fiaure 3. Mean (& SEM) paw lick latencies (s) for animals
administered 0.5nM (n=7), 1 .OnM (n=8), 2.0nM (n=7)
naltrindole or vehicle (n=8) throughout al1 eight days of
hot-plate exposure. ' p c 0.05 versus vehicle treated animals.
NALTRINDOLE
0.5nM 1 .OnM 2.0nM
DOSE
Figure 4. Mean (& SEM) area under the curve (AUC) for animals
administered naltrindole or vehicle, calculated over al1 eight
days of hot-plate exposure.
NOR-SNI v E H c L E r 0.5nM + l.OnM + 2.0nM
D A Y S
Fiaure 5. Mean (k SEM) paw lick latencies (s) of animals
adrninistered 0.5nM (n=9), 1 .OnM (n=7), 2.0nM (n=8)
nor-binaltorphimine or vehicle (n=8) throughout al1
eight days of hot-plate exposure.
Figure 6 presents the mean AUC for al1 groups calculated over the eight days of
experimentation. A one way (Group) ANOVA indicated no significant group
differences on this measure, F c 1 .O, p 2 .05.
NOR-ENI
0SnM 1 .OnM 2 . 0 n ~
DOSE
W r e 6. Mean (& SEM) area under the curve (AUC) for animals
administered nor-binaltorphimine or vehicle, calculated
over al1 eight days of hot-plate exposure.
DISCUSSION a,. The results from the present experiments add further to the evidence
demonstrating that animals exposed to a novel environment display less
reactivity to nociceptive stimulation. When vehicle-treated animals were
exposed repeatedly to the testing apparatus, the latency ta lick their hind paw
decreased, implying that pain reactivity was low during the initial hot plate tests,
and increased over subsequent exposures to the hot plate apparatus. This
increase in pain sensitivity has been proposed to be the result of the habituation
of novelty-induced hypoalgesia (NIH) (Rochford & Stewart, 1987).
It has been shown previously that the nonspecific opiate receptor
antagonist naloxone attenuates the rate with which pain reactivity increases
over repeated hot plate exposures, suggesting that opiate receptor blockade
can inhibit the rate of habituation of NIH (Rochford & Stewart, 1987; Rochford &
Dawes, 1993). Moreover, naloxone does not affect pain sensitivity during the
initial hot plate tests, suggesting that NIH is nonopiod in nature, and that this
nonopioid substrate is modulated by an opioidergic substrate involved in
learning. The aim of the present study was to investigate the role of the p, 6 and
K opiate receptor subtypes in this habituation process.
Experiments 1 and 2 revealed that ICV administration of the pspecific
antagonist CTOP and the gspecific antagonist naltrindole inhibited the increase
in pain reactivity observed over repeated hot plate exposure, suggesting that
these ligands were able to attenuate the rate of habituation of NIH. These
experiments also yielded evidence that p receptor blockade attenuates the
habituation of novelty-induced hypoalgesia more readily than 6 receptor
blockade. Administration of 1 nM CTOP was able to inhibit the habituation of
novelty-induced hypoalgesia, whereas a dose of 2 nM naltrindole was required
to achieve the same effect.
Experiment 3 suggested that the K receptor does not appear to be involved
in the habituation of NI H. This experiment demonstrated that administration of
the specific K receptor antagonist NOR-BNI, in the same dose range that the
other antagonists were adrninistered in Experiments 1 and 2, had no effect on
pain sensitivity throughout the entire course of hot plate exposures. One
objection to this conclusion could be that NOR-BNI has a weaker affinity to it's
receptor relative to the other antagonists. As such it could be argued that the
dose range used for NOR-BNI was not ideal. However, we have shown that
administration of NOR-BNI in doses ranging frorn 5 to 70 nM also failed to effect
NIH, suggesting that this is not a problem.
This pattern of results suggests that opioid substrates may attenuate the
habituation of NIH through two distinct receptor subtypes. Opioid ligands are
notorious for being relatively non-selective for specific opiate receptor subtypes.
This consideration raises the possibility that the attentuation of the habituation
of NIH rnay be mediated through a single opiate receptor subtype. That is,
CTOP may have inhibited the habituation of NIH through its actions at 6
receptors, or, naltrindole may have exerted this effect through its ability to block
p receptors. It is worth noting, however, that these ligands were able to exert
their effects at low (1 .O - 2.0) nanornolar concentrations. Moreover, available
evidence suggests that at these doses, CTOP and naltrindole behave as highly
specific antagonists for their respective receptors (Hawkins et al., 1989;
Emmerson et al., 1994; Rogers et al., 1990; Negus et al., 1993). Consequently,
we do not believe that these ligands inhibited the habituation through an action
at a receptor distinct from the receptor for which they are selective.
Recent evidence has suggested that p and 6 opiate receptors are not
hornogenous, but may consist of different receptor subtypes. It is now generally
conceded that both the p and the 6 receptors consist of at least two subtypes,
labelled the p, and ~ i , and the 6, and 6, subtypes (Fang et al., 1994; Tiseo &
Yaksh, 1993; Sofuoglu et al., 1992; Portoghese et al., 1992; Porreca &
Yamamura, 1994; Shah et al., 1994; Koch & Bodnar, 1993). To complicate
matters further, it has recently been suggested that y and 6 receptors may
coexist to form a ~ i / 6 receptor complex (Heyman et al., 1989a & 1989b; Porreca
et al., 1987). It has been shown that subanalgesic doses of [Met5]-enkephalin
and [Leu5]-enkephalin, endogenous ligands selective for the 6 receptor,
decrease and increase respectively, the antinociceptive potency of the p-
selective agonist morphine (Vaught et al., 1982; Barrett & Vaught, 1982; Vaught
& Takemori, 1979; Lee et al., 1980; Larson et al., 1980). This effect is also
observed with some exogenous ligands selective for the 6 receptor, such as ([D-
Pen2, 0-Penq-enkephalin (DPDPE). However, other ligands selective for the 8
receptor, such as [D-Ala2, Leu5,Cys6]enkephalin (DALCE) have no eff ect on p-
receptor mediated antinociception (Porreca et al., 1992; Heyman et al., 1987;
Sheldon et al., 1989). These contrasting results have led to the hypothesis that
these ligands may be acting at distinct 6 receptor subtypes. Porreca and
colleagues (1987) distinguish the 6 receptors that are able to modulate p-
mediated antinociception and those that do not as the 6 uncomplexed and the
pl6 complexed receptors respectively.
At present, the precise roles played by independent and coupled p and 6
receptors is a matter of heated debate (Traynor & Elliott, 1993; Sheldon et al.,
1989; Schoffelmeer et al., 1993). There is evidence to suggest that
independent p. and 6 opioid receptors can be distinguished from the p/S
receptor complex by cellular location and function (Jackish et al.. 1986;
Schoffelmeer et al., 1988; 1993; Mulder et al., 1991). Independent p and 6
receptors may be located on presynaptic nerve terminals where they regulate
the inhibitory effects of endogenous opioids on depolarization-induced
norepinephrine and acetylcholine release respectively. The pl6 receptor
complexes may be located postsynaptically where they have been shown to
regulate the inhibition of dopamine D, receptor mediated adenylate cyclase
activity in rat striatum (Schoffelmeer et al., 1987; 1988; 1993). It has also been
suggested that independent and coupled S receptors may correspond to the 6,
and 8, subtypes respect ively.
Available evidence suggests that CTOP does not influence the p/S complex.
and that naltrindole has a 200-fold higher affinity for the independent 6 receptor
(Schoffelmeer et al., 1992). These data provide some evidence that the ability
of these antagonists to attenuate the habituation of NIH is probably not
mediated through effects at the putative Ct/6 receptor complex, but by
independent p and 6 receptors. One objection to this conclusion could stem
from the finding that CTOP and naltrindole were both found to exert the same
effect; that is, both were found to attenuate the habituation of NIH. As
rnentioned above, it has been suggested that independent and 6 receptors
rnay exert opposite effects insofar as activation of independent 6 receptors has
been shown to inhibit p-receptor mediated antinociception (Porrecca et al,
1987). One resolution to this dilemma could stem from the suggestion that the
antagonistic effects exerted by 6 receptors on p. receptors rnay be dependent
upon the response system under investigation. That is, whereas an opposing
functional relationship may exist between independent p and 6 receptors
mediating antinociception, this conclusion does not necessarily imply a similar
kind of functional relationship between the p and 6 receptors mediating the
habituation of NIH. Indeed, it is well recognized that the systems subserving
cognitive function (i.e., learning and memory) are anatomically distinct from
those mediating antinociception. As such, it is not unreasonable to hypothesize
that the f unctional relationsh ip between diff erent receptors mediating cognitive
function may be distinct from that mediating changes in pain reactivity.
There is a substantial body of evidence demonstrating that administration of
endogenous and exogenous opiate ligands can influence the rate and
expression of learning, particularly in those paradigms in which aversive stimuli
are employed to promote learning (e.g., avoidance learning, Jaffe & Blanco,
1994; Jodar et al., 1995; Janak et al., 1994; Liljequist, 1981 ; Patterson et al.,
1989). Endogenous opiate peptides are released during stressful experiences
(Amir et al., 1980; Bodnar et al., 1980). The effects of opiate receptor agonists
and antagonists within these learning paradigms have been found to be dose-
dependent (Jodar et al., 1 995; Schulties & Martinez, 1 992; llyutchenok &
Dubrovina, 1995; Braida et al., 1994; Aloyo et al., 1993; Castellano & Puglisi-
Allegra, 1983). For instance, the endogenous opioids B-endorphin, dynorphin,
[Metlenkephalin and [Leulenkephalin have each been shown to display a U-
shaped dose response curve in a variety of aversive learning tasks, suggesting
that these substances can exert either memory enhancing or memory inhibiting
effects dose-dependently (Martinez & Rigter, 1980; Martinez et al., 1 984; 1 988;
Schulties et al., 1988; Schulteis & Martinez, 1992; Colombo et al., 1992; Janak
et al., 1994; Jodar et al., 1995; Patterson et al., 1989).
The present results add further to the evidence implicating endogenous
opiate substrates in learning insofar as they indicate that blockade of p and 6
receptors can attenuate the rate of acquisition of habituation learning. Several
independent laboratories have demonstrated that opiate receptor ligands can
profoundly influence learn ing processes that modulate pain transmission.
However, it is not clear whether this effect occurs because opiate ligands
influence the learning processes directly, or whether this effect occurs through
an indirect mechanism. For instance, Fanselow and colleagues have
demonstrated that exposure to a CS previously associated with footshock
administration can evoke a conditioned hypoalgesic response which is
apparently opiate-rnediated in that it can be attenuated by naloxone
administration (Fanselow & Bolles, 1979; Fanselow, 1984; Calcagnetti et al.,
1987; Fanselow et al., 1988; Fanselow et al., 1991; Kim et al., 1993). It has also
been found that administration of naloxone or of specific p receptor antagonists
during the conditioning trials (Le., when the CS-footshock pairings are
administered) can augment the magnitude of conditioning (Fanselow et al.,
1988). Fanselow has argued that this enhancement is not atrributable to the
hypothesis that p receptor blockade facilitates the learning processes directly
involved in forming the CS-footshock association. Rather, he argues that
because the conditioned hypoalgesia is opioid rnediated, opiate receptor
blockade increases the perceived magnitude of the footshock US. The
increase in US intensity results in more robust conditioning. Therefore, opioid
substrates are not directly modulating learning per se, but indirectly influence
the expression of the learning through an increase in pain sensitivity.
A related finding has been reporteci when noxious thermal stimulation,
rather than footshock, is used as the US (Foo & Westbrook, 1991 ; Westbrook et
al., 1991; Greeley et al., 1988; Walker et al., 1991). To illustrate, Greeley et al.
(1988) obtained a form of conditioned analgesia in rats when they administered
naloxone prior to hot-plate testing. In these studies the temperature of the hot
plate was set at 49.5O C, and animals were maintained on the plate for a 30 sec
period, independent of when they licked their paws. Both vehicle- and
naloxone-treated groups displayed relatively short paw lick latencies on the first
hot plate test. Over subsequent tests, the paw lick latencies in naloxone-treated
animals increased dramatically, whereas those for the vehicle-treated group did
not. In other words, animals in the naloxone group acquired a conditioned
hypoalgesic response over successive exposures to the hot-plate apparatus.
Greeley et al (1988) argued that naloxone administration increased the
perceived severity of the noxious thermal stimulation imposed by hot plate
testing. This effectively increased the intensity of the US, and resulted in a
robust conditioned hypoalgesic response.
Both the phenornena described above demonstrate that opiate receptor
blockade can produce an effect that is not necessarily the result of an action
directly on processes involved in learning. In contrast, we are suggesting that
the effects of CTOP and naltrindole observed in the present experiments were
the results of a direct effect of these ligands on processes mediating
habituation. This conclusion can be justified only by showing that CTOP and
naltrindole did not influence the perceived intensity of the noxious thermal
stimulation to which animals were exposed in the present study. In support of
this conclusion, it is to be noted that CTOP and naltrindole did not influence
pain reactivity during the initial hot plate tests. Moreover, recall, once again that
Rochford & Stewart (1987) have shown that naloxone administration can
maintain elevated paw lick latencies when animals are exposed to a
nonfunctional hot plate. Thus, exposure to nociceptive stimulation is not a
requirement for the development of the effect. As such, it is difficult to argue that
naloxone administration altered the perceived intensity of noxious thermal
stimulation. Finally, it should be noted that the pattern of results observed in the
present experirnents are not best described within a conditioning framework.
Greeley et al (1988) assumed the operation of a Pavlovian learning mechanism
precisely because they observed an acquisition curve for the conditioned
hypoalgesic response displayed by naloxone-treated animals. In the present
experiments, opiate receptor antagonists did not increase paw lick latencies,
rather they prevented the reduction in latencies observed in vehicle-treated
animals. The pattern of results in vehicle-treated animals suggests the
operation of an habituation mechanism, as such the fact that the rate of
reduction in paw lick latencies over repeated plate exposures could be
attenuated by CTOP and naltrindole suggests that these ligands interfered
directly with those processes involved in the habituation of novelty-induced
h ypoalgesia.
Whereas the arguments made above suggest that p and 6 opiate receptors
are implicated in mediating the habituation of novelty-induced hypoalgesia, an
equally important question concerns whether opiates substrates may be
involved in the mediation of the novelty-induced hypoalgesic response itself.
As will be discussed further below, there is good evidence to suggest that
novelty-induced hypoalgesia is mediated, at least in part, through activation of
noradrenergic substrates. It is to be noted, however, that this evidence does not
autornatically exclude the possibility that opiate mechanisms may also mediate
the effect. As alluded to in the introduction, novelty-induced hypoalgesia is
resistent to naloxone administration, in that this ligand does not influence paw
lick latencies durhg the first few hot plate tests. The present experiments
dernonstrated that CTOP and naltrindole also do not influence pain reactiivity
during the initial tests. Moreover, Rochford et a1 (1 993) have shown that
novelty-induced hypoalgesia does not display cross tolerance to morphine
analgesia. These findings would suggest that novelty-induced hypoalgesia is
mediated predominantly by nonopiate substrates. Currently available data do
not, however, permit us to conclude that novelty-induced hypoalgesia is
mediated exclusively by nonopiate mechanisms. The reason for this stems from
the fact that resistence to opiate receptor blockade and resistence to the
development of cross tolerance with morphine do not invariably force the
conclusion that a given hypoalgesic response is nonopiate mediated. Recall
that Watkins et al (1992) have shown that forms of stress-induced analgesia that
are resistent to naloxone administration and do not display cross tolerance to
morphine can be attenuated by coadministration of pairs (Le., p. and 6 or p and
K) of opiate receptor antagonists. Thus, in order for us to conclude confidently
that opiate mechanisms are not involved in the mediation of novelty-induced
hypoalgesia, it will be necessary to assess whether coadministration of pairs of
opiate antagonists reduce the elevated paw lick latencies observed during the
initial exposures to the hot plate apparatus.
Independent of whether opiate mechanisms may or may not mediate
novelty-induced hypoalgesia, it is now clear that other, nonopiate, transmitter
systems are involved in generating the antinociception observed when animals
are exposed to novel environrnents. Stressful events, including exposure to
novelty, have been shown to increase the activity of the noradrenergic neurons
in the locus coeruleus (Tanaka et al., 1982; Abercrombie & Jacobs, 1988). More
importantly, it has been shown that novelty-induced hypoalgesia can be
inhibited by administration of the a, adrenergic receptor agonist clonidine and
potentiated by the a, adrenerg ic receptor antagonist yohirn bine (Rochford,
1992; Rochford & Dawes, 1993). Clonidine and yohimbine have been shown to
in hibit and en hance, respectively, noradrenergic activity (Aghajanian et al.,
1977; Anden et al., 1970; Dubocovich, 1984; Karege & Gaillard, I W O ;
Rasmussen & Jacobs, 1986). Thus, yohim bine may potentiate novelty-induced
hypoalgesia by augmenting novelty-induced activation of noradrenergic
neurons; clonidine would attenuate the effect by inhibiting noradrenergic
neurotransm ission.
Electrophysiological and biochemical evidence has amply demonstrated
that noradrenergic substrates are under inhibitory opiate control. Thus,
administration of either endogenous or exogenous opiate agonists have been
shown to reduce noradrenergic neuron fi ring rate and noradrenalin release
neurons (Gergen et al., 1996; Nishikawa & Shimizu, 1990; Carr & Gregg, 1 995;
Simmons et al., 1992; llles 8 Norenberg, 1990). Naloxone administration
prevents this inhibition (Tanaka et al., 1982; Abercrombie & Jacobs, 1988).
Thus, it has been proposed that naloxone may retard the habituation of novelty-
induced hypoalgesia by preventing opioid inhibition of noradrenergic
neurotransmission (Rochford & Dawes, 1992; Rochford et al., 1 993).
In the present experirnents, the p-selective antagonist CTOP and the 6-
selective antagonist naltrindole were found to prevent the habituation of
novelty-induced hypoalgesia, whereas the K-selective antagonist NOR-BNI was
without effect. According to the model presented above, therefore, it would
appear that opiate inhibition of noradrenergic activity is mediated through the p
and the 6, but not the K, opiate receptor subtypes. In support of this conclusion,
both p and 6 selective agonists have been found to inhibit noradrenergic
activity, whereas K-selective agonists do not possess this activity (Carr & Gregg,
1995; Matsumoto et al., 1994; Sevcik et al., 1993). It must be noted, however,
that the evidence implicating the involvement of the 6 receptor in inhibiting
noradrenergic activity is not strong, and it has been suggested that opiates
inhibit noradrenergic activity exclusively through an action at p-receptors (Illes &
Norenberg, 1990). Thus, further work will be needed to confirm the role of the 6
receptor in the attenuation of the habituation of novelty-induced hypoalgesia.
One way in which this issue can be addressed further is by contrasting the
effects of p- and Gselective agonists on the rate of habituation of novelty-
induced hypoalgesia. If both receptors are involved in the habituation of
novelty-induced hypoalgesia, then administration of both p- and Gselective
agonists should facilitate such habituation.
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