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Immunoreactive Calcium-Binding GABA Interneurons in IL
MSCON
Immunoreactive Cell Types
Imm
unor
eact
ice
Cell
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CB CR DBL CB CR DBL Layers 2/3 Layers 5/6
Early-life stress (ELS) has been implicated in increasing anxiety and aggression in rats, possibly by means of modulating inhibitory GABA interneurons colocalized with cannabinoid receptors in the prefrontal cortex (PFC). We sought to investigate the developmental effects of maternal separation (MS) and cannabinoid type 1 (CB1) receptor antagonism on anxiety and social behaviors in male and female Sprague-Dawley rats. With the use of retrospective analysis, we wished to discover if juvenile social play behaviors could be used to predict those seen in adults. During adulthood, rats were exposed to the CB1 antagonist, rimonabant (Rim), or vehicle (Veh) prior to elevated-plus maze (EPM), marble burying (MB), and social interaction. With immunohistochemistry (IHC), we investigated whether changes in calbindin (CB) and calretinin (CR) expressing GABAergic interneurons in the medial prefrontal cortex (mPFC) may mechanistically underlie behavioral deficits. Sex-differences in adult social behavior resulted from MS, most notably an increase in aggression and evasion in adults. Rim treatment uncovered a trend of decreased anxiety in MS, but an increase in controls (CON). Rim reduced social and explorative behaviors in males, but increased the crossovers and stretch-attend (SA) postures in females in EPM. Juvenile social play behaviors were predictive of some behaviors in adulthood. A moderate trend was observed for the interaction of condition, cell type, region, and cortical layer. This trend, along with prior research, suggests that differences in anxiety and social behavior seen in adults are differentially affected by Rim treatment dependent on changes in GABAergic interneuron subpopulations resulting from early-life stress.
Method
Developmental Sex-differences in Anxiety and Social Behavior may be mediated by Calcium-Binding GABAergic Interneurons and differentially affected by Early Life Stress and Cannabinoid Type 1 Receptor Antagonism
Patrick M. Einhorn
Figure 8. Microscope images of GABAergic Interneurons in the mPFC. Calretinin neurons appear stained in green and Calbindin neurons appear in red. Those double labeled for both appear in both colors and have been marked above with a arrow.
Experiment 2: Distribution of Calbindin (CB) and Calretinin (CR) expressing GABAergic Interneurons in layers 2/3 and 5/6 of the Anterior Cingulate (ACC), Prelimbic (PL), Infralimbic (IL) subregions of the mPFC of adolescent males (N=10, 5 MS and 5 CON).
Receptors were labeled for CB (Swant; 1:500) and CR (Merck; 1:10,000) and immuno-positive neurons were counted using unbiased stereology (StereoInvestigator).
Laboratory of Developmental Neuropharmacology, Department of Psychiatry, McLean Hospital/Harvard Medical School, Belmont, MA 02478, USA
Experiment 1: Male and female Sprague-Dawley rat pups (N=36) were maternally separated (MS) for four hours/day and kept at a thermoneutral temperature or animal facility reared (CON) between postnatal days (P) 2 and P20. Subjects were then acutely treated with a Cannabinoid Type 1 (CB1) receptor antagonist, Rimonabant [RIM), 3 mg/kg, i.p.) or vehicle (Veh; DMSO) 15 minutes prior to adult anxiety and social behavior testing. Anxiety testing consisted of single, counterbalanced trials of Marble Burying (MB) trial and Elevated Plus-Maze (EPM).As seen in figure 1a., social behavior with novel, same-sex conspecifics was examined in juveniles (social play) and again in adulthood (social investigation).
Subjects. Male, Sprague-Dawley rats (n = 46; 3 age groups [Juvenile: P25, Adolescent: P40, and Adult: P104-114])
PL
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IL
Immunohistochemistry
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Immunoreactive Calcium-Binding GABA Interneurons in mPFC Subregions
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Immunoreactive Cell Types
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Immunoreactive Calcium-Binding GABA Interneurons in ACC
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Immunoreactive Cell Types
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CB CR DBL CB CR DBLLayers 2/3 Layers 5/6
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Immunoreactive Calcium-Binding GABA Interneurons in PL
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Immunoreactice Cell Types
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^6)
CB CR DBL CB CR DBL
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Anxiety Behavior
MS+Rim MS+Veh CON+Rim CON+Veh05
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Elevated Plus Maze
Subject Conditon and Treatment
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MS+Rim MS+Veh CON+Rim CON+Veh02468
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Marble Burying Behav-ior
Subject Condition and Treatment
Mar
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Bur
ried
Figure 1. (A) Number of subjects and their conditions, as well as, social investigation pairings. (B) Timeline documenting course of experimentation.
AB
Figure 2. Sample section of mPFC and subregions investigated for immunohistochemistry.
Figure 9. Distribution of total immunoreactive GABAergic Interneuron cell type densities in mPFC subregions. As seen above, double labeled neurons (DBL) in all regions were much less numerous than those marked for CB or CR alone.
Figure 10. Immunoreactive cell densities in the ACC. No significant changes in immunoreactive cell markers were observed within the ACC. On average, GABAergic interneurons distribution was less dense in layers 5/6.
Figure 11. Immunoreactive cell densities in the PL. No significant differences were observed in the PL.
Figure 12. Immunoreactive cell densities in the IL. A strong trend (p=0.11) was noted in the density of CR neurons in layers 2/3 of the IL, however this failed to reach significance.
Figure 13(a-d). Anxiety Behavior Measures.
EPM testing revealed that, on average, MS subjects spent less time in the open arms of EPM, though this failed to reach significance (13A). Similarly, MS+Rim males appeared to show lessened anxiety as a result of Rim treatment when compared to CON+Rim (13A).
Conversely, Rim treatment in females induced a nonsignificant anxiogenic effect by decreasing time spent in the open arms (13A). Analysis of arm crossovers revealed that females were significantly more active than males in the EPM (13B, p=0.02). Also, Rim treatment significantly reduced crossovers in females (13B, p=0.03).Rim treatment revealed a strong trend of reduced frequency of stretched-attend postures, a measure of hesitancy or risk-assessment (13C, p=0.08). This trend was significant when taken only in regard to females (13C, p=0.02).
A B
C D
Marble Burying revealed no significant interaction of ELS condition or treatment. However, treatment with Rim in males revealed a strong trend of Rim treatment reducing MB in MS subjects but increasing MB in controls (13D, p=0.08).
Male
Female
Sex Juvenile Social Behavior
Adult Behavior Relationship
♂ Pinning Approaching (+), r = 0.598, p = 0.003
♂ Boxing Crawling Under/Over Conspecific
(+), r = 0.484, p = 0.023
♂ Social Sniffing
Approaching (-), r = -0.536, p = 0.01
♂ Social Sniffing
Boxing (-), r = -0.444, p = 0.034
♀ Boxing Crawling Under/Over Conspecific
(+), r = 0.772, p = 0.042
♀ Boxing Anogenital Exploration (+), r = 0.849, p = 0.016
♂+♀ Chasing Marble Burying (+), r = 0.534, p = 0.04
♂+♀ Chasing Self-Grooming (-), r = -0.50, p = 0.41
♂+♀ Pinning Approaching (-), r = 0.545, p = 0.029
Pounce Pin
Chase Bo
xBit
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Pounce Pin
Chase Bo
xBit
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Social
Sniff
Nape A
ttack
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10152025303540
Male (P25) Social Play
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Social Behaviors
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Pounce Pin Chase Box Bite Social Sniff0
10
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Female (P25) Social Play
MSCON
Social Behaviors
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Pounce Pin Chase Box Bite Social Sniff
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Social Play Behaviors Displayed in Adult Males
CON+VehCON+RimMS+VehMS+Rim
Behaviors
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Crawl O
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Self-G
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Social
Grooming
Tail M
anipu
lation
02468
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Adult Male Social Behaviors
CON+VehCON+RimMS+VehMS+Rim
Behaviors
Beha
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Fre
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Pounce Pin Chase Box Bite Social Sniff0
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Social Play Behaviors Displayed in Adult Females
CON+VehCON+RimMS+VehMS+Rim
Behaviors
Beha
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Genita
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ration
Anog
enita
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Crawl O
ver/U
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Appro
achFol
low
Evasiv
e Beh
avior
Self-G
rooming
Social
Grooming
Tail M
anipu
lation
02468
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Adult Female Social Behaviors
CON+VehCON+RimMS+VehMS+Rim
Behaviors
Beha
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Fre
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Table 1. Juvenile social behaviors predictive of adult behaviors in untreated controls
As seen in Table 1., correlational analysis revealed that multiple juvenile social behaviors were predictive of adult behaviors in untreated controls. To our knowledge, this is the first time that juvenile behaviors have been isolated as potential predictive markers for adult behaviors. Sex-differences in these behaviors further highlight the need for female subjects in all fields of new research.
Figure 3. Juvenile Male Social Play Behaviors. MS subjects exhibited significantly more pounces and bites than controls, indicating that MS increased aggression in juveniles.
Figure 4. Juvenile Female Social Play Behaviors. No significant differences between female MS and CON subjects was present as juveniles, however, a trend of increased aggression was seen in MS subjects.
Approach Social Grooming0
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Sex-differences in Adult Social Behaviors
MaleFemale
Behaviors
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Figure 5. Sex-differences in Juvenile Social Behaviors.As juveniles, females engaged in significantly more pins than males, regardless of condition. Males, however, exhibited significantly more social sniffs than females.
Figure 6. Sex-differences in Adult Social Behaviors.As adults, females showed significantly more approaches to novel conspecific than males, however, females also exhibited significantly fewer instances of social grooming.
Experiment 1. & 2. Statistical Analysis.A significance level of p<0.05 was used for all analyses.
Figure 7 (a-d). Adult Social Behaviors by Sex across Condition and Treatment.
As seen in figure 7 (a-d) above, a Treatment X Condition X Sex interaction was observed in chasing behavior (F(1,28)= 6.072, p=0.02). A Treatment X Sex interaction indicated significant changes in anogenital explorations (F(1,28)= 4.157, p=0.051) and social grooming (F(1,28)= 6.97, p=0.013); post-hoc analysis revealed that males receiving Rim engaged in fewer anogenital explorations and social grooms than Veh males (p=0.01 and p=0.004, respectively). As seen in figure 7, acute Rim treatment in adult rats significantly decreased chases (F(1,28)=6.072, p=0.02), approaches (F(1,28)= 8.58, p=0.007), and social grooming (F(1,28)=5.706, p=0.024).
MS produced significant changes in the frequency of pounces (F1,28)= 14.209, p=0.001), chases (F(1,28)= 6.072, p=0.02), genital explorations (F(1,28)= 4.517, p=0.043), evasive behaviors (F(1,28)= 9.713, p=0.004), and tail manipulations (F(1,28)= 20.324, p<0.001). Sex differences existed in chasing (F(1,28)= 6.072, p=0.02), approaching (F(1,28)= 7.409, p=0. 011), self-grooming (F(1,28)= 3.035, p=0.092) and social grooming (F(1,28)= 4.303, p=0.047) behaviors. A significant interaction was found between treatment and condition for chases (F(1,28)= 6.072, p=0.02).
Social Behavior
Predictability of Juvenile Social Play
Conclusions
Experiment 1. The results of experiment 1. demonstrate important differences in the effects of neonatal MS and also Rim treatment in rats, which are summarized as follows: (1) females exhibited increased time in the open arms of EPM (2) a trend of reduced time spent in closed-arms of EPM in males treated with Rim, when compared to MS+Veh and CON+Rim, (3) Rim treatment increased crossovers and reduced SA in females in EPM, (4) Treatment with Rim may have reduced MB in MS, but increased MB in CON, (5) a trend of MS increasing aggressive behaviors in juvenile male, but decreasing aggression in juvenile females, (6) MS subjects exhibited increased aggressive and evasive behaviors in adult social interaction, (7) treatment with Rim reduced social and explorative behaviors in adult males, (8) Juvenile behaviors were predictive of some adult behaviors.
Experiment 2. The results of the present study demonstrate important differences in the effects of neonatal MS on prefrontal GABAergic interneuron subpopulations, which are summarized as follows: (1) a trend of difference in immunoreactive cell density by Condition X (Marker X Region X Layer), and (2) significant differences of immunoreactive CBP expressing cells across mPFC regions and layers.Experiments 1. & 2.Significant findings as well as strong trends indicate that MS, and also potentially sex, may differentially alter the anxiogenic/anxiolytic effects of Rim treatment, when compared to controls. It appears likely that this many be due in part to the action of Rim altering transmission at presynaptic terminal of calcium-binding protein expressing (CB & CR) GABAergic interneurons colocalized with CB1 receptors in the mPFC.
Abstract
