Stereological analyses of reward system nuclei in maternally deprived/separated alcohol drinking rats

Abstract

The experience of early life stress can trigger complex neurochemical cascades that influence emotional and addictive behaviors later in life in both adolescents and adults. Recent evidence suggests that excessive alcohol drinking and drug-seeking behavior, in general, are co-morbid with depressive-like behavior. Both behaviors are reported in humans exposed to early life adversity, and are prominent features recapitulated in animal models of early life stress (ELS) exposure. Currently, little is known about whether or how ELS modulates reward system nuclei. In this study we use operant conditioning of rats to show that the maternal separation stress (MS) model of ELS consumes up to 3-fold greater quantities of 10% vol/vol EtOH in 1-h, consistently over a 3-week period. This was correlated with a significant 22% reduction in the number of dopaminergic-like neurons in the VTA of naïve MS rats, similar to genetically alcohol-preferring (P) rats which show a 35% reduction in tyrosine hydroxylase (TH)-positive dopaminergic neurons in the VTA. MS rats had a significantly higher 2-fold immobility time in the forced swim test (FST) and reduced sucrose drinking compared to controls, indicative of depressive-like symptomology and anhedonia. Consistent with this finding, stereological analysis revealed that amygdala neurons were 25% greater in number at P70 following MS exposure. Our previous examination of the dentate gyrus of hippocampus, a region involved in encoding emotional memory, revealed fewer dentate gyrus neurons after MS, but we now report this reduction in neurons occurs without effect on the number of astrocytes or length of astrocytic fibers. These data indicate that MS animals exhibit neuroanatomical changes in reward centers similar to those reported for high alcohol drinking rats, but aspects of astrocyte morphometry remained unchanged. These data are of high relevance to understand the breadth of neuronal pathology that ensues in reward loci following ELS.

Keywords: Alcohol-preferring rats; Amygdala; Dentate gyrus; Drug addiction; Early life stress; Excessive alcohol drinking; Maternal deprivation; Maternal separation; P rats; Stereology; VTA.

Figure 1. Anterior-posterior extent of anatomical regions used for stereological measurements

A) Sections of amygdala, includes the basal, central, medial, lateral nuclei from bregma −1.3 to −4.2 mm. B) Sections of the VTA includes the rostral VTA, parainterfascicular, paranigral, and parabrachial pigmented nuclei from bregma −4.56 to −6.84 mm. C) Sections of the DG, includes rostral most DG with all three layers included for astrocyte analysis from bregma −1.72 mm to −6.84 mm. The red outline is a representative contour to demarcate the region of interest to be counted as was outlined during stereological analysis. Nissl sections for A and C were from the brainmaps.org interactive atlas, and the bregma levels and regional borders were identified with Paxinos Atlas.

Figure 2. Increased alcohol drinking and reduction of VTA neurons in MS adult rats, similar to P rats

A) Compared to control SD rats (blue), MS rats (gray) trained to lever press for 10% alcohol on an FR4 schedule exhibit sustained increased levels of alcohol drinking during 14 days of access, n=10 per group. B) Stereological analysis of P70 adult rat VTA showed a reduction in the number of dopamine-like neurons in animals exposed to early postnatal (P2–P21) MS, n=6 per group. C) TH-immunoreactivity in the VTA of alcohol-preferring (P) rats revealed a reduction of large TH+ dopaminergic neurons, n=8 per group. D) Representative images of TH+ immunoreactivity in the VTA of P and NP animals. Data are expressed as mean ± SEM. *, p<0.05; **, p<0.01, p<0.001.

Figure 3. Depression symptomatology, anhedonia, and increased amygdala neurons in MS adult rats

A) Immobility in the forced swim test of control SD rats (blue) compared to MS rats (gray) show increased immobility in MS, n=16 per group. B) Sucrose (3% w/v) intake via bottle drinking of MS over 3 consecutive days is reduced compared to control, n=10 per group. C) Stereological analysis of naïve P70 adult rat amygdala (n=6 per group) showed increased neuron number in animals exposed to MS during the pre-weaning period. Data are expressed as mean ± SEM. **, p<0.01 ***, p < 0.001.

Figure 4. Stereological analysis of astrocyte number and astrocyte fiber length in hippocampal dentate gyrus of MS and control rats

A) Mean astrocyte counts or B) total astrocyte fiber length did not differ between MS or control groups, n=6 per group. D) Representative example of GFAP+ astrocytes in MS and CTL animals processed adjacently on the same embedding sheet, and imaged at the same illumination levels. Arrows show Astrocyte cell bodies counted in the infra- and supra-granular DG space. Bottom panels are enlarged region of insets (white square); red circles represent the spaceball probe intersected with either thin or thick fibers. Data are expressed as mean ± SEM. *, p<0.05 by ANOVA.

Figure 5. Schematic representing the use of Isotropic sphere (spaceball) probe for fiberlength analysis

A) View through the Z axis of a section with two astrocytes represented. The virtual sphere (spaceball) is in the shaded region A′. A′) Enlargement of the virtual spaceball sphere with optical sections (a–e) to analyze the fibers as they cross the perimeter of the sphere at that level. B) Images of each optical plane along the z-axis of the sphere. At any given plane, fibers are only counted if they are in focus as they traverse the perimeter of an optical Z plane. At plane d, the red astrocyte fiber is long enough to traverse the perimeter of the sphere twice, whereas the yellow astrocyte does not cross at that plane; therefore 2 instances are counted. C) Image of one of the sections of the specimen used in this study. The x represents where in-focus fibers cross the perimeter of the optical plane (circle) of the space ball probe which was 10μm in diameter.