Neonatal 5,7-DHT Lesions Cause Sex-Specific Changes in Mouse Cortical Morphogenesis

Both monoaminergic and cholinergic afferent projections to the neocortex putatively modulate cortical morphogenesis and plasticity. Previously we showed that neonatal,electrolytic lesions: the cholinergic nucleus basalis magnocel!ularis (nBM) projections to the neocortex result in significant decreases-of cortical layer width that correlate with cognitive alterations. Such electrolytic lesions, performed for lack of a selective neurotoxin in mice, may affect mono- aminergic fibers of passage. Here, we investigate the effects of neonatal 5,7 dihydroxytryptamine (5,7-DHT) focal injections into the nBM region on cortical laminar morphology in adult male and female mice. 5,7-DHT lesions on the first postnatal day resulted in significant cortical depletion of both serotonin and norepinephrine that attenuated with age. Generally, cortical layer widths increased in response to the lesion; the effects were layer, region, and sex specific. Previous reports from our laboratories described longterm behavioral alterations after comparable focal, neonatal 5,7-DHT lesions. The studies described here provide an anatomical basis for such behavioral alterations. Our data suggest that monoaminergic and cholinergic projections to the cortex may have opposite effects on the developing cortical neuropil. Jointly, our morphological and behavioral findings may have important implications for a variety of developmental disorders in humans and provide some insights into sex differences in the penetrance of these disorders

Effects of Environmental Enrichment on Spatial Memory and Neurochemistry in Middle-Aged Mice

The present study compared the effects of environmental enrichment on spatial memory, glutamic acid decarboxylase (GAD) activity, and synaptophysin levels in middle-aged male and female mice. Prior to testing, a subset of 18-month-old male and female C57BL/6 mice was housed with two to three toys and a running wheel in the home cage for up to 29 d. Adult mice (7 mo) of both sexes and the remaining middle-aged mice were group (social) housed, but not exposed to enriching objects. After the enrichment period, all mice were tested in a 1-day version of the Morris water maze, in which both spatial and nonspatial memory were assessed. Immediately after testing, the hippocampus and frontoparietal cortex were dissected, and GAD activity and synaptophysin levels were measured. Environmental enrichment reduced the age-related impairment in spatial acquisition and retention; relative to adult social controls, middle-aged enriched mice were unimpaired, whereas middle-aged social controls were impaired. This reduction was similar in middle-aged males and females. Enrichment did not affect cued memory in either sex. Although hippocampal GAD activity was increased by enrichment in males, all other neurochemical measurements were unaffected by enrichment or aging in either sex. These data suggest that environmental enrichment initiated at middle age can reduce age-related impairments in spatial memory in males and females, although the underlying neurobiological mechanisms of this effect remain unknown

Acetyl-L-carnitine improves behavior and dendritic morphology in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder affecting 1 in 10,000 girls. Approximately 90% of cases are caused by spontaneous mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Girls with RTT suffer from severe motor, respiratory, cognitive and social abnormalities attributed to early deficits in synaptic connectivity which manifest in the adult as a myriad of physiological and anatomical abnormalities including, but not limited to, dimished dendritic complexity. Supplementation with acetyl-L-carnitine (ALC), an acetyl group donor, ameliorates motor and cognitive deficits in other disease models through a variety of mechanisms including altering patterns of histone acetylation resulting in changes in gene expression, and stimulating biosynthetic pathways such as acetylcholine. We hypothesized ALC treatment during critical periods in cortical development would promote normal synaptic maturation, and continuing treatment would improve behavioral deficits in the Mecp2(1lox) mouse model of RTT. In this study, wildtype and Mecp2(1lox) mutant mice received daily injections of ALC from birth until death (postnatal day 47). General health, motor, respiratory, and cognitive functions were assessed at several time points during symptom progression. ALC improved weight gain, grip strength, activity levels, prevented metabolic abnormalities and modestly improved cognitive function in Mecp2 null mice early in the course of treatment, but did not significantly improve motor or cognitive functions assessed later in life. ALC treatment from birth was associated with an almost complete rescue of hippocampal dendritic morphology abnormalities with no discernable side effects in the mutant mice. Therefore, ALC appears to be a promising therapeutic approach to treating early RTT symptoms and may be useful in combination with other therapies

Carnitine concentrations in blood plasma collected at PN 47.

<p>All values represent mean ± SEM. The * indicates values significantly different from WTS values. WTS: wild type saline-treated; WTA: wild type ALC-treated; NS: null saline-treated; NA: null ALC-treated.</p

Motor and cognitive functions are modestly improved in null ALC-treated mice compared to saline-treated null mice during early treatment.

<p>General activity levels (<b>A</b>) were assessed at PN 21, PN 29, and PN 42. Saline-treated null mice were hypoactive compared to saline-treated wild type mice at all ages (<i>p</i>'s<0.05). Activity in ALC-treated null mice increased to a level intermediate between saline-treated WT and null mice on PN 21 and 29 (<i>p</i>'s>0.05), but was significantly impaired on PN 43. Deficits in motor coordination (<b>B</b>), assessed at PN 44, were unaffected by ALC treatment in null mice. Cognitive function was measured at PN 28 using an object recognition task (<b>C</b>). Object memory was intact in all mice 1 h after object training and in both wild type groups 24 h after training. Saline-treated nulls exhibited no memory for the object 24 h after training, while object memory in ALC-treated null mice was intermediate between saline-treated null and saline-treated WT levels. Cognitive function was measured on an associative fear conditioning task at PN 46 (<b>D</b>). All groups performed the contextual fear portion of the task similarly, while both groups of null mice were impaired on the cued fear portion of the task. ALC treatment did not affect performance on either portion of the task. All values represent the mean ± SEM. ODI: object discrimination index; WTS: wild type saline-treated; WTA: wild type ALC-treated; NS: null saline-treated; NA: null ALC-treated. † NS vs. WTS, WTA, NA (<i>p</i><0.05) # NS vs. WTS, WTA (<i>p</i><0.05) ‡ NS and NA vs. WTS and WTA (<i>p</i>'s<0.05) * <i>p</i><0.05 between indicated groups.</p

Reduced dendritic length and complexity are mostly rescued in ALC-treated null mice.

<p>Representative drawings of Golgi-Cox stained neurons analyzed in the dentate gyrus of the hippocampus in (<b>A</b>) WT saline-treated, WT ALC-treated, null saline-treated, and null ALC-treated mice. (<b>B</b>) Area of the cell body was not affected by genotype or treatment. (<b>C</b>) Total dendritic length was significantly reduced in saline-treated nulls compared to both WT groups (<i>p</i>'s<0.001) and was rescued with ALC treatment in null mice (saline-treated vs. ALC-treated nulls; <i>p</i> = 0.016). (<b>D</b>) Dendritic complexity was decreased in saline-treated null mice compared to both groups of WT mice indicated by fewer dendrite crossings between 75 and 150 µm from the cell body. ALC treatment in null mice had a significant effect on the number of dendritic crossings in close proximity to the cell body. All values represent the mean ± SEM. AU = arbitrary units. WTS: wild type saline-treated; WTA: wild type ALC-treated; NS: null saline-treated; NA: null ALC-treated. † NS vs. WTS, WTA, NA (<i>p</i><0.05) # NS vs. WTS, WTA (<i>p</i><0.05) ‡ NS and NA vs. WTS and WTA (<i>p</i>'s<0.05).</p

General health, motor and cognitive functions are predominately normal in <i>Mecp2</i> heterozygous females at the ages examined.

<p>Weight (<b>A</b>) and grip strength (<b>B</b>) were not statistically different between the four groups of female mice. Locomotor activity (<b>C</b>) measured over the 12 hour dark cycle was similar between saline-treated WT females and both groups of heterozygous females (saline and ALC-treated). ALC-treated WT females were significantly more active than heterozygous females (saline and ALC-treated), but not saline-treated WT females on PN 29 and PN 42. Motor coordination measured on the rotorod at PN 43 (<b>D</b>) was significantly impaired in saline-treated heterozygous females compared to WT females (saline-treated and ALC-treated). ALC treatment improved performance of heterozygous females to a level intermediate between saline-treated heterozygous and WT females. Performance on both contextual and cued fear conditioning (<b>E</b>) was similar between all four groups of female mice at PN 46.</p

General health and metabolic function are improved in ALC-treated null mice compared to saline-treated nulls.

<p>(<b>A</b>) Weight (g) was measured daily between postnatal day (PN) 1 and PN 45 in all mice. Saline-treated nulls weighed significantly less than all other groups between PN 22 and 35 (<i>p</i>'s<0.05). Between PN 36 and 45, both saline-treated and ALC-treated null mice weighed significantly less than saline-treated and ALC-treated WT mice (<i>p</i>'s<0.05). (<b>B</b>) Forepaw grip strength (kg) was assessed on PN 22, PN 30, and PN 43. Grip strength was lower in saline-treated nulls compared to saline-treated WTs on all PN's examined (<i>p</i>'s<0.003). In ALC-treated nulls, grip strength was improved to saline-treated WT levels on PN 22, intermediate between saline-treated null and saline-treated WT levels on PN 30, and significantly impaired compared to saline-treated WT mice on PN 43. Metabolic function was assessed by measuring carbon dioxide (CO₂) production (<b>C</b>) and oxygen (O₂) consumption (<b>D</b>) on PN 22, PN 30, and PN 43. CO₂ and O₂ levels were significantly decreased in saline-treated null mice compared to both WT groups (saline-treated and ALC-treated) on PN 43 (<i>p</i>'s<0.03). CO₂ levels were significantly improved in ALC-treated nulls compared to saline-treated null mice (<i>p</i><0.005) and O₂ levels were intermediate between saline-treated null and saline-treated WT levels on PN 43. All values represent the mean ± SEM. WTS: wild type saline-treated; WTA: wild type ALC-treated; NS: null saline-treated; NA: null ALC-treated. † NS vs. WTS, WTA, NA (<i>p</i><0.05) # NS vs. WTS, WTA (<i>p</i><0.05) ‡ NS and NA vs. WTS and WTA (<i>p</i>'s<0.05) * <i>p</i><0.05 between indicated groups.</p