Distinct profiles of anxiety and dysphoria during spontaneous withdrawal from acute morphine exposure

ABSTRACT The negative motivational aspects of withdrawal include symptoms of both anxiety and depression, and emerge after termination of chronic drug use as well as after acute drug

exposure. States of acute withdrawal are an inherent part of intermittent drug use in humans, but the contribution of acute withdrawal to the development of addiction has received limited

systematic investigation, because of a lack of preclinical models for withdrawal states that emerge spontaneously after acute drug exposure. Here, we have characterized a spontaneous

increase in the magnitude of the acoustic startle reflex (ie, spontaneous withdrawal-potentiated startle) that emerges after acute morphine administration in rats, and compared the time

course of startle potentiation and place conditioning. We find that startle potentiation seems to be related to a decrease in opiate receptor occupancy and reflects an anxiety-like state

with a pharmacological profile similar to other signs of opiate withdrawal. Spontaneous startle potentiation emerges before the rewarding effects of morphine have subsided, even though

naloxone administration after a single morphine exposure causes both startle potentiation and conditioned place aversion (CPA). These results show that negative emotional signs of withdrawal

develop after just one exposure to morphine, and are likely a recurrent aspect of intermittent drug use that may contribute to the earliest adaptations underlying the development of

addiction. Furthermore, the dissociation between spontaneous startle potentiation and CPA suggests anxiogenic and dysphoric manifestations of opiate withdrawal may be mediated by distinct

neural mechanisms that are progressively engaged as withdrawal unfolds. SIMILAR CONTENT BEING VIEWED BY OTHERS AN ENDOGENOUS OPIOID CIRCUIT DETERMINES STATE-DEPENDENT REWARD CONSUMPTION

Article 13 October 2021 OXYTOCINERGIC INPUT FROM THE PARAVENTRICULAR NUCLEUS TO THE NUCLEUS ACCUMBENS CORE MODULATES METHAMPHETAMINE-CONDITIONED PLACE PREFERENCE Article Open access 23 May

2025 MESOLIMBIC DOPAMINE RELEASE PRECEDES ACTIVELY SOUGHT AVERSIVE STIMULI IN MICE Article Open access 27 April 2023 INTRODUCTION The negative motivational aspects of withdrawal from chronic

drug exposure contribute to the maintenance of established drug addiction (Koob and Le Moal, 2008), but negative emotional states also emerge after acute drug exposure (Breiter et al, 1997;

Kirby and Stitzer, 1993; Van Dyke and Byck, 1982). These episodes of acute withdrawal are a recurrent and integral component of human drug use (Baker et al, 2004), emerging after occasional

drug use or when ongoing drug intake is interrupted by sleep or periods when drug supply is limited (Dole et al, 1966; Haertzen and Hooks, 1969). Alleviation of acute withdrawal may

motivate further drug use, and changes in neural activity during acute withdrawal could contribute to drug-induced alterations in physiology and brain function (Houshyar et al, 2003;

Houshyar et al, 2004). Withdrawal from acute opiate exposure can be precipitated by opiate receptor antagonists (for review, see Harris and Gewirtz, 2005), but in the context of human opiate

abuse, withdrawal emerges spontaneously in the absence of an antagonist. Despite this fact, surprisingly few preclinical models have been developed to study the spontaneous emergence of

withdrawal after acute opiate exposure. This study describes distinct profiles of anxiety and dysphoria in rats that emerge spontaneously after acute exposure to morphine. Withdrawal from

chronic drug use produces symptoms of both anxiety and depression, including restlessness, irritability, dysphoria, and anhedonia (American Psychiatric Association, 2000; Haertzen and Hooks,

1969). The acoustic startle reflex is a validated measure of anxiety in both animals and humans (Lang et al, 2000), and is elevated in rodents during spontaneous withdrawal from acute

morphine exposure (ie, withdrawal-potentiated startle; Harris and Gewirtz, 2004a). Other spontaneous signs of acute morphine withdrawal in rodents, including conditioned place aversion (CPA)

(Bechara et al, 1995) and increased thresholds for intracranial self-stimulation (ICSS) (Liu and Schulteis, 2004), may reflect states of dysphoria or anhedonia associated with depression

(Barr et al, 2002; Carlezon and Chartoff, 2007; Land et al, 2008b). Given the growing number of experimental dissociations between anxiety- and depression-like behavior in rodents (Bosch et

al, 2008; Land et al, 2008a; Nestler and Carlezon, 2006; Sahuque et al, 2006; Wallace et al, 2009), as well as distinctions between clinical disorders of anxiety and depression (American

Psychiatric Association, 2000; Goldberg, 2008; Kessler et al, 2008; Krueger, 1999), it is important to distinguish between these specific negative affective components of opiate withdrawal,

as they may not necessarily coincide with one another. To address these issues, we have further characterized spontaneous withdrawal-potentiated startle and compared its time course with

that of spontaneous CPA. We show that spontaneous withdrawal-potentiated startle seems to be related to a decrease in opiate receptor occupancy and has an anxiety-like pharmacological

profile that resembles other measures of opiate withdrawal. However, startle potentiation emerges while rats still exhibit conditioned place preference (CPP), showing an increase in

anxiety-like behavior before the rewarding effects of morphine have subsided. In contrast, withdrawal-potentiated startle and CPA develop concurrently when withdrawal is precipitated by

naloxone (an opiate receptor antagonist). These results indicate that anxiogenic and dysphoric manifestations of acute morphine withdrawal reflect changes in distinct neural systems. These

negative emotional states accompany the earliest stages of drug exposure, are likely a recurrent feature of intermittent drug use in humans, and thus may contribute significantly to the

development of addiction. MATERIALS AND METHODS SUBJECTS Male Sprague–Dawley rats (Harlan) were housed in groups of 4–5 in metal cages with a 12 h light–dark cycle (light on 0800–2000 hours)

and free access to food and water except during testing. Rats were allowed to acclimate to housing conditions for 2 weeks after arrival, were gently handled for two consecutive days before

any testing, and weighed 250–350 _g_ at the beginning of each experiment. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and

were approved by the University of Minnesota Institutional Animal Care and Use Committee. DRUGS Morphine sulfate was provided by the National Institute on Drug Abuse (Rockville, MD).

Naloxone, chlordiazepoxide, and R,S-propranolol hydrochlorides were obtained from Sigma (St. Louis, MO). LY235959 was obtained from Tocris (Ellisville, MO). All drugs were dissolved in 0.9%

saline (except propranolol, which was dissolved in water) and injected (i.p.) in a volume of 1 ml/kg body weight. Over the course of these studies, we shifted to s.c. administration of

morphine and naloxone to be consistent with the majority of other work in this field (Houshyar et al, 2003; Houshyar et al, 2004; Schulteis et al, 1994). We directly compared i.p. and s.c.

morphine injections in several experiments and found no significant differences between routes of administration (data not shown), so results from both routes of administration have been

pooled. All drug doses are expressed as the weight of the salt. ACOUSTIC STARTLE Acoustic startle was tested in four identical plastic cages (17 × 8.5 × 11 cm) resting on compression springs

and located within individual ventilated sound-attenuating chambers. Cage movement resulted in displacement of a piezoelectronic accelerometer (Model ACH-01, Measurement Specialties, Valley

Forge, PA) attached to each cage. Voltage output from the accelerometer was filtered and amplified by a custom-built signal processor, digitized on a scale of arbitrary units ranging from

0–1000 (National Instruments SCB100 and PCI-6071E boards), and recorded using Matlab (The MathWorks, Natick, MA). Startle amplitude was defined as the peak accelerometer voltage during the

first 200 ms after onset of the startle stimulus. High-frequency speakers (Radio Shack Supertweeters, range=5–40 kHz) located 10 cm beside each cage delivered the startle stimuli, which were

50-ms bursts of filtered white noise (low pass: 22 kHz, rise-decay <5 ms) at intensities of 95 or 105 dB. Ventilating fans elevated background noise to ∼60 dB. Acoustic startle was

tested on each of 2 days before drug exposure. For each test session, rats were placed in the startle chambers for a 5-min acclimation period, and then presented with 40 startle stimuli (20

each at 95 or 105 dB in semi-random order) with a 30-s inter-stimulus interval. Average startle amplitudes from the second day were used to match animals into experimental groups with

similar overall mean startle amplitudes. Each day of drug testing began with a baseline startle session before any drug injections. Several experiments involved startle testing over multiple

days using Latin Square or crossover designs; details are provided in figure legends. All drug injections were given in the colony, and rats remained in their home cage between drug

injections and startle tests. PLACE CONDITIONING Our place conditioning apparatus and procedure were developed according to published recommendations (Bardo and Bevins, 2000; Carlezon, 2003;

Cunningham et al, 2006). The apparatus consists of a rectangular plastic cage (40 × 20 × 20 cm) divided into two sides by a central partition. Each side has a distinct floor texture and

wall color: metal bars paired with white walls, and wire mesh paired with black striped walls. Each rat's position within the apparatus was monitored by an overhead video camera

connected to a computer running AnyMaze software (Stoelting, Wood Dale, IL). Rats were transported to the place conditioning room and allowed to acclimate for at least 10 min before every

experimental session. Each experiment began with a 10-min baseline session in which rats were free to move between both sides of the apparatus. The rats used in these studies spent an

average of 320 s (53.4%) on the bar side during the baseline session; two rats with >75% baseline preference for one side were excluded from further study. The side of the apparatus

paired with drug treatment was counterbalanced within each experiment, yielding an unbiased procedure in which rats spend ∼50% of the baseline session on the side to be paired with drug.

Daily conditioning sessions began 24 h after the baseline session. Details concerning the number, duration, and order of conditioning sessions are provided in figure legends. Twenty-four

hours after the last conditioning session, a 10-min test session was conducted in which rats were free to move between both sides of the apparatus. We chose to express place conditioning

results in terms of percent time spent on the drug-paired side, rather than using a difference score measured in seconds, because percentage measures are relatively independent of the length

of the testing session and thus facilitate comparisons across studies. DATA ANALYSIS Startle data were collapsed across both intensities (95/105 dB) before statistical analysis (Harris and

Gewirtz, 2004a), as the magnitude of withdrawal-potentiated startle was not affected by stimulus intensity (data not shown). In each experiment, we first conducted one-way analysis of

variance (ANOVA) to verify similar baseline startle amplitude between experimental groups; there were no differences in baseline startle between groups in any experiment (data not shown).

Changes in startle after morphine administration were calculated as percent change from baseline on the same day (Walker and Davis, 2002b). For experiments that utilized a crossover design,

baseline startle amplitude was similar on both days of testing, so an average baseline value was used to calculate percent change on each individual day. An area under the curve measure for

total withdrawal severity was calculated for each individual subject by adding together percent change in startle across all time points tested; mean and standard error were then calculated

for all subjects in each group. All data were analyzed using factorial ANOVA, with repeated measures on within-subject factors. For main effects or interactions involving repeated measures,

the Huynh–Feldt correction was applied to control for potential violations of the sphericity assumption. Significant interactions were followed with tests for simple effects (Keppel and

Wickens, 2004). When appropriate, significant main effects were followed with polynomial trend analysis. All statistical analysis was conducted using SPSS (version 13.0) with a Type I error

rate of _α_=0.05 (two-tailed). Group sizes for each experiment are indicated in figure legends. RESULTS STARTLE TIME COURSE DURING SPONTANEOUS AND NALOXONE-PRECIPITATED WITHDRAWAL Startle

was tested 2–5 h after acute administration of morphine (10 mg/kg), with some rats receiving naloxone (2.5 mg/kg) just before the 2:00 startle test (Figure 1a). Startle was significantly

potentiated 4–5 h after injection of morphine alone, consistent with our earlier report that also showed startle returns to baseline 6 h after this dose of morphine (Harris and Gewirtz,

2004a). Naloxone caused an immediate but transient potentiation of startle at 2:00, with no change from baseline 3–5 h after morphine injection (Naloxone × time interaction: F3,78=12.19,

_p_<0.001). This dose of naloxone was selected based on our earlier work (Harris et al, 2004b) in an effort to completely displace morphine from the opiate receptor. There were no

significant changes in startle after injection of saline or naloxone alone (Figure 1b) (Naloxone × time interaction, F3,66 <1). It is noteworthy that the peak magnitude of spontaneous

withdrawal (4:00: 61.0±11.6%) was significantly larger than the peak magnitude of naloxone-precipitated withdrawal (2:00: 29.8±9.0%) (_t_28=2.23, _p_=0.034). This difference was more

pronounced when comparing total withdrawal severity, measured as area under the curve across all time points tested (spontaneous: 102.2±19.5%; precipitated: 24.9±21.0%) (_t_28=2.67,

_p_=0.013]. The difference in total withdrawal severity was partly driven by the absence of spontaneous startle potentiation at later time points after naloxone administration (Figure 1b).

MORPHINE RE-EXPOSURE DELAYS STARTLE POTENTIATION If startle potentiation represents a withdrawal effect, it should be blocked by re-exposure to morphine (Figure 2a). Indeed, the startle

potentiation normally observed 4 h after an initial morphine injection was prevented by a second injection of morphine 3 h after initial injection (Figure 2b) (initial injection × second

injection interaction: F1,32=11.07, _p_=0.002). We conducted a second startle test in the same animals 7 h after the initial injection (Figure 2c). Both groups that received a second

injection of morphine (ie, 4 h earlier) showed significant startle potentiation at this time (main effect of second injection: F1,32=76.11, _p_<0.001; interaction: F1,32 <1). Thus,

morphine re-exposure does not prevent startle potentiation, but delays its onset until opiate receptor occupancy eventually decreases. PHARMACOLOGICAL PROFILE OF SPONTANEOUS

WITHDRAWAL-POTENTIATED STARTLE We next examined whether anxiolytic drugs attenuate spontaneous withdrawal-potentiated startle. Both chlordiazepoxide (a benzodiazepine) and propranolol (a

β-adrenergic receptor antagonist) prevent other forms of startle potentiation (Walker and Davis, 2002a) at the same doses used here (10 mg/kg), and also decrease anxiety-like behavior in a

number of other behavioral paradigms (Cole and Koob, 1988; Harris and Aston-Jones, 1993a, 1993b; Knoll et al, 2007; Rodriguez-Romaguera et al, 2009). Administration of chlordiazepoxide

prevented spontaneous withdrawal-potentiated startle (Figure 3a) (morphine × chlordiazepoxide interaction: F1,30=5.49, _p_=0.026), as did administration of propranolol (Figure 3b) (morphine

× propranolol interaction: F1,20=5.91, _p_=0.025). Neither anxiolytic drug affected startle amplitude in the absence of morphine. NMDA receptor antagonists also prevent signs of opiate

withdrawal in rodents (Harris et al, 2008; Kawasaki et al, 2005; Rasmussen, 1995). We examined the effects of LY235959, a competitive NMDA receptor antagonist shown to attenuate precipitated

morphine withdrawal (Jones et al, 2002), using doses (1–3 mg/kg) that prevent tolerance to morphine analgesia (Bilsky et al, 1996) and sensitization to morphine-induced locomotion (Mendez

and Trujillo, 2008). LY235959 produced a dose-dependent attenuation of startle potentiation (Figure 4) (morphine × LY235959 interaction: F2,33=14.27, _p_<0.001). There was a significant

linear effect of LY235959 dose after morphine injection (_p_=0.012), but no effect after saline injection (_p_=0.56). STARTLE POTENTIATION AFTER THE FIRST MORPHINE EXPOSURE As the preceding

experiments utilized Latin Square and crossover designs, sometimes involving multiple exposures to morphine, we sought to clarify whether startle was potentiated after the very first

exposure to morphine. We pooled control data from the preceding experiments in which startle was tested 4 h after an animal's first exposure to morphine (_n_=33) or saline (_n_=31), and

found a highly reliable potentiation of startle after morphine injection (51.2±6.6%) that was significantly greater than the change in startle after saline injection (5.3±4.6%; _t_62=5.62,

_p_<0.001). PLACE CONDITIONING TIME COURSE AFTER ACUTE MORPHINE EXPOSURE A delayed CPA has been reported after acute exposure to morphine (Bechara et al, 1995). We next determined the

time course of place conditioning after injection of 10-mg/kg morphine (Figure 5). Each group spent ∼50% time on the drug side during the baseline session, confirming the unbiased nature of

our place conditioning procedure. ANOVA indicated a significant session × time interaction (F6,87=4.89, _p_<0.001). As expected, CPP was observed immediately after morphine injection

(0:00) (_t_11=2.85, _p_=0.016), and was maintained at 2:00 (_t_11=4.29, _p_=0.001) and 4:00 (_t_10=3.70, _p_=0.004). The 4:00 time point is when we observe peak startle potentiation after

this same dose of morphine (cf. Figure 1), indicating that rats are still experiencing a state of reward when anxiety-like behavior emerges. There was no effect of place conditioning at

6:00, and a non-significant tendency for CPA at 8:00 (_t_22=1.46, _p_=0.16). A trend toward CPP was also observed at 10:00 (_t_11=2.18, _p_=0.052). There was a significant fit to

fourth-order polynomial trend across time (_p_=0.003), suggesting the emergence of aversion after the initial preference. PLACE CONDITIONING AND STARTLE DURING NALOXONE-PRECIPITATED

WITHDRAWAL Because spontaneous withdrawal-potentiated startle and CPA emerged at different times after acute morphine injection, we asked whether these two behavioral effects could be

dissociated under other conditions. As 2.5-mg/kg naloxone produces startle potentiation when administered 2 h after 10-mg/kg morphine (cf. Figure 1), we examined whether naloxone causes CPA

under these same conditions (Figure 6a). ANOVA indicated a significant session × group interaction (F2,28=10.04, _p_=0.001). Exposure to morphine alone caused CPP (_t_8=2.49, _p_=0.038),

whereas naloxone had no effect in the absence of morphine (_t_7 <1). However, naloxone administration 2 h after morphine caused CPA (_t_13=3.90, _p_=0.002). Earlier studies have shown

that naloxone still causes CPA when administered 24 h after a single morphine injection (Araki et al, 2004; Parker and Joshi, 1998), and we also replicated this effect (Figure 6b). At the 24

 h time point, ANOVA indicated a trend towards a session × group interaction (F2,44=2.15, _p_=0.13). Planned comparisons showed that administration of morphine followed by naloxone caused

CPA (_t_14=2.47, _p_=0.027), whereas there was no effect of either morphine alone (_t_15 <1) or naloxone alone (_t_15 <1). The effect of naloxone on acoustic startle has not been

examined 24 h after a single exposure to morphine. We found that naloxone still produced startle potentiation 24 h after a single morphine injection (Figure 7) (_t_10=2.57, _p_=0.028),

similar to its effect 2 h after acute morphine injection. Startle potentiation and CPA thus develop concurrently when naloxone is administered either 2 or 24 h after a single morphine

injection. DISCUSSION Our results show that anxiety-like behavior (ie, startle potentiation) emerges spontaneously after a single exposure to morphine, seems to be related to a decrease in

opiate receptor occupancy, and shares a pharmacological profile with other forms of opiate withdrawal. Startle potentiation develops before the rewarding effects of morphine have subsided,

clearly dissociating increased anxiety-like behavior from decreased reward system activity. This study represents the first direct demonstration that anxiogenic and dysphoric manifestations

of opiate withdrawal may be mediated by distinct neural mechanisms, which are progressively engaged during withdrawal after acute exposure to morphine. THE NATURE OF SPONTANEOUS

WITHDRAWAL-POTENTIATED STARTLE Spontaneous withdrawal-potentiated startle emerged and peaked 4 h after injection of 10-mg/kg morphine, consistent with our earlier report (Harris and Gewirtz,

2004a). This corresponds to a time at which morphine levels in blood and brain have declined substantially (Barjavel et al, 1995; Hipps et al, 1976), and the direct behavioral and

neurochemical effects of morphine have already peaked and are returning to baseline (Babbini and Davis, 1972; Barjavel et al, 1995; Di Chiara and Imperato, 1988; Hipps et al, 1976). This

suggests startle elevation emerges as morphine metabolism leads to falling drug levels and reduced opiate receptor occupancy. Startle was potentiated by naloxone administration 2 h after

morphine, whereas morphine re-exposure delayed the onset of startle potentiation, suggesting a link between startle potentiation and decreased opiate receptor occupancy. Spontaneous

withdrawal-potentiated startle was also blocked by chlordiazepoxide and propranolol, two anxiolytic drugs shown earlier to attenuate increases in startle amplitude caused by conditioned fear

cues and exposure to bright light (de Jongh et al, 2002; Risbrough et al, 2003; Walker and Davis, 2002a). Chlordiazepoxide prevents other forms of anxiety-like behavior in rodents (eg,

Knoll et al, 2007), whereas propranolol has been shown to reduce affective signs of opiate withdrawal (Harris and Aston-Jones, 1993a, 1993b). NMDA receptor antagonists also alleviate signs

of opiate withdrawal (Harris et al, 2008; Kawasaki et al, 2005; Rasmussen, 1995). LY235959, a competitive NMDA receptor antagonist that reduces precipitated morphine withdrawal (Jones et al,

2002), produced a dose-dependent attenuation of startle potentiation. These results clearly indicate that startle elevation shares a pharmacological profile with other measures of opiate

withdrawal. RELATIONSHIP BETWEEN STARTLE POTENTIATION AND PLACE CONDITIONING A delayed CPA has been observed after administration of morphine (Bechara et al, 1995), as well as other opiates

(Pain et al, 2008) and other addictive drugs (Ettenberg and Bernardi, 2007; Morse et al, 2000; Pliakas et al, 2001). We assessed the time course of place conditioning after administration of

10-mg/kg morphine, and found that CPP persisted up to 4 h after morphine injection (see also White et al, 2005). This time course closely parallels the elevation of extracellular dopamine

levels in the nucleus accumbens (Di Chiara and Imperato, 1988), consistent with the role of nucleus accumbens dopamine in generating morphine CPP (Fenu et al, 2006). A tendency for CPA

emerged 8 h after morphine injection. Other studies have reported robust CPA 11–16 h after acute exposure to 20-mg/kg morphine (Bechara et al, 1995; Vargas-Perez et al, 2007), which is

likely related to the differences in morphine dose and the number and timing of conditioning sessions. There was a significant fourth-order polynomial trend across time, suggesting the acute

rewarding effects of morphine were followed by delayed aversive effects. We speculate that the tendency toward CPP at 10 h could reflect alleviation of an aversive withdrawal state. We were

surprised to find CPP 4 h after 10-mg/kg morphine, the same time point at which we observed peak startle potentiation. To determine whether a similar dissociation was observed under other

conditions, we examined the effect of naloxone on acoustic startle and place conditioning at different times after a single morphine injection. We found that injection of 2.5-mg/kg naloxone

generated CPA (as well as startle potentiation) 2 h after morphine injection. These results are consistent with human studies showing that naloxone can precipitate withdrawal symptoms as

soon as 45 min after acute morphine administration (Heishman et al, 1989). Several signs of withdrawal (Eisenberg, 1982; Gellert and Sparber, 1977), including CPA (Araki et al, 2004; Parker

and Joshi, 1998), are still observed when naloxone is administered 24–48 h after a single exposure to morphine. We observed both startle potentiation and CPA when naloxone was administered

24 h after one morphine injection, showing an additional similarity between startle potentiation and other measures of withdrawal. A dissociation between the emergence of startle

potentiation and CPA was only observed when withdrawal was allowed to unfold spontaneously. These results provide an important example in which antagonist-precipitated withdrawal does not

precisely recapitulate the conditions of spontaneous withdrawal. Precipitated withdrawal is a useful experimental tool for controlling the timing of withdrawal and studying states of

dependence. However, in the context of human opiate abuse, withdrawal develops spontaneously in the absence of an opiate receptor antagonist. Our findings highlight the importance of further

developing models of spontaneous opiate withdrawal in rodents, to examine potential similarities and distinctions between spontaneous and precipitated withdrawal states. In future studies,

it will be important to examine the opiate receptor subtypes mediating different behavioral changes during spontaneous and precipitated withdrawal from acute morphine exposure. Specific

antagonists of the mu-opioid receptor (MOR) can precipitate signs of withdrawal after chronic morphine exposure (Le Guen et al, 2003; Maldonado et al, 1992), suggesting that loss of MOR

occupancy may cause spontaneous withdrawal. However, morphine also has a lower affinity for the kappa-opioid receptor (KOR) (Goldstein and Naidu, 1989), and some effects of morphine can be

mediated by KOR activation (Nobre et al, 2000; Sante et al, 2000; Yamada et al, 2006). As KOR agonists produce signs of anxiety, dysphoria, and anhedonia in humans and rodents (Land et al,

2008b; Motta et al, 1995; Nestler and Carlezon, 2006; Pfeiffer et al, 1986; Sante et al, 2000; Shippenberg et al, 2007), signaling cascades triggered by KOR activation could also contribute

to spontaneous withdrawal. On the other hand, KOR antagonists can in some cases exacerbate the severity of opiate withdrawal (Spanagel et al, 1994). The potential contributions of MOR and

KOR could be addressed in future studies using techniques that directly measure receptor occupancy, such as autoradiography, to examine changes in the occupancy of MOR and KOR across

multiple brain structures in the hours after acute morphine administration. POTENTIAL NEURAL SUBSTRATES FOR ANXIETY AND DYSPHORIA During spontaneous withdrawal from acute morphine exposure,

startle potentiation emerges before the rewarding effects of morphine have subsided, and thus before the onset of CPA. Our data suggest that startle potentiation may be caused by decreased

opiate receptor occupancy. In contrast, CPA may instead represent an opponent process to the acute rewarding effects of morphine (Vargas-Perez et al, 2007). As such, CPA may reflect a

decrease below baseline activity in the mesolimbic dopamine system. This type of change has been shown during withdrawal from chronic opiate exposure (Diana et al, 1995; Rossetti et al,

1992; Spanagel et al, 1994), as well as acute amphetamine exposure (Barr et al, 2002), although we are not aware of similar studies performed after acute morphine administration. Adaptations

within the reward system are thought to play a role in depression (Nestler and Carlezon, 2006), and depression-like changes during opiate withdrawal may be manifested as CPA (Vargas-Perez

et al, 2007) and elevated ICSS thresholds (Liu and Schulteis, 2004). In contrast, startle potentiation emerges while the reward system is still active, but its activity has decreased from

peak levels (Di Chiara and Imperato, 1988). This suggests the anxiety-like manifestations of opiate withdrawal may be closely tied to a relative decrease in hedonic state (ie, a negative

slope), rather than an absolute decrease below baseline. Portions of the extended amygdala, including the bed nucleus of the stria terminalis and central nucleus of the amygdala, play a

general role in states of withdrawal (Koob and Le Moal, 2008) and anxiety (Walker et al, 2003), and are specifically involved in antagonist-precipitated withdrawal from acute morphine

administration (Cabral et al, 2009; Criner et al, 2007; Harris et al, 2006). This involvement could result from a direct and local effect of morphine, or may be secondary to morphine-induced

elevations in extracellular dopamine (Carboni et al, 2000). The specific role of these circuits in different emotional manifestations of spontaneous withdrawal will be an important topic

for future research. This framework may help explain why baseline startle amplitude is not elevated during precipitated withdrawal from chronic opiate administration (Fendt and Mucha, 2001;

Kalinichev and Holtzman, 2003; Mansbach et al, 1992). These conditions produce dramatic decreases in mesolimbic dopamine system activity (Diana et al, 1995; Pothos et al, 1991; Rossetti et

al, 1992) and brain reward function (Schulteis et al, 1994). The resulting depression-like state could obscure or overwhelm the expression of anxiogenic manifestations of withdrawal. Indeed,

human patients diagnosed with depressive illness do not exhibit increases in startle amplitude under conditions that normally produce startle potentiation in control subjects (Dichter and

Tomarken, 2008; Forbes et al, 2005; Lang and McTeague, 2009). IMPLICATIONS FOR ADDICTION Our results clearly indicate that withdrawal is a complex and multifaceted construct. We have

described distinct time courses for two specific emotional manifestations of acute withdrawal (ie, startle potentiation and CPA). The spontaneous evolution of other signs of acute withdrawal

may parallel one of the time courses we have described, or may follow other unique time courses. For example, spontaneous increases in ICSS threshold have been reported 24 h after acute

morphine exposure (Liu and Schulteis, 2004). We also note that spontaneous hyperalgesia has been observed after a single exposure to heroin (Laulin et al, 1998) or morphine (Sweitzer et al,

2004). The time course of hyperalgesia is complex, as it emerges and dissipates in the hours after opiate administration, then reappears 24 h later and lasts several days (Laulin et al,

1998). Thus, the various emotional and physical manifestations of spontaneous withdrawal likely result from a cascade of numerous neurobiological events, which develop and evolve as a

function of time. It is important to note that anxiety-like signs of spontaneous withdrawal may represent one of the earliest manifestations of the withdrawal syndrome, developing before CPA

and changes in ICSS threshold. As spontaneous startle potentiation was delayed by re-exposure to morphine, relief or prevention of anxiety may be particularly important for motivating

continued drug use. The relief of anxiety states may provide primary negative reinforcement for ongoing drug use, perhaps by maintaining dopaminergic tone within the extended amygdala, as D1

receptor antagonism in the amygdala can enhance cocaine intake, even while dopamine levels in NAc remain elevated (Hurd et al, 1997). In addition, anxiety-like states may motivate drug use

because they predict the subsequent emergence of depression-like states, thus serving as secondary negative reinforcers. Finally, anxiety-like states generated by stressful experience could

also contribute to stress-induced relapse, particularly given the common neural circuitry involved in stress-induced reinstatement (Shaham et al, 2003) and potentiated acoustic startle

(Walker et al, 2003). Events that occur during spontaneous withdrawal may also contribute to some of the unique effects of intermittent opiate exposure. For example, intermittent injections

of morphine produce physiological changes similar to those caused by chronic stress (Houshyar et al, 2003; Houshyar et al, 2004), changes not observed when morphine is administered

continuously. The acute withdrawal state that follows each intermittent morphine injection may contribute to this stress-like profile. Indeed, changes in brain activity during spontaneous

withdrawal could contribute to any difference between the consequences of continuous and intermittent opiate exposure. As human drug abuse is routinely interrupted by drug-free periods

(Baker et al, 2004; Dole et al, 1966), it will be important to examine whether events that occur during spontaneous withdrawal contribute to adaptations in brain function during intermittent

drug exposure. These results add to a growing number of dissociations between anxiety- and depression-like behavior under a variety of experimental conditions (Bosch et al, 2008; Land et

al, 2008a; Nestler and Carlezon, 2006; Sahuque et al, 2006; Wallace et al, 2009), and raise important considerations for future research. First, signs of withdrawal develop spontaneously

after just one exposure to morphine, and are likely expressed after each intermittent exposure to an opiate. This means withdrawal is not unique to the termination of chronic drug use, but

is an intrinsic component of drug taking that may play an important but often neglected role in the development of addiction. Second, anxiety-like manifestations of withdrawal emerge while

the animal is still experiencing a state of reward. As dysphoria and other depression-like manifestations of withdrawal likely reflect decreases below baseline in reward system activity,

symptoms of anxiety and depression may develop at different times as withdrawal unfolds, which could have important treatment implications. Therapeutic interventions that ameliorate symptoms

of anxiety and depression, such as KOR antagonists (Knoll et al, 2007; Land et al, 2008a; Land et al, 2008b; Nestler and Carlezon, 2006), may prove particularly effective. A clearer

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thank Bonnie LaCroix and Malaak Moussa for expert technical assistance, and Dr Andrew Harris and members of the Gewirtz and Thomas labs for helpful comments. This work was supported by

funding from the University of Minnesota Graduate School (to PER) and Grants from NIDA (DA007234 and DA023750 to PER, DA019666 to MJT, and DA018784 to JCG), the Whitehall Foundation (to

MJT), and NARSAD (to JCG). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Graduate Program in Neuroscience and Departments of Neuroscience and Psychology, University of Minnesota,

Minneapolis, MN, USA Patrick E Rothwell, Mark J Thomas & Jonathan C Gewirtz Authors * Patrick E Rothwell View author publications You can also search for this author inPubMed Google

Scholar * Mark J Thomas View author publications You can also search for this author inPubMed Google Scholar * Jonathan C Gewirtz View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Jonathan C Gewirtz. ADDITIONAL INFORMATION DISCLOSURE/CONFLICT OF INTEREST The authors declare that, except for income

received from their primary employers, no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional

service, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE Rothwell, P., Thomas, M. & Gewirtz, J. Distinct Profiles of Anxiety and Dysphoria during Spontaneous Withdrawal from Acute Morphine Exposure.

_Neuropsychopharmacol_ 34, 2285–2295 (2009). https://doi.org/10.1038/npp.2009.56 Download citation * Received: 12 March 2009 * Revised: 25 April 2009 * Accepted: 26 April 2009 * Published:

03 June 2009 * Issue Date: September 2009 * DOI: https://doi.org/10.1038/npp.2009.56 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

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* withdrawal * anxiety * dysphoria * acoustic startle * place conditioning