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DopamineD1receptoractivityisinvolvedintheincreasedanxietylevelsobservedinSTZ-induceddiabetesinrats

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DOI:10.1016/j.bbr.2016.06.060

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Behavioural Brain Research 313 (2016) 293–301

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

opamine D1 receptor activity is involved in the increased anxietyevels observed in STZ-induced diabetes in rats

aniela Rebolledo-Solleiroa, Luis Fernando Ontiveros Araizaa, Laura Broccolib,nita C. Hanssonb, Luisa Lilia Rocha-Arrietac, Raúl Aguilar-Robleroa,inerva Crespo-Ramíreza, Kjell Fuxed, Miguel Pérez de la Moraa,∗

Division of Neuroscience, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, MexicoNeuroanatomy Research Group, Institute for Psychopharmacology at Central Institute for Mental Health, Medical Faculty Mannheim, University ofeidelberg, GermanyDepartment of Pharmacobiology, Instituto de Investigación y Estudios Avanzados (CINVESTAV) Sede Sur, Mexico City, MexicoDivision of Cellular and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institutet, Sweden

i g h l i g h t s

Anxiety-like behavior is seen in rats made diabetic by streptozotocin treatment.Amygdaloid D1 receptor blockade reduces streptozotocin induced anxiety-like behavior.Streptrozotocin treatment elicits overexpression of amygdaloid D1 receptors.

r t i c l e i n f o

rticle history:eceived 21 April 2016eceived in revised form 26 June 2016ccepted 29 June 2016vailable online 30 June 2016

eywords:nxietyiabetesopaminemygdala

ntercalated islandshock/Probe Burying Test

a b s t r a c t

Epidemiological surveys have indicated that anxiety disorders are more frequent in diabetic patients thanin the general population. Similar results have been shown in animal studies using the streptozotocin(STZ)-induced diabetes model. The mechanisms underlying this relationship are not clearly understood,but it has been suggested that alterations in the dopaminergic neurotransmission, which plays an impor-tant role in the amygdaloid modulation of fear and anxiety, may be involved. The aim of this study wasto ascertain whether or not the amygdaloid DA D1 receptors are involved in the increase of anxiety-likebehavior observed in “diabetic” animals. Adult Wistar male rats were injected with STZ (50 mg/kg, i.p.)in two consecutive days and subjected to the Shock-Probe Burying Test 10 days after the beginning oftreatment. STZ-treated rats showed a significant increase in immobility/freezing behavior whereas noeffects were elicited in latency to bury, burying behavior itself and the number of shocks received duringtesting as compared with non-diabetic controls. These results suggest the triggering of a passive copingresponse in the STZ-treated rats. Interestingly, immobility/freezing behavior was reversed following the

intra-amygdaloid dopamine D1 receptor blockade by the local microinfusion of SCH23390 (100 ng/side).Autoradiographic experiments showed a selective increase of [3H]-SCH23390 binding in the ventral inter-calated paracapsular islands of STZ-treated rats when compared to the non-treated control group. Ourresults suggest that a hyperdopaminergic state involving DA D1 receptors within the amygdala may havea role in the increase of anxiety observed in diabetic rats.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

There is strong evidence that type-1 diabetes mellitus (T1DM)ncreases the risk for affective and anxiety disorders. Using a

∗ Corresponding author at: Apartado Postal 70-253, México, DF 04510, Mexico.E-mail address: mperez@ifc.unam.mx (M. Pérez de la Mora).

ttp://dx.doi.org/10.1016/j.bbr.2016.06.060166-4328/© 2016 Elsevier B.V. All rights reserved.

meta-analytical approximation, [1] showed evidence for an asso-ciation between anxiety disorders and hyperglycemia in diabeticpatients. In the last decade, it has also been established that theprevalence of anxiety disorders (generalized anxiety disorder, ago-raphobia, social phobia, and post-traumatic stress disorder) in

diabetic patients is higher than in the general population [28,9,32]supporting the idea of a relationship between diabetes and anxiety.

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Emotional alterations in diabetes have also been studied in ani-al models with a special focus on depression and anxiety. One

f the most popular and well-characterized methods to induce ayperglycemic condition is the streptozotocin-induced diabetesSID) model. Rodents treated with streptozotocin (STZ) exhibitypoinsulinemia [25,20,24], hyperglycemia, glycosuria, polyuria25], polyphagia and polydypsia [4] among other symptoms thatharacterize T1DM in humans. They also show a marked loss ofody weight [25,3,24]. Using this animal model, it has been demon-trated that hyperglycemic rodents exhibit increased anxiety-likeehaviors in different paradigms, such as the social interaction test,he open-field test (OFT), the zero maze, [42], the Elevated-Plus

aze (EPM) [48,42] and the hole-board test [27].Anatomically, a number of brain regions have been involved

n the modulation of anxiety. The amygdala, however, seems tolay a major role in this modulation since relevant sensory infor-ation from the environment reaches its lateral and basolateral

mygdaloid (BLA) nuclei and after being locally processed is finallyonveyed to the central nucleus (CeA) where a proper anxietyesponse is implemented [10,35]. From a biochemical point of view,t is interesting that dopaminergic mechanisms seem to have a

ajor influence on the amygdaloid modulation of anxiety (for aeview see [41]).

The mechanisms underlying the relationship between anxietynd diabetes are not clearly understood, but it has been suggestedhat alterations in the dopaminergic neurotransmission, may benvolved. Post mortem studies have demonstrated that the contentf dopamine (DA) in diabetic brain was increased in the medialypothalamus, caudate putamen, and medial and lateral pallidus30]. Likewise, [15] demonstrated that in STZ-treated rats the mRNAf tyrosine hydroxylase, the rate-limiting enzyme for DA and nora-renaline synthesis, was up-regulated in the locus coeruleus and inhe ventral tegmental area. Using the SID model both an increase43] and a decrease [50,31] in dopamine turnover has been reportedn the hippocampus and the striatum, respectively. It has alsoeen shown that rats rendered hyperglycemic by STZ treatmentresented a significant decrease in DA content in the hypotha-

amus and the brainstem [44] and that there also exists in theat a decrease in the extracellular levels of dopamine, as mea-ured by microdialysis from hippocampus of awake freely movingTZ-treated [53,43] and spontaneously diabetic [53] rats. In addi-ion, both an increase in [3H]spiperone binding, a DA D2 receptorntagonist, to striatal membranes [33], and an enhancement of3H]dopamine binding together with an up-regulation of mRNA forA D1 and D2 receptors in the hippocampus has also been pub-

ished [43]. Although these data show evidence of dopaminergicystem alterations in STZ-treated rats within distinct brain regions,he role of this neurotransmitter system within the amygdala hasot yet been studied in diabetic individuals. The aim of this work iso shed some light on the possible role of the amygdaloid dopamin-rgic system in the modulation of the increased anxiety responsesbserved in rodents in this pathological condition.

. Methods

.1. Animals

Male Wistar rats 250–300 g from Instituto de Fisiología Celu-ar, Universidad Autónoma de México, were housed in a controllednvironment (temperature 22 ◦C, lights on 07:00–19:00 h) withater and food (Purina chow) ad libitum. The experiments were

onducted according to the guidelines established by the local Mex-can Ethics Committee in agreement with NIH publication 80–23,evised 1996. Efforts were taken to minimize the suffering of thenimals throughout all experimental procedures.

rain Research 313 (2016) 293–301

2.2. Streptozotocin-induced diabetes

For the behavioral experiments animals were injected with adose of 100 mg/kg STZ i.p. (diluted in citrate buffer pH 4.8) adminis-tered on two consecutive days (50 mg/kg) (n = 24), as described by[52]. Non-diabetic animals received vehicle (Citrate Buffer 0.1 M)only (n = 24). Behavioral evaluations were made 10 days after thistreatment, as reported by [43]. Rats with serum glucose levelsabove 250 mg/dL were considered diabetic [6].

2.3. Behavioral procedures

Animals were handled for 5 min once a day during 4 consecu-tive days. Rats were kept overnight in the experimental room inorder to maintain their basal anxiety levels and minimize stress-ful stimuli. The day of the experiment, each rat was removed fromits home cage and put in the adequate apparatus for behavioralevaluation in the following order: Shock Probe/Burying and OpenField Tests (see below). Behavioral experiments were conductedbetween 10:00 and 16:00 h in a sound-attenuated room equippedwith video-recording facilities. The devices used for the evaluationof behavior were placed beneath the video camera, and behaviorwas recorded in the absence of any observer. In all the experimentsrats were assigned to each group in a randomized manner and wereused only once.

2.4. Shock-Probe/Burying Test

The test was carried out essentially as described by [49] and[14]. See also [40,41]. The test was conducted in an acrylic cage(27 × 16 × 23 cm) with its floor covered with a uniform layer (5 cm)of fine sawdust. The cage was equipped with an electrified probe(7 cm long, 0.5 cm thick) which protruded from one of its walls, 5 cmabove the bedding and through which the rats received an electricshock (0.3 mA) any time they come into contact with the probe.The current was generated by a constant-current shock generator(LaFayette Instruments, Inc). During the test four parameters wererecorded: the total amount of time that the rat spends burying theprobe with the fore-paws (burying behavior), the total amount oftime that the rat spends lying or sitting motionless carrying outsmall and slow lateral head movements or doing only those move-ments required for breathing (immobility/freezing behavior), theburying behavior latency (represented by the time from the firstshock to the start of the burying behavior), and the number ofshocks received by the rat during the test.

2.5. Open field test

In order to analyze locomotor activity, we evaluated the ratsin an Open Field Test (OFT) immediately after the Shock/ProbeBurying Test. Locomotion in the OFT was carried out in an arena(40 × 40 × 30 cm) divided into 16 squares (10 × 10 cm) as previ-ously described [22]. Each rat was placed in the center of the arenaand was allowed to explore it for 5 min. Locomotion was regis-tered using a specialized software (Omni-Alba/AOBiomed, Mexico),which recorded light beam interruptions produced by the animal,while exploring the arena.

2.6. Amygdaloid DA D1 receptor binding assay

After injection of STZ as described above animals (n = 5/group)

were sacrificed 10 days after by decapitation and the brainswere rapidly removed, frozen in dry ice and stored at −70◦ Cuntil use. Vehicle treated rats (n = 6) were handled in a similarway. Frozen brains were sectioned at 18 �m thickness on the

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As seen in Table 1 STZ treatment resulted in a body weight loss10 days after the beginning of treatment and in a highly significantincrease in blood glucose levels.

Table 1Body weight and blood glucose levels in non-fasted STZ-induced diabetic rats.

Vehicle STZ (100 mg/Kg)

D. Rebolledo-Solleiro et al. / Behavio

oronal plane using a cryostat (CM-1510-3 Leica Instruments, Nuss-och, Germany). Sections were collected on cleaned-gelatin-coated

icroscope slides (6 sections/slide) and stored at −70◦ C until theay of incubation. In vitro autoradiography was performed on par-llel sections for labelling DA D1 receptors with [3H]-SCH2339010 nM, final concentration). Non-specific binding was measuredn the presence of 1 �M SKF 83566, as described previously by46] Brain sections were incubated for 15 min at room tempera-ure in 50 mM Tris HCl buffer (pH 7.4), containing 5 mM MgCl2 and

mM EDTA, 0.1 mM bacitracin and 0.1% bovine serum albumin.ncubation in [3H]-SCH23390 lasted 120 min at 30 ◦C in a humid-fied chamber and was terminated with three consecutive buffer

ashes (2 min each at 4 ◦C). Finally, the slides were rinsed (3 s) inistilled water at 4 ◦C and the sections were quickly dried under aentle stream of cold air.

FUJI imaging plates (Storage Phosphor Screen BAS-IP TR2025 Ecreen, GE Healthcare Life Sciences, Pittsburgh, USA) were exposedo sections for 6–7 days and afterwards scanned in a phosphorim-ger (Fuji Phosphorimager Typhoon FLA 700, GE Healthcare Lifeciences, Pittsburgh, USA). The MCID program (InterFocus Imag-ng Ltd, Cambridge, UK) was used for densitometry analysis. The3H]-quantitation standard curve (Amersham, GE Healthcare Lifeciences, Pittsburgh, USA) was used to interpolate the measuredptical densities (photostimulable luminescence per mm2) of theissue equivalent DA D1 receptors densities from sections intoCi/mg. Binding in femtomoles per milligram (fmol/mg) was cal-ulated based on the specific activity of the radioligand and theaturation binding equation (B = Bmax*[R]/(Kd +[R]), solving formax, Bmax = maximal bound receptor/transporter, Kd = receptorffinity in nM) and data were expressed as fmol/mg proteinmean ± SEM). Brain regions were identified according to the rattlas of [37] within levels −1.80 to −2.8 mm from Bregma. Onlyections with well identified amygdaloid regions were included inhe statistical analysis.

.7. Surgical and microinjection procedures

For implantation of permanent cannulae into the amygdala, theats were anesthetized with ketamine hydrochloride (170 mg/kg,.p.) and placed in a stereotaxic frame (Kopf Instruments, Tujunga,A, USA) with the incisor bar set at −3.3 mm. The body temperatureas maintained at 37◦ C using a CMA/150 Temperature Controller

CMA/Microdialysis, Stockholm, Sweden). Bilateral stainless steeluide cannulas 0.46 mm o.d. (C315G, Plastics One, Roanoke, USA)ere positioned near the rostrolateral main intercalated islands

coordinates AP: −2.12 mm, L: ±4.5 mm, V: −7.7 mm) from bregmaccording to the atlas of [37]. Guide cannulae were affixed withtainless steel crews and dental acrylic cement (Laboratorios Arias,exico City) and sealed with a dummy cannula (C315DC, Plas-

ics One). Benza-Biotic L. A. (Laboratorios Tornel, Mexico) wasiven to prevent infection. After 5 days of recovery, the ratsere injected with STZ (50 + 50 mg/kg, i.p.) as mentioned above

nd behavioral evaluations in the Shock-Probe/Burying Test andhe OFT were made 10 days after the beginning of the injec-ion with STZ. Control rats received vehicle (Citrate Buffer) only.n the day of the experiment animals were divided at random

nto three different experimental conditions: control group (vehi-le + saline microinjection, n = 18); STZ + Saline group (STZ + salineicroinjection, n = 15) and STZ + SCH23390 (STZ + microinjection

f SCH23390, n = 18). Either SCH23390 (Tocris, Ellisville, USA), aA D1 receptor antagonist (100 ng per side diluted in 0.250 �l

aline) or saline vehicle (0.250 �l per side) was injected bilaterally

ia an injection cannula (0.2 mm outer diameter, C3151, Plasticsne) which protruded 1 mm beyond the end of the guide cannula.CH23390 or saline were injected over a period of 5 min, using

CMA/Microinjection Pump (CMA/Microdialysis). Cannulae were

rain Research 313 (2016) 293–301 295

kept in place for 30 s after the injection to prevent backflow of thesubstance and the behavioral tests started immediately after theinjection. The doses used here for SCH23390 were based on thework of [39], where they demonstrated that low doses of this com-pound (30–120 ng) have anxiolytic effects in the Black and WhiteBox Test.

2.8. Histological evaluation

In order to verify the placements of the cannulae implanted,at the end of the OFT, the animals were deeply anesthetized withsodium pentobarbital (65 mg per rat; PISA Agropecuaria, Mexico)and 0.2 �l of a diluted solution of Pontamine sky blue (Sigma) wasmicroinjected bilaterally via their injection cannulae. Brains wereremoved and postfixed by 10% formaldehyde for 1 or 2 weeks.Placement of the cannulae was verified on coronal sections (50micrometers) made with a cryostat (CM-1510-3, Leica Instruments,Nussloch, Germany) and counterstained with cressyl violet. Onlyrats with both cannulae tips within the amygdala (BLA and/or CeA)were included in this study.

2.9. Glucose levels

Following decapitation blood was collected from the trunk ofnon-fasted rats in heparinized tubes and glucose levels were mea-sured using Accucheck® reactive strips.

2.10 Statistical analysis.Kolmogorov-Smirnov test was used to assess whether exper-

imental groups exhibited a normal distribution. Parametricstatistics, either unpaired two-tail Student’s t-test or one-wayANOVA followed by the Tukey’s post-hoc test, was used to com-pare body weight and blood glucose levels among groups. Sincein the behavioral experiments not all the groups showed a nor-mal distribution their results were evaluated using non-parametricstatistics. Accordingly, Mann-Whitney U test was used to comparethe effects of the STZ-treated rats against its corresponding controlgroup. Effects of either STZ + saline or STZ + SCH23390 treatmentagainst the vehicle + saline control group were compared usingthe Kruskal-Wallis test followed by the Dunn as a post-hoc test.For DA D1 receptor autoradiography all data were expressed asmean ± SEM. The data met assumptions of normality and homo-geneity of variances and treatment effects were analyzed using theone-way analysis of variance (ANOVA) test in each amygdala region[46,7]. Significance was set at P < 0.05. Statistical parameters werecomputed using GraphPad Prism 5 and Statistica software.

3. Results

3.1. Effects of STZ treatment on body weight and blood glucoselevels

Body weight (g) 260 ± 3 210 ± 4***

Blood glucose (mg/dL) 120 ± 3 460 ± 4***

Mean ± SEM. Two-tailed, unpaired T-test, n = 24.*** P < 0.001.

296 D. Rebolledo-Solleiro et al. / Behavioural Brain Research 313 (2016) 293–301

Fig. 1. Effects of STZ treatment in rats submitted to the Shock-Probe/Burying test. A significant increase was noticed in immobility/freezing whereas non-significant effectswere elicited on the other behaviors studied. (a) Burying behavior latency. (b) Total time spent in burying behavior. (c) Number of shocks received. (d) Time spent inimmobility/freezing behavior. Bars represent medians are shown with their respective interquartile range (boxes). Whiskers indicate the highest and lowest values withint

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Fig. 2. Effects of STZ treatment on locomotor activity of rats in the Open-Field test.The number of events (number of beam interruptions) was significantly reduced in

he sample. Mann-Whitney two-tailed test, * P < 0.05 (n = 24 animals/group).

.2. Effects of STZ treatment in anxiety-like behavior andocomotion

STZ-treated rats showed a significant increase in immobil-ty/freezing behavior (Fig. 1d) in the Shock/Probe Burying test

hereas non-significant effects were observed in latency to buryFig. 1a) and the total time of burying behavior (Fig. 1b) as well asn the number of shocks received during testing (Fig. 1c) as com-ared with non-diabetic controls. Locomotor activity as measured

n the Open-Field test was significantly reduced in STZ-treated ratsompared with the vehicle group (Fig. 2).

.3. Dopamine D1 receptor binding in STZ-treated rats

Results on DA D1 receptor autoradiography under saturatedonditions from different amygdaloid subregions is summarizedn Table 2 and Fig. 3. In all amygdaloid regions [3H]-SCH23390

inding was numerically increased but did only reach statisticalignificance in the ventral intercalated paracapsular islands (IvP,(1,5) = 7.0; p < 0.05).A trend towards significance was also observedn the medial amygdaloid nucleus (MeA, F(1,8) = 3.754, p = 0.089).

the Open-Field test. Bars represent medians with the respective interquartile range(boxes). The whiskers indicate the highest and lowest values within the sample.Mann-Whitney two tailed test, ** P < 0.01 (n = 24 animals/group).

3.4. Effects of the bilateral intra-amygdaloid microinjection ofSCH23390 on body weight and blood glucose levels in STZ treatedrats following their exposure to the Shock-Probe Burying test

As seen in Table 2, a significant reduction in body weight andan increase in blood glucose levels were found in STZ-treated ratsas compared to their vehicle-treated controls. Similar effects ofthe bilateral intra-amygdaloid administration of STZ + SCH23390

D. Rebolledo-Solleiro et al. / Behavioural Brain Research 313 (2016) 293–301 297

Table 2Body weight and blood glucose levels in STZ-induced diabetic rats injected with SCH23390 into the amygdala.

Vehicle + Saline STZ (100 mg/Kg) + Saline STZ (100 mg/Kg) + SCH23390 (100 ng/side)

Body weight (g) 280 ± 5 250 ± 7*** 230 ± 5***

Blood glucose (mg/dL) 130 ± 5 550 ± 21*** 500 ± 20***

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ean ± SEM. One-way ANOVA followed by Post-hoc Tukey,*** P < 0.001 (vs. Vehicle + Saline).

ere observed on any of these parameters as compared to theirespective saline control groups.

.5. Effects of the intra-amygdaloid microinjection of SCH23390n the behavior of STZ-treated rats in the Shock-Probe Buryingest and on their locomotion in the Open-Field test

Cannulae placements within the amygdala are shown in Fig. 4.annulae tips were found within the rostral amygdala mostly

ocated between the BLA and CeA were the dopamine DA D1eceptor-rich main and medial intercalated paracapsular islandsre distributed. Behaviorally, a significant increase in immobil-ty/freezing behavior (KW stat(2.62) = 15.52; P < 0.01) was observedn STZ-treated rats when they were tested in the Shock-Probe Bury-ng test (Fig. 5d) without showing any changes in the latency toury (KW stat(2,62) = 4.30; P > 0.05) (Fig. 5a), the burying behavior

tself (KW stat(2.62) = 4.25; P > 0.05) (Fig. 5b) or the number of shockseceived (KW stat(2,62) = 7.64; P > 0.05) during testing (Fig. 5c) as

ompared with the control group. Immobility/freezing behavioras however prevented by the intra-amygdaloid microinfusion

f SCH23390 (Fig. 5d). Microinjection of SCH23390 within themygdala did not produce any effects on the locomotion (KW stat

ig. 3. Increased DA D1 receptor binding in the ventral intercalated paracapsular islandepresentation of measured areas and (right) quality of [3H]-SCH23390 autoradiography

f [3H]-SCH23390 binding within the amygdala of STZ- and vehicle treated control raescribed in Materials and Methods, with [3H]-SCH23390 (10 nM). Unspecific binding wccording to [8]. BLA: basolateral amygdaloid nucleus; CeA: central amygdaloid nucleusorsal intercalated islands; Ilp: lateral intercalated islands; Imp: medial intercalated islaxpressed as mean ± SEM. Statistical analysis was performed by region-wise one-way AN

(2.62) = 4.01; P > 0.05) of the animals when measured in the OpenField test, as compared with the control and the STZ + saline groups(Fig. 6).

4. Discussion

The current work was aimed to study by using thestreptozotocin-induced diabetes model whether or not amygdaloidDA D1 receptors may be involved in the increase of anxiety-likebehavior observed in diabetes-like states. The main finding of thispaper is that the blockade of amygdaloid DA D1 receptors preventsthe increase in anxiety (immobility/freezing) observed in strepto-zotocin treated rats.

The streptozotocin-induced diabetes model has been widelyused as an animal model of type-1 Diabetes Mellitus (T1DM)because of the ability of STZ to destroy �-pancreatic cells, generat-ing a severe hyperglycemia in experimental subjects [25,47]. Ourresults give evidence for this hyperglycemic condition and show

that the rats have a significant loss of body weight and exhibitsevere polydypsia (data not shown). These latter results indicatethat the animals studied in our experiments presented a condi-tion similar to the T1DM in humans, as reported by other authors

s after STZ treatment in Wistar rats. Upper row (left, midle) showing a schematicon a coronal amygdala rat section. Lower row showing quantitative measurementsts. Coronal sections (18 �m, Bregma level −1.80 to −2.8 mm) were incubated as

as determined in the presence of the D1 receptor antagonist SKF 83566 (1 �M); BMA: basomedial amygdaloid nucleus; IN: main intercalated island; Idp: medialnds; Ivp: ventral intercalated islands; MeA: medial amygdaloid nucleus. Data areOVA, n = 3-6/group, *p < 0.05.

298 D. Rebolledo-Solleiro et al. / Behavioural B

Fig. 4. Schematic representation of the sites of cannula implantation within theamygdala as verified by histological examination. Sections of amygdala were takenaccording to the rat brain atlas of [37] at levels: −1.8, −1.88, −2.12, −2.30,−2.56, −2.80, −3.14 and −3.30 mm from bregma, White circles: control group(vehicle + saline); gray circles: STZ + saline group; black circles: STZ + SCH23390group. Abbreviations (see −1.80 level): Aco: anterior cortical amygdaloid nucleus;BAOT: bed nucleus accessory olfactory tract; BLA: basolateral amygdaloid nucleus,ant; BMA: basomedial amygdaloid nucleus; CeC: Central nucleus, lateral divi-sion, capsular; CeM: central nucleus medial division; Cpu: caudate putamen; CxA:cas

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ortex-amygdala transition zone; Den: dorsal endopiriform nucleus; LaDL: lateralmygdaloid nucleus, dorsolateral; MeAD: medial amygdaloid nucleus, dorsal; SI:ustancia innominate; Ven: ventral endopiriform nucleus.

25,4,3,24]. It is important to mention that STZ is transported intohe �-cells through the glucose transporter GLUT-2, which is notresent in the hematoencephalic barrier [29]. Thus, the possibilityhat the STZ may have direct effects in the brain after its systemicdministration may be excluded. It may therefore be assumed thathe behavioral effects observed in our experiments were producedy the metabolic/hyperglycemic condition caused by the STZ treat-ent.Using the streptozotocin-induced diabetes model we were

ble to give novel evidence for a significant increase in immo-ility/freezing behavior [51,11] in STZ-treated rats when theyere evaluated in the Shock/Probe Burying test. Interestingly, no

ffects were observed in burying behavior. These results suggest

rain Research 313 (2016) 293–301

the preferential triggering of passive coping responses [11] in thehyperglycemic rats which may be related to the deteriorated phys-ical state of the animals which follows 10 days of the STZ treatmentand to the use of energy-saving behavioral strategies to cope withharmful situations. In support of this physically deteriorated statea decrease in body weight and locomotion was found in this study.Our results are also in line with studies carried out in STZ-treatedrats which report avoidance of the open arms in the Elevated Plus-Maze [48,42], a reduced exploration of the central space in theOpen-Field test [42] and a diminution in the number of head-dipsinto the holes of the Hole-Board test [27], which in these paradigmsrepresent passive coping strategies to avoid danger. Furthermore,the possibility that the anxiety-like behavior found in this workhad been produced as a consequence of unspecific effects of the STZtreatment seems unlikely since it was fully prevented by the amyg-daloid infusion of SCH23390, a DA D1 antagonist. Also, this DA D1antagonist treatment did not counteract the locomotor inhibitionfound in the STZ-treated rats.

The reason why STZ-induced reduction in locomotion wasobserved in intact but not in cannulated animals is not entirelyclear. However, besides the fact that the experimental variabilitybetween both experiments may have had a role in these differ-ences, we have to underline that high levels of stress have beenreported in chronically implanted animals [26] and that increasedlocomotion has been found in stressed animals [16]. In addition,a chronic state of stress exists in diabetes [54], characterized byan increased activity of the hypothalamus-pituitary-adrenal (HPA)axis [5,13].

Thus, it is feasible that a reduction in locomotion may be pro-duced, as shown in this work, when saline-treated groups fromcannulated rats are compared with non-cannulated saline treatedgroups. In contrast, streptozotozin-treated animals showing highlevels of stress will hardly increase their stress levels by cannula-tion and will display a similar locomotor activity to non-cannulatedanimals as also found in this work. Under these conditions it is notsurprising that streptozotozin treatment in non-cannulated rats(Fig. 2) resulted in a decrease in locomotion when STZ-treated ratswere compared with their own controls whereas such effects werenot evident in cannulated rats.

Accumulating evidence suggests that DA D1 receptor-mediatedmechanisms have a major role in the amygdaloid modulation ofanxiety [38,12,41] since the intra-amygdaloid administration ofdopamine D1 receptor agonists and antagonists have straight-forward effects on both conditioned and unconditioned tests ofanxiety. Accordingly, intra-amygdaloid infusion of SCH23390 hasanxiolytic effects in the White and Dark Box [39] and Elevated Plus-Maze [2], interferes with both fear-potentiated startle Lamont andKokkinidis [55] and conditioned immobility/freezing [17] and hasa blocking effect on second order conditioning [35]. In turn, theintra-amygdaloid administration of SKF82958, a DA D1 receptoragonist, potentiates conditioned immobility/freezing [17]. On thisscenery, our behavioral results suggest that an enhanced dopamineD1 receptor activity within the amygdala may be involved in theincreased anxiety observed in the diabetic population since thebilateral amygdaloid administration of SCH23390, a selective DA D1receptor antagonist [23] fully prevented the increase in immobil-ity/freezing behavior elicited by the STZ treatment. In line with thispossibility, in spite of the problem to have high quality amygdalasections depicting equivalent paracapsular islands for both controland STZ-treated rats, an increase in [3H]-SCH23390 binding withinthe Ivp was found in this work following STZ treatment.

The reason why DA D1 receptor was upregulated within Ivp

and not in other intercalated paracapsular islands is not clear.However, converging evidence indicates that there exists con-siderable cytoarchitectonical and functional heterogeneity amongthe intercalated paracapsular islands [8,36] including a differential

D. Rebolledo-Solleiro et al. / Behavioural Brain Research 313 (2016) 293–301 299

Fig. 5. Effects of the bilateral microinjection of SCH23390 on the behavior of STZ-treated rats treated in the Shock/Probe Burying test. Blockade of DA D1 receptor activity bythe bilateral intra-amygdaloid administration of SCH23390 prevented immobility/freezing behavior without altering any other behavioral parameter. (a) Burying behaviorlatency. (b) Total time spent in burying behavior. (c) Number of shocks received. (d) Time sinterquartile range (boxes). The whiskers indicate the highest and lowest values within thDunn test.

Fig. 6. Effects of the microinjection of SCH23390 in rats treated with STZ and submit-trp

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ed to the Open Field Test. Bars represent medians with the respective interquartileange (boxes). The whiskers indicate the highest and lowest values within the sam-le. N: vehicle + saline = 18; STZ + saline = 15; STZ + SCH23390 = 18. Dunn test.

ctivation pattern to different fear states. Thus, while Ivp seems toe specifically involved in fear expression [21,8] the main inter-alated island is specifically activated during extinction retrieval8].

Since a similar dopamine D1 receptor up-regulation has alsoeen found following STZ treatment both in hippocampus [43] andtriatum [31] it is most likely that in our experiments the increasesn dopamine D1 receptor binding have also been developed as aonsequence of the STZ treatment. In the hippocampus [43] an

ncreased dopamine D1 receptor mRNA expression was associ-ted with an enhanced [3H]-dopamine binding and a decreased3H]-dopamine affinity, and in the striatum [31] only changes inopamine D1 receptor affinity were found. The mechanisms under-

pent in immobility/freezing behavior. Bars represent medians with their respectivee sample. **P < 0.01. N: vehicle + saline = 18; STZ + saline = 15; STZ + SCH23390 = 18.

lying the increase in [3H]-SCH23390 binding found in this worktherefore remain to be clarified in future experiments. However,since the Kd value for [3H] SCH23390 has been reported to be 0.3 nM[45] and the concentration of hot ligand was 3.3 times higher thanits Kd value, an increase in the binding likely reflects an increase inthe number of binding sites (Bmax). Finally, it is worth to considerthat the lack of effects of the bilateral intra-amygdaloid administra-tion of SCH23390 on blood glucose levels rules out the possibilitythat the behavioral effects observed following its intra-amygdaloidadministration had been produced as a consequence of an improve-ment in the metabolic condition of the animals.

The mechanism by which anxiety is elicited in STZ-treated ratsis far from clear. It is, however, feasible that in view of the largedeterioration of the metabolic homeostasis in STZ-treated animalsnumerous metabolic/hormonal signals reach many brain areas.They may directly or indirectly influence the amygdala and itsanxiogenic output. It seems clear, however that chronic hyper-glycemia may play a major role in the anxiogenic state via DAmechanisms since it has been demonstrated that dopamine, asmeasured after microdialysis is elevated in amygdala after feed-ing or following the peripheral administration of glucose, and thatamygdaloid dopamine levels decreases after insulin treatment [19].In line with this, a decrease in STZ-induced anxiety-like behav-ior has been reported in several models of anxiety after insulinadministration [18].

Converging experimental evidence suggests that dopamine, byactivating DA D1 receptors within the intercalated paracapsular

islands, may increase the anxiogenic output of the amygdala byreleasing BLA principal neurons from their cortical feedforwardinhibition exerted by the lateral GABAergic paracapsular islands

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ich in DA D1 receptors [34,41]. In addition, dopamine acting on up-egulated DA D1 receptors on Ivp GABA neurons may also increasehe anxiogenic output of the amygdala by removing the inhibitionf the CeA exerted by GABAergic neurons located at the capsulateraleA [8]; (for a review see [36]). It seems then possible, based on theurrent findings, that hyperglycemia and/or an insulin deficit alongith all its neuroendocrine consequences e.g. HPA dysregulation,

mong many others may, create an anxiogenic hyperdopaminergictate within the amygdala through a potential up-regulation of themygdaloid DA D1 dopamine receptors. It remains for further stud-es to clarify the impact of a dysregulated neuroendocrine systemn the amygdaloid modulation of anxiety.

onclusions

Our studies demonstrate the development of an anxiogenictate following STZ treatment which may in part involve an up-egulation of amygdaloid DA D1-receptor mechanisms. This opensp the possibility that blockade of amygdaloid dopamine D1 recep-ors may be used as a therapeutic alternative for the control of thencreased anxiety observed in diabetic patients.

cknowledgements

This work was supported by an international collaborativeesearch grant from the Swedish Research Council (348-2014-396) with Mexico (M. Pérez de la Mora) In addition, this studyas supported in part by the grants IN200508 and IN203111 fromirección General de Asuntos del Personal Académico, UNAM, theundesministerium für Bildung und Forschung (e:Med program,KZ: 01ZX1311A (ref: Spanagel R et al. (2013) A systems medicineesearch approach for studying alcohol addiction. Addiction biology8(6):883–896)), the Deutsche Forschungsgemeinschaft (DFG cen-er grant SFB1134). We also thank Consejo Nacional de Ciencia yecnología (CONACYT) for the support of this work through granto. 220173. This study was performed as part of the work of D.R-

to obtain her Ph.D. degree within the program of Doctorado eniencias Biomédicas of the UNAM. The assistance of the Comput-

ng Unit of the Instituto de Fisiología Celular, Universidad Nacionalutónoma de México (UNAM) and in particular to Francisco Pérez-ugenio is fully appreciated. We all authors declare that there areo actual or potential conflict of interest.

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