Posted: April 13th, 2023

Genetics, Cocaine

196  | wileyonlinelibrary.com/journal/cns CNS Neurosci Ther. 2021;27:196–205.
1 | INTRODUCTION
Addiction is a mental disorder defined by compulsive and chronic drug
taking. The development of drug abuse after an extended period of drug
taking has been suggested to depend on a shift from early recreational
drug use to later compulsive drug abuse. The neurobiological mechanisms
leading to this shift remain unknown. Furthermore, compared with a limited drug history, extended drug exposure was assumed to have a higher
probability to lead to prolonged neuronal alterations, resulting in a higher
risk of drug relapse and a further extension of drug abuse.1-4
The insular cortex (IC), or insula for short, plays an important
role in drug addiction. Both human5-7 and animal studies8
showed
Received: 30 July 2020  |  Revised: 19 September 2020  |  Accepted: 23 September 2020
DOI: 10.1111/cns.13469
ORIGINAL ARTICLE
Limited versus extended cocaine intravenous selfadministration: Behavioral effects and electrophysiological
changes in insular cortex
Yi-Xiao Luo1,2 | Donald Huang1 | Changyong Guo1 | Yao-Ying Ma1,2,3
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd
Luo and Huang are contributed equally to this work.
1
Department of Pharmacology and
Toxicology, Indiana University School of
Medicine, Indianapolis, IN, USA
2
Department of Psychology, Behavioral
Neuroscience Program, State University of
New York, Binghamton, NY, USA
3
Stark Neurosciences Research Institute,
Indiana University School of Medicine,
Indianapolis, IN, USA
Correspondence
Yao-Ying Ma, Department of Pharmacology
and Toxicology, Indiana University School of
Medicine, 635 Barnhill Drive, Indianapolis,
IN 46202, USA.
Email: ym9@iu.edu
Funding information
Brain & Behavior Research Foundation
grant, Grant/Award Number: 24989;
National Institute on Alcohol Abuse
and Alcoholism, Grant/Award Number:
R01AA025784
Abstract
Aims: Limited vs extended drug exposure has been proposed as one of the key factors
in determining the risk of relapse, which is the primary characteristic of addiction
behaviors. The current studies were designed to explore the related behavioral
effects and neuronal alterations in the insular cortex (IC), an important brain region
involved in addiction.
Methods: Experiments started with rats at the age of 35 days, a typical adolescent
stage when initial drug exposure occurs often in humans. The drug-seeking/taking
behaviors, and membrane properties and intrinsic excitability of IC pyramidal neurons
were measured on withdrawal day (WD) 1 and WD 45-48 after limited vs extended
cocaine intravenous self-administration (IVSA).
Results: We found higher cocaine-taking behaviors at the late withdrawal period
after limited vs extended cocaine IVSA. We also found minor but significant effects
of limited but not extended cocaine exposure on the kinetics and amplitude of action
potentials on WD 45, in IC pyramidal neurons.
Conclusion: Our results indicate potential high risks of relapse in young rats with
limited but not extended drug exposure, although the adaptations detected in the IC
may not be sufficient to explain the neural changes of higher drug-taking behaviors
induced by limited cocaine IVSA.
KEYWORDS
cocaine, drug seeking, drug taking, whole-cell patch-clamp, insula cortex
| LUO et al.  197
that IC lesions interrupt addictive behaviors, suggesting that the
IC or the IC-mediated interoceptive system is sensitized in addicted subjects as a consequence of addiction history and a cause
of relapse. However, a different picture was suggested by neuroimaging studies, in which reduced activity9,10 and shrunken gray
matter5,11 in the IC were detected in individuals with substance
abuse history. These apparently contradictory lines of evidence
may indicate that the IC can both promote drug use (eg, via increased perception of craving for drugs) and weaken the processes
that prevent drug use (such as decision-making and the evaluation
of negative consequences).12 The IC consists of 3 subregions, that
is, posterior granular insular, anterior agranular insula (AAI), and
the intermediate dysgranular insula).13-15 Particular attention in
addiction research has been payed to the AAI due to its rich chemoarchitecture, including the presence of dopamine and opioid
receptors.6,16-20
The present study used a rat model of cocaine intravenous
self-administration (IVSA) to explore the behavioral and electrophysiological consequences of limited vs extended IVSA after short
(1 day) or prolonged (45 days) withdrawal periods (designated as
WD1 and WD45, respectively). Experiments started with rats at the
age of 35 days, a typical adolescent stage when initial drug exposure
occurs often in humans.21,22
2 | MATERIALS AND METHODS
2.1 | Experimental subjects
All procedures were performed in accordance with the United
States Public Health Service Guide for Care and Use of Laboratory
Animals and were approved by the institutional Animal Care and
Use committee at the State University of New York, Binghamton
and Indiana University School of Medicine. Experiments were
conducted on male Sprague Dawley rats, bred in-house using
breeders originally derived from Envigo (USA). With the day of
birth being deemed as postnatal day (P) 0, rats were allowed to
develop normally until P21-23 when animals were weaned and
pair-housed in standard Plexiglas bins unless receiving cannula
implantation procedure. Rats were maintained on a 7 am/7 pm light/
dark schedule with ad libitum access to food and water. Rats were
allowed 5-7 days to acclimate to colony conditions and handled to
habituate them to human contact prior to experimentation. Similar
to our previous studies, rats received catheter implantation on
postnatal ~35 days. Seven days later, rats received one overnight
session, followed by 5 of 1-hour daily sessions (considered
as limited training procedure) or 21 of 6-hour daily sessions
(considered as extended training procedure) of cocaine IVSA
training.23,24 In total, 75 rats were used in this study, among which
8 rats were excluded due to patency failure of the catheter (n = 6)
and behavioral outliers (n = 2). From the 67 remaining subjects,
21 were trained by limited (n = 11) or extended (n = 10) cocaine
IVSA procedures, followed by cocaine-seeking test on WD1 and
WD45, and cocaine-taking test on WD46-48 (Figure 1A). The
other 46 rats were trained by limited (n = 9 for saline and n = 14
for cocaine) or extended (n = 10 for saline and n = 13 for cocaine)
IVSA procedures and whole-cell patch-clamp recordings were
obtained either 1 or 45 days after the last IVSA session.
2.2 | Intravenous self-administration of cocaine
2.2.1 | Catheter implantation
As described previously,23-27 a silastic catheter was inserted into
the right jugular vein, and the distal end was led subcutaneously to
the back between the scapulae. Catheters were constructed from
silastic tubing (~8 cm; inner diameter 0.020 in, outer diameter 0.037
in) connected to a One Channel Rat Button (Instech Labs). Rats
were allowed to recover for ~7 days. During recovery, the catheter
was flushed daily with 1 mL/kg body weight of heparin (10 U/mL)
and gentamicin antibiotic (5 mg/mL) in sterile saline to help protect
against infection and catheter occlusion.
2.2.2 | Self-administration apparatus
Experiments were conducted in operant-conditioning chambers
enclosed within sound-attenuating cabinets (Med Associates).
Each chamber contained two levers randomly assigned as active vs
inactive levers, a food dispenser, and the conditioned stimulus (CS)
light 9 cm above each lever. No food or water was provided in the
chambers during the training or testing sessions.
2.2.3 | Intravenous cocaine selfadministration training
~7 days after catheter implantation, cocaine self-administration
training began with an overnight session at 7 pm to 7 am on the
following day. The daily training session, either 1 hour per day for
5 days as limited cocaine access or 6 hours per day for 21 days as
extended cocaine access, started on the day after. The same training
protocol was used in overnight and daily sessions. Rats were placed in
the self-administration chamber on a fixed ratio (FR) 1 reinforcement
schedule with the house light on. Active lever press resulted in a
cocaine infusion (0.75 mg/kg over 2-4 seconds) and illumination
of a CS light above the active lever for 20 seconds with the house
light off. In contrast, the inactive lever press led to no outcome but
was also recorded. Rats that received at least 60 cocaine rewards
in the overnight session were allowed to move to daily selfadministration of cocaine ~24 hr after the overnight training on an
FR1 reinforcement schedule. Animals that did not meet this standard
(n = 1) were removed from subsequent self-administration training.
198  |    LUO et al.
2.2.4 | Cocaine-seeking test
Rats previously assigned for behavioral test were tested on both
WD1 and WD45 for their cocaine-seeking behaviors. The cocaine-seeking test lasted for 1 hour, during which rats were reexposed to the operant chamber with the exact same conditions
as during cocaine IVSA training except no cocaine IV delivery was
programmed after active lever press. The active lever press during
1 hour seeking test was analyzed as an index of cocaine-seeking
level (Figure 1D).
2.2.5 | Cocaine-taking test
Multiple doses of cocaine were evaluated in the cocaine-taking test
as described before.28 Briefly, a 2-hour period was divided into four
30-minute segments, which allowed the assessment of four pseudorandomized doses of cocaine (0.1, 0.3, 1.0, and 3.0 mg/kg per injection) in a 2-hour session. Two adjacent 30-minute sessions were
spaced by a ~5-minute interval. During each 30-minute session,
the exact same conditions, although the dose of cocaine per injection was different, were applied to rats, which means rats received
FIGURE 1 Effects limited vs extended cocaine IVSA on cocaine-seeking and cocaine-taking behaviors during the withdrawal period. A,
Timeline of cocaine-seeking and cocaine-taking test with specified age of days at each stage. Rats were trained by limited (upper line) or
extended (lower line) cocaine IVSA procedures. P, postnatal days. B, The daily # of cocaine IV infusion and daily # of active lever press by
rats exposed to limited cocaine IVSA procedure. C, The daily # of cocaine IV infusion and daily # of active lever press by rats exposed to
extended cocaine IVSA procedure. D, Cocaine-taking behaviors on WD1 and WD45. E, Cocaine-taking behaviors tested by multiple dose
of cocaine (ie, 0.1, 0.3, 1.0, 3.0 mg/kg per infusion) on WD46-48. F, cocaine (0.1 mg/kg per infusion)-taking behaviors of individual rat are
represented by the circles in the columns. Data were analyzed by one-way ANOVA (each curve in B, C) and two-way ANOVA with repeated
measures (D,E), followed by Bonferroni post-hoc tests or Student’s t test (F). n = 11 in limited cocaine IVSA group; n = 10 in extended
cocaine IVSA group. *P < .05; **P < .01, comparations between day 1 vs any of other days during the IVSA training (C), WD1 vs WD45 (D), or
limited vs extended IVSA groups (E,F)
| LUO et al.  199
cocaine IV delivery after active lever press, followed by a 20-second
timeout during which the cue light signals were presented and no cocaine delivery was allowed. This 2-hour daily session was done once
per day for 3 consecutive days (ie, WD46-48). The average number
of cocaine infusions from 3 30-minute sessions with a specific dose
of cocaine was analyzed as an index of cocaine-taking level.
2.3 | Brain slice whole-cell patch-clamp recordings
Standard procedures were used for preparing slices and
whole-cell patch-clamp recordings as detailed in our previous
publications.23,24,27 Before sacrifice, the rats were anesthetized
with isoflurane and subsequently transcardially perfused with 4°C
cutting solution (in mM: 135 N-methyl-D-glucamine, 1 KCl, 1.2
KH2PO4, 0.5 CaCl2, 1.5 MgCl2, 20 choline-HCO3, 11 glucose, pH
adjusted to 7.4 with HCl, and saturated with 95% O2/5% CO2).
The rat was decapitated, and then the brain was removed and
glued to a block before slicing using a Leica VT1200s vibratome
in 4°C cutting solution. Coronal slices of 250-µm thickness were
cut such that the preparation contained the signature anatomical
landmarks (eg, the rhinal fissure, the anterior commissure, and
the corpus callosum) that clearly delineate the AAI area. After
allowing at least 1 hour for recovery, slices were transferred from
a holding chamber to a submerged recording chamber where it
was continuously perfused with oxygenated ACSF maintained at
30 ± 1°C.
Standard whole-cell current- or voltage-clamp recordings were
obtained with a MultiClamp 700B amplifier (Molecular Devices), filtered at 3 kHz, amplified 5 times, and then digitized at 20 kHz with
a Digidata 1550B analog-to-digital converter (Molecular Devices).
The recording electrodes (3-5 MΩ) were filled with (in mmol/L): 108
KMeSO3, 20 KCl, 0.4 K-EGTA, 10 HEPES, 2.5 Mg-ATP, 0.25 Na-GTP,
7.5 phosphocreatine (Na2), 1 L-glutathione, 2 MgCl2, pH 7.3. The
recording bath solution contained (in mM): 119 NaCl, 2.5 KCl, 2.5
CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, saturated with 95% O2/ 5% CO2 at 30 ± 1°C. Details for whole-cell patchclamp recordings can be found in one of our previous publications.27
Cells were patched in voltage clamp mode and held at −70 mV. Cell
membrane capacitance (Cm), input resistance (Rm), and time constant (τ) were calculated by applying a depolarizing step voltage
command (5 mV) and using the membrane test function integrated in
the pClamp10 software. Then, recordings were switched to current
clamp mode. Resting membrane potential was measured and then
adjusted to −70 mV through injection of positive current (50-100 pA).
The intrinsic excitability was examined using a series of depolarizing
current pulses and by constructing input-output (I-O) functions.
2.4 | Data acquisition and analysis
Data were collected either 2 days or 21 days after the last IVSA
session. All results are shown as mean ± SEM. Each experiment
was replicated in at least 10 rats for in vivo behavioral tests and at
FIGURE 2 Effects of limited vs extended cocaine IVSA on the kinetics of action potentials. A, A sample trace showing the second
action potential induced by 300 pA injection current was analyzed for the spike kinetics. B, A sample trace of action potential showing the
measurement of rise time, decay time, and amplitude. C, Sample trace of action potential from the pyramidal neurons in the IC 1 d (left)
and 45 d (right) after limited saline (denoted “S”)/ cocaine (denoted “C”) IVSA training. D, E, Summarized data showing that, relative to the
saline controls, limited cocaine treatment significantly prolonged the rise time (D), decay time (E), and half-amplitude duration (F) of action
potential 45 d but not 1 d after IVSA training. G, Summarized data showing that limited cocaine treatment significantly decreased the
amplitude of action potential on WD45, relative to WD1. H, Sample trace of action potential from the pyramidal neurons in the IC 1 d (left)
and 45 d (right) after extended saline (in gray)/ cocaine (in black) IVSA training. I-L, Summarized data showing no difference between saline
controls vs extended cocaine-treated rats in rise time (I), decay time (J), half-amplitude duration (K), or amplitude (L) of action potential on
both 1 d and 45 d after IVSA training. Data were analyzed by two-way ANOVA, followed by Bonferroni post-hoc tests. n/m, the number of
cells/animals for data collection. *P < .05
200  |    LUO et al.
least 4 rats (usually 2-4 cells per animal were recorded per group)
for in vitro electrophysiological recordings. Sample size is presented as n, referring to the number of rats in behavioral tests, or
presented as m/n, where “m” refers to the number of cells examined and “n” refers to the number of rats used for electrophysiology experiments. Statistical significance was assessed using
the Student t test (Figure 1F), one-way ANOVA with repeated
measures (each individual curve in Figure 1B,C), two-way ANOVA
(Figures 2D-G,I-L and 4A-D), or two-way ANOVA with repeated
measures (Figures 1D,E and 3B,D,F,H), followed by Bonferroni
post-hoc tests. The analytical unit is the animal in behavioral data
(Figure 1), or both the cell and the animal in the in vitro slice recording data (Figure 2-4).
3 | RESULTS
3.1 | Effects of limited vs extended cocaine IVSA on
cocaine-seeking and cocaine-taking behaviors during
the withdrawal period
Adolescent rats (n = 21), implanted with catheter on postnatal day
35 and recovered for ~1 week, were trained by limited (ie, 1 hour
per day for 5 days) or extended (ie, 6 hours per day for 21 days)
cocaine IVSA procedure, followed by a period of forced abstinence
during which the rats were just housed in the home cage, except on
WD1 and WD45 when they underwent the cocaine-seeking test,
and on WD46-48 when they received the cocaine-taking test in
the operant chambers as shown in Figure 1A. A flat curve of the
daily cocaine infusion (F4,40 = 1.4, P = .24) and the number of active
lever presses (F4,40 = 2.2, P = .09) was observed during the 5 day
of limited cocaine IVSA training sessions (Figure 1B). An increasing number of daily cocaine infusions (F20,180 = 24.9, P < .01) and
active lever presses (F20,180 = 8.0, P < .01) were observed during
21-day extended cocaine IVSA sessions (Figure 1C). During the
withdrawal period, the cocaine-seeking behaviors were increased
on WD45, compared with that on WD1, regardless of the limited
vs extended cocaine history (limited/extended F1,18 = 0.2, P = .20;
WD1/WD45 F1,18 = 62.4, P < .01; limited/extended × WD1/WD45
interaction F1,18 = 2.0, P = .17; Figure 1D). Interestingly, rats with
a history of limited cocaine IVSA showed an upward shift of doseresponse curve (ie, the dose of cocaine and the cocaine-taking
response), compared with that from the extended group (limited/
extended × cocaine dose interaction F3,57 = 3.0, P = .04; limited/
extended F1,19 = 7.9, P = .01; cocaine dose F3,57 = 60.4, P < .01;
Figure 1E), partially demonstrated at the dose of 0.1 mg/kg cocaine
FIGURE 3 Effects of limited vs extended cocaine IVSA on spike number of pyramidal neurons in the IC induced by current injections.
A, B, Example traces (A) and summarized data (B) showing no significant effects of limited cocaine IVSA on spike number 1 d after IVSA
training. C, D, Example traces (C) and summarized data (D) showing no significant effects of limited cocaine IVSA on spike number 45 d after
IVSA training. E, F, Example traces (E) and summarized data (F) showing no significant effects of extended cocaine IVSA on spike number
1 d after IVSA training. G, H, Example traces (G) and summarized data (H) showing no significant effects of extended cocaine IVSA on spike
number 45 d after IVSA training. Data were analyzed by two-way ANOVA with repeated measures, followed by Bonferroni post-hoc tests.
n/m, the number of cells/animals for data collection
| LUO et al.  201
per infusion (t19 = 2.5, P = .02; Figure 1F). Drug-seeking and drugtaking behaviors after a prolonged withdrawal period have been
well accepted as reliable indices of relapse.29-31 Thus, we conclude
that the extended cocaine IVSA procedure used in the current
study did not increase the risk for relapse. Interestingly, the limited
cocaine IVSA procedure may increase the relapse risk due to significantly increased drug-taking behaviors on WD45.
3.2 | Effects of limited vs extended cocaine IVSA
on the kinetics and amplitude of evoked action
potentials in insula pyramidal neurons
Whole-cell current clamp recordings in IC-containing coronal
slices were performed in rats 1 day or 45 days after IVSA of saline
or cocaine. The kinetics and amplitude of the action potential were
analyzed by measuring the 2nd action potential induced by 300 pA
current injection (Figure 2A). The insula pyramidal neurons from
rats with a history of limited IVSA procedure, that is, 1 hour per day
for 5 days, displayed significantly increased rise time (Figure 2D,
saline/cocaine × WD1/WD45 interaction F1,65 = 4.1, P = .04, saline/cocaine F1,65 = 21.3, P < .01, and WD1/WD45 F1,65 = 0.1,
P = .80, cell-based; saline/cocaine × WD1/WD45 interaction
F1,19 = 4.4, P = .049, saline/cocaine F1,19 = 12.1, P < .01, and WD1/
WD45 F1,19 = 0.1, P = .66, animal-based), decay time (Figure 2E,
saline/cocaine × WD1/WD45 interaction F1,65 = 4.2, P = .04, saline/cocaine F1,65 = 30.0, P < .01, and WD1/WD45 F1,65 = 4.9,
P = .03, cell-based; saline/cocaine × WD1/WD45 interaction
F1,19 = 4.5, P = .047, saline/cocaine F1,19 = 27.3, P < .01, and WD1/
WD45 F1,19 = 4.5, P = .047, animal-based), half-amplitude duration
(Figure 2F, saline/cocaine × WD1/WD45 interaction F1,65 = 4.7,
P = .03, saline/cocaine F1,65 = 36.4, P < .01, and WD1/WD45
F1,65 = 3.0, P = .08, cell-based; saline/cocaine × WD1/WD45 interaction F1,19 = 4.6, P = .045, saline/cocaine F1,19 = 24.9, P < .01,
and WD1/WD45 F1,19 = 2.7, P = .12, animal-based), and decreased
amplitude of the evoked action potential (Figure 2G, saline/cocaine × WD1/WD45 interaction F1,65 = 14.1, P < .01, saline/cocaine F1,65 = 6.9, P = .01, and WD1/WD45 F1,65 = 9.0, P < .01,
cell-based; saline/cocaine × WD1/WD45 interaction F1,19 = 10.8,
P = .01, saline/cocaine F1,19 = 6.4, P = .02, and WD1/WD45
F1,19 = 8.4, P = 0.01, animal-based) 45 days, but not 1 day, after
cocaine IVSA, compared with saline controls. However, no significant differences in rise time (Figure 2I, saline/cocaine × WD1/
WD45 interaction F1,68 = 0.23, P = .63, saline/cocaine F1,68 = 0.5,
P = .50, and WD1/WD45 F1,68 = 1.6, P = .22, cell-based; saline/
cocaine × WD1/WD45 interaction F1,19 = 0.4, P = .53, saline/cocaine F1,19 = 1.3, P = .27, and WD1/WD45 F1,19 = 0.5, P = .49,
animal-based), decay time (Figure 2J, saline/cocaine × WD1/
WD45 interaction F1,68 = 0.2, P = .67, saline/cocaine F1,68 = 1.0,
P = .32, and WD1/WD45 F1,68 = 1.4, P = .24, cell-based; saline/
FIGURE 4 Effects of limited vs
extended cocaine IVSA on RMP and
action potential threshold. A, Summarized
data showing no significant effects of
limited IVSA on RMP in insula pyramidal
neurons. B, Summarized data showing
no significant effects of limited IVSA
on action potential threshold in insula
pyramidal neurons. C, Summarized data
showing no significant effects of extended
IVSA on RMP in insula pyramidal
neurons. D, Summarized data showing
no significant effects of limited IVSA
on action potential threshold in insula
pyramidal neurons. Data were analyzed by
two-way ANOVA, followed by Bonferroni
post-hoc tests. n/m, the number of cells/
animals for data collection
202  |    LUO et al.
cocaine × WD1/WD45 interaction F1,19 = 0.4, P = .54, saline/cocaine F1,19 = 0.8, P = .37, and WD1/WD45 F1,19 = 1.5, P = .25,
animal-based), half-amplitude duration (Figure 2K, saline/cocaine × WD1/WD45 interaction F1,68 = 0.1, P = .75, saline/cocaine F1,68 = 1.2, P = .28, and WD1/WD45 F1,68 = 0.4, P = .51,
cell-based; saline/cocaine × WD1/WD45 interaction F1,19 = 0.3,
P = .57, saline/cocaine F1,19 = 1.1, P = .30, and WD1/WD45
F1,19 = 0.6, P = .44, animal-based), and action potential amplitude
(Figure 2L, saline/cocaine × WD1/WD45 interaction F1,68 = 2.1,
P = .15, saline/cocaine F1,68 < 0.1, P = .88, and WD1/WD45
F1,68 = 0.5, P = .49, cell-based; saline/cocaine × WD1/WD45 interaction F1,19 = 2.3, P = .15, saline/cocaine F1,19 = 0.1, P = .42,
and WD1/WD45 F1,19 = 0.4, P = .55, animal-based) were detected
in rats with an extended IVSA (ie, 6 hr per day for 21 days) of saline vs cocaine either 1 day or 45 days after the last IVSA training
session, demonstrating significant kinetic changes of APs after a
prolonged withdrawal period from limited but not extended IVSA
of cocaine.
3.3 | Effects of limited vs extended cocaine IVSA on
intrinsic excitability of insula pyramidal neurons
The number of spikes evoked by depolarizing current injections
ranging from 50 to 400 pA were also analyzed. Our data showed
that, relative to saline controls, cocaine IVSA procedure did not
affect the number of action potentials evoked 1 day vs 45 days
after the last IVSA session (Figure 3). Specifically, neither
significant effects of limited cocaine IVSA on spike number were
detected 1 day after IVSA training (Figure 3B, saline/cocaine × I
inj
interaction F7,266 = 0.5, P = .85, and saline/cocaine F1,38 < 0.1,
P = .82, cell-based; saline/cocaine × I
inj interaction F7,77 = 0.5,
P = .83, and saline/cocaine F1,11 < 0.1, P = .89, animal-based)
or 45 days after IVSA training (Figure 3D, saline/cocaine × I
inj
interaction F7,224 = 0.2, P = .98, and saline/cocaine F1,32 = 0.1,
P = .77, cell-based; saline/cocaine × I
inj interaction F7,56 = 0.3,
P = .95, and saline/cocaine F1,8 = 0.1, P = .77, animal-based), nor
significant effects of extended cocaine IVSA on spike number were
detected 1 day after IVSA training (Figure 3F, saline/cocaine × I
inj
interaction F7,273 = 0.8, P = .60, and saline/cocaine F1,39 = 0.4,
P = .55, cell-based; saline/cocaine × I
inj interaction F7,70 = 1.0,
P = .46, and saline/cocaine F1,10 = 0.4, P = .54, animal-based)
or 45 days after IVSA training (Figure 3H, saline/cocaine × I
inj
interaction F7,266 = 0.2, P = .98, and saline/cocaine F1,38 = 0.1,
P = .79, cell-based; saline/cocaine × I
inj interaction F7,63 = 0.2,
P = .97, and saline/cocaine F1,9 = 0.1, P = .80, animal-based).
Further data analyses showed no significant difference of
RMPs in rats with a history with limited (Figure 4A, saline/cocaine × WD1/WD45 interaction F1,70 = 2.2, P = .14, saline/cocaine
F1,70 = 1.1, P = .30, and WD1/WD45 F1,70 = 0.1, P = .79, cellbased; saline/cocaine × I
inj interaction F1,19 = 2.4, P = .14, saline/
cocaine × F1,19 = 0.9, P = .35, and WD1/WD45 F1,19 = 0.1, P = .80,
animal-based) or extended (Figure 4C, saline/cocaine × WD1/
WD45 interaction F1,77 = 3.3, P = .08, saline/cocaine F1,77 = 3.1,
P = .08, and WD1/WD45 F1,77 = 1.0, P = .34, cell-based; saline/cocaine × I
inj interaction F1,19 = 3.0, P = .10, saline/cocaine × F1,19 = 3.1, P = .09, and WD1/WD45 F1,19 = 1.0, P = .38,
animal-based) cocaine vs saline IVSA. Finally, limited (Figure 4B,
saline/cocaine × WD1/WD45 interaction F1,70 = 0.1, P = .79,
saline/cocaine F1,70 < 0.1, P = .97, and WD1/WD45 F1,70 = 0.2,
P = .67, cell-based; saline/cocaine × I
inj interaction F1,19 = 0.1,
P = .78, saline/cocaine × F1,19 < 0.1, P = .95, and WD1/WD45
F1,19 = 0.1, P = .71, animal-based) or extended (Figure 4D, saline/
cocaine × WD1/WD45 interaction F1,77 = 0.3, P = .58, saline/cocaine F1,77 = 3.0, P = .09, and WD1/WD45 F1,77 = 1.6, P = .21,
cell-based; saline/cocaine × I
inj interaction F1,19 = 0.4, P = .54,
saline/cocaine × F1,19 = 2.7, P = .11, and WD1/WD45 F1,19 = 1.6,
P = .22, animal-based) cocaine IVSA did not significantly influence
the threshold of action potential. Thus, the membrane excitability
of AAI pyramidal neurons was not affected by either limited or
extended cocaine IVSA history.
4 | DISCUSSION
Our data demonstrated no differences of drug-seeking behaviors
after limited vs extended cocaine IVSA, although both limited and
extended IVSA rats showed higher drug-seeking behaviors on
WD45, compared with WD1, which indicates the duration of withdrawal period can significantly increase the drug-seeking behaviors as evidenced before.32-35 Importantly, limited cocaine IVSA
significantly increased cocaine-taking behaviors compared with
the extended cocaine IVSA group as supported by the upward
shift of the dose-response curve of cocaine taking at the later
withdrawal stage. Thus, limited, compared with extended, cocaine
history, may lead to a potential high risk of relapse to cocaine.
Notably, this behavioral outcome dispels the current belief that
extended drug exposure has more robust effects on drug-seeking
and drug-taking behaviors. For example, extended access to drugs
significantly increased drug-taking behaviors in the withdrawal
period36-38 and compulsive drug-seeking behaviors could only
be established in rats by extended but not limited cocaine IVSA
procedure39. It is critical to point out the developmental stages
when drug exposure and drug withdrawal occur. Relative to adults,
adolescent rats took cocaine more readily, were more sensitive to
lower doses, and showed greater escalation of cocaine intake.40
Thus, our IVSA training was done at the adolescence stage. On
another hand, it has been fashionable in addiction research to run
drug exposure with extended daily sessions (≥6 hours) during a
long period (eg, at least 10 days).41-44 However, studies on limited
vs extended drug exposure in adolescent animals were still lacking.
Most, if not all, of the previous studies comparing the prolonged
effects of limited vs extended drug exposure on drug-seeking/taking behaviors used adult animals, whereas rats were adolescent
when the current experiments started. We found high risk of relapse in rats with limited but not extended drugs at the adolescent
| LUO et al.  203
stage. Thus, it is assumed the developmental stage of drug experience matters more than the amount of drug in the young brain.
This is based on several observations: First, at the initial
drug-taking stage, young brains may be more sensitive to the
effects of drugs. This vulnerability of adolescents may be fully
masked by chronic drug administration.21,22 According to clinical
observations, the onset of drug use during adolescence appears
to lead to a prolonged sensitivity to drug relapse and dependence
in humans.45,46 Second, withdrawal experience of the young brain
may trigger more intensive neuronal alterations that facilitate the
drug-seeking and drug-taking behaviors even after a prolonged
withdrawal period. Our previous works demonstrated active neuronal adaptations by the passage of the withdrawal period.23,24,47
The current limited cocaine IVSA procedure involves at least two
factors, which significantly increase the risk of drug-taking behaviors at the late withdrawal time. One is the limited exposure of
cocaine, which is enough for young brains to be reshaped with
the abusive potential of drugs. The other is the withdrawal period
starting from the adolescent stage when the brain is highly malleable. Our current conclusion, that is, high risk of relapse associated
with limited drug exposure at the adolescent stage, followed by a
withdrawal period, is not contradictory but an essential complement to the established addiction theory.
Low drug taking observed in rats with a history of extended cocaine IVSA might be attributed to their poor learning and memory,
which resulted in a poor retrieval of the previously learned association
between the action (ie, lever press) and output (cocaine IV delivery and
cue light) in the seeking/taking test during the withdrawal period. This
can be excluded by the comparable drug-seeking behaviors between
rats pre-treated by limited vs extended cocaine IVSA on either WD1
or WD45, and comparable drug-taking behaviors at 10 mg/kg cocaine
per injection on WD46-48, indicating the retrieval of previous acquired
operant behaviors was intact. Also, the poor locomotion could be a
consequence of extended cocaine IVSA, which can be excluded by the
comparable number of inactive lever presses in rats treated by limited
vs extended cocaine IVSA (eg, active lever presses in 1-hour drug-seeking test on WD45: limited, 10.1 ± 3.4; extended, 8.7 ± 2.7, P = .34).
Our data showed escalated drug intake in rats with extended
IVSA procedure, consistent with previous observations.48 However,
there are some exceptions; data from rhesus monkeys showed no
escalated intake over time after extended access to cocaine IVSA.49
We found that rats with escalated drug-taking behaviors during the
extended cocaine IVSA procedure did not show high risk of relapse.
Together with the previous report that the presence of escalation
did not affect reinstatement,48 we assume that the presence of escalation has limited predictability on the risks of relapse. Escalation of
cocaine taking was produced by continuous cocaine IVSA.50
Although the critical involvement of the IC in addiction-related
behaviors has been widely accepted, little was known about the intrinsic excitability of IC pyramidal neurons in rats which experienced
limited vs extended IVSA. We found some minor but significant effects of limited but not extended cocaine exposure on the kinetics
and amplitude of action potentials on WD 45 after limited cocaine
IVSA, in pyramidal neurons from the AAI. Although our original hypothesis was that, compared with the saline controls, extended, but
not limited, cocaine exposure may lead to significant changes (eg,
modified intrinsic excitability) in pyramidal neurons in the AAI, we
did not see any effects of extended cocaine IVSA on this electrophysiological readout in insular pyramidal neurons. These findings
indicate that limited cocaine exposure has some, probably priming,
effects on the capacity of IC neurons to generate APs and these effects are compensated after extended cocaine exposure. It is worth
to note that the slower kinetics and smaller amplitude of action potentials observed on WD45 after limited cocaine IVSA could be also
observed in unhealthy cells. Our cells in different groups were similar in terms of access resistance and holding current evaluated by
Membrane Test in voltage clamp, indicating comparable quality of
cells among groups. Thus, the observed changes in kinetics and amplitude of action potentials might be attributable to the adaptations
of ion channels (eg, Na+
and K+
).
The involvement of the IC in cocaine-related effects is well
supported by previous literature. First, molecular alterations in
the IC were detected 21 days but not 1 day after 5-day cocaine
exposure. Specifically, the cocaine challenge produced a decrease
in zif 268 expression after the 21-day, but not 1-day, withdrawal
period.51 Second, by using an extended procedure of cocaine access, the anterior IC was found to mediate the maintenance of escalated cocaine-taking behaviors but not the initial development of
cocaine-taking behaviors.52 Third, different subregions of IC may
be involved in cocaine-seeking behaviors triggered by different
factors. Agranular IC was involved in drug context-induced reinstatement of cocaine-seeking behavior without altering locomotor
activity.53 Anterior dorsal agranular IC participated in cue-associated cocaine-seeking behaviors,54 whereas rostral agranular IC was
reported to be related to cocaine-seeking behaviors intensified by
negative affect.55 Last but not least, human studies also demonstrated changes in the IC, as it was reported by human imaging
studies that cocaine dependence is related to altered functional interactions of the IC with prefrontal networks.56
5 | CONCLUSIONS
Compared with the extended cocaine IVSA, adolescent limited
cocaine IVSA increased drug-taking behaviors, indicating the importance of the developmental stage of initial drug exposure and
the withdrawal period. The minor neuronal adaptations in the AAI
may not be the primary neuronal substrate involved in high risk of
relapse induced by adolescent brief exposure to cocaine. Future
studies may refine the neuronal and molecular substrate explorations in the IC by investigating other IC subregions, other types
of neurons such as GABAergic interneurons, changes in synaptic
transmission, etc. Obviously, other brain regions, such as nucleus
accumbens and mPFC, may be explored to identify the key neurological events which cause the limited drug exposure-induced high
drug-taking behaviors.
204  |    LUO et al.
ACKNOWLEDGMENTS
This work was supported by NIH grants (R01AA025784) and Brain &
Behavior Research Foundation grant #24989.
CONFLICT OF INTEREST
The authors report no conflicts of interest.
DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of
this study are available (1) within this article and (2) from the
corresponding author upon reasonable request.
ORCID
Yao-Ying Ma https://orcid.org/0000-0003-3735-8412
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How to cite this article: Luo Y-X, Huang D, Guo C, Ma Y-Y.
Limited versus extended cocaine intravenous selfadministration: Behavioral effects and electrophysiological
changes in insular cortex. CNS Neurosci Ther. 2021;27:196–
205. https://doi.org/10.1111/cns.13469

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