Correlation Between Intracranial Calcification and Extracranial Stenosis of the Internal Carotid Artery.

Intracranial artery calcification is a marker of vascular atherosclerosis and has a high prevalence worldwide. Both atherosclerosis of the internal carotid artery at the carotid sinus in the neck and intracranial calcification have been associated with ischemic stroke. The relationship between the two has not been well studied. The present study investigated how carotid sinus narrowing could relate to calcification located in the distal intracranial artery at the cavernous carotid. We examined a population not selected for cerebral disease. This retrospective study contained 179 subjects aged 18 years and older from the Hawaii Diagnostic Radiology database. Extracranial internal carotid artery stenosis was determined using the absolute diameter, North American Symptomatic Carotid Endarterectomy Trial, and common carotid artery methods. Calcification was scored using the modified Woodcock method. A positive correlation between intracranial calcification and extracranial carotid stenosis was found using all three methods. Individuals with intracranial calcification were more likely to be older, have a smaller internal carotid artery diameter, and have a greater percent stenosis at the internal carotid artery than those without intracranial artery calcification (p<0.001 for all). These results may help refocus interest in calcification in studies of cerebral vasculature and its correlation with extracranial carotid stenosis.

Clinically Used Hormone Formulations Differentially Impact Memory, Anxiety-Like, and Depressive-Like Behaviors in a Rat Model of Transitional Menopause.

A variety of U.S. Food and Drug Administration-approved hormone therapy options are currently used to successfully alleviate unwanted symptoms associated with the changing endogenous hormonal milieu that occurs in midlife with menopause. Depending on the primary indication for treatment, different hormone therapy formulations are utilized, including estrogen-only, progestogen-only, or combined estrogen plus progestogen options. There is little known about how these formulations, or their unique pharmacodynamics, impact neurobiological processes. Seemingly disparate pre-clinical and clinical findings regarding the cognitive effects of hormone therapies, such as the negative effects associated with conjugated equine estrogens and medroxyprogesterone acetate vs. naturally circulating 17ß-estradiol (E2) and progesterone, signal a critical need to further investigate the neuro-cognitive impact of hormone therapy formulations. Here, utilizing a rat model of transitional menopause, we administered either E2, progesterone, levonorgestrel, or combinations of E2 with progesterone or with levonorgestrel daily to follicle-depleted, middle-aged rats. A battery of assessments, including spatial memory, anxiety-like behaviors, and depressive-like behaviors, as well as endocrine status and ovarian follicle complement, were evaluated. Results indicate divergent outcomes for memory, anxiety, and depression, as well as unique physiological profiles, that were dependent upon the hormone regimen administered. Overall, the combination hormone treatments had the most consistently favorable profile for the domains evaluated in rats that had undergone experimentally induced transitional menopause and remained ovary-intact. The collective results underscore the importance of investigating variations in hormone therapy formulation as well as the menopause background upon which these formulations are delivered.

Age Impacts the Burden That Reference Memory Imparts on an Increasing Working Memory Load and Modifies Relationships With Cholinergic Activity.

Rodent aging research often utilizes spatial mazes, such as the water radial-arm-maze (WRAM), to evaluate cognition. The WRAM can simultaneously measure spatial working and reference memory, wherein these two memory types are often represented as orthogonal. There is evidence, however, that these two memory forms yield interference at a high working memory load. The current study systematically evaluated whether the presence of a reference memory component impacts handling of an increasing working memory load. Young and aged female rats were tested to assess whether aging impacts this relationship. Cholinergic projections from the basal forebrain to the hippocampus and cortex can affect cognitive outcomes, and are negatively impacted by aging. To evaluate whether age-related changes in working and reference memory profiles are associated with cholinergic functioning, we assessed choline acetyltransferase activity in these behaviorally-tested rats. Results showed that young rats outperformed aged rats on a task testing solely working memory. The addition of a reference memory component deteriorated the ability to handle an increasing working memory load, such that young rats performed similar to their aged counterparts. Aged rats also had challenges when reference memory was present, but in a different context. Specifically, aged rats had difficulty remembering which reference memory arms they had entered within a session, compared to young rats. Further, aged rats that excelled in reference memory also excelled in working memory when working memory demand was high, a relationship not seen in young rats. Relationships between cholinergic activity and maze performance differed by age in direction and brain region, reflecting the complex role that the cholinergic system plays in memory and attentional processes across the female lifespan. Overall, the addition of a reference memory requirement detrimentally impacted the ability to handle working memory information across young and aged timepoints, especially when the working memory challenge was high; these age-related deficits manifested differently with the addition of a reference memory component. This interplay between working and reference memory provides insight into the multiple domains necessary to solve complex cognitive tasks, potentially improving the understanding of complexities of age- and disease- related memory failures and optimizing their respective treatments.

Contrasting effects of individual versus combined estrogen and progestogen regimens as working memory load increases in middle-aged ovariectomized rats: one plus one does not equal two.

Most estrogen-based hormone therapies are administered in combination with a progestogen, such as Levonorgestrel (Levo). Individually, the estrogen 17ß-estradiol (E2) and Levo can improve cognition in preclinical models. However, although these hormones are often given together clinically, the impact of the E2 + Levo combination on cognitive function has yet to be methodically examined. Thus, we investigated E2 + Levo treatment on a cognitive battery in middle-aged, ovariectomized rats. When administered alone, E2 and Levo treatments each enhanced spatial working memory relative to vehicle treatment, whereas the E2 + Levo combination impaired high working memory load performance relative to E2 only and Levo only treatments. There were no effects on spatial reference memory. Mitogen-activated protein kinases/extracellular signal-regulated kinases pathway activation, which is involved in memory formation and estrogen-induced memory effects, was evaluated in 5 brain regions implicated in learning and memory. A distinct relationship was seen in the E2-only treatment group between mitogen-activated protein kinases/extracellular signal-regulated kinases pathway activation in the frontal cortex and working memory performance. Collectively, the results indicate that the differential neurocognitive effects of combination versus sole treatments are vital considerations as we move forward as a field to develop novel, and to understand currently used, exogenous hormone regimens across the lifespan.

Cognitive changes across the menopause transition: A longitudinal evaluation of the impact of age and ovarian status on spatial memory.

Cognitive changes that occur during mid-life and beyond are linked to both aging and the menopause transition. Studies in women suggest that the age at menopause onset can impact cognitive status later in life; yet, little is known about memory changes that occur during the transitional period to the postmenopausal state. The 4-vinylcyclohexene diepoxide (VCD) model simulates transitional menopause in rodents by depleting the immature ovarian follicle reserve and allowing animals to retain their follicle-deplete ovarian tissue, resulting in a profile similar to the majority of perimenopausal women. Here, Vehicle or VCD treatment was administered to ovary-intact adult and middle-aged Fischer-344 rats to assess the trajectory of cognitive change across time with normal aging and aging with transitional menopause via VCD-induced follicular depletion, as well as to evaluate whether age at the onset of follicular depletion plays a role in cognitive outcomes. Animals experiencing the onset of menopause at a younger age exhibited impaired spatial memory early in the transition to a follicle-deplete state. Additionally, at the mid- and post- follicular depletion time points, VCD-induced follicular depletion amplified an age effect on memory. Overall, these findings suggest that age at the onset of menopause is a critical parameter to consider when evaluating learning and memory across the transition to reproductive senescence. From a translational perspective, this study illustrates how age at menopause onset might impact cognition in menopausal women, and provides insight into time points to explore for the window of opportunity for hormone therapy during the menopause transition period. Hormone therapy during this critical juncture might be especially efficacious at attenuating age- and menopause- related cognitive decline, producing healthy brain aging profiles in women who retain their ovaries throughout their lifespan.

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17ß-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions.

Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17ß-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of behavior. These distinct associations imply that cognition, anxiety-like, and depressive-like behaviors are modulated by unique serotonergic neurocircuitry, opening the possibility of novel avenues of targeted treatment for different types of cognitive and affective disorders.

Estrogen receptor-ß regulates human tryptophan hydroxylase-2 through an estrogen response element in the 5' untranslated region.

In the dorsal raphe nucleus, 17ß-estradiol (E2) increases the expression of the brain-specific, rate-limiting enzyme for serotonin biosynthesis, tryptophan hydroxylase-2 (Tph2). Although estrogen receptor beta (ERß) has been localized to Tph2 neurons, little is known about the transcriptional regulation of the Tph2 gene by estrogen. Since the ERß agonist, diarylpropionitrile (DPN) also increases Tph2 expression, we tested the hypothesis that E2 regulates the Tph2 promoter through direct interactions with ERß. A serotonergic cell line, B14, which endogenously expresses ERß was transiently transfected with a fragment of the human TPH2 5'-untranslated region (5'-UTR) cloned into a luciferase reporter vector (TPH2-luc). Treatment with E2 or DPN caused a dose-dependent increase of TPH2-luc activity. In contrast, E2 conjugated to bovine serum albumin, which is cell membrane impermeable, had no effect on TPH2-luc activity. An estrogen receptor (ER) antagonist blocked E2 or DPN-induced TPH2-luc activity suggesting a classical ER mechanism. In silico analysis revealed an estrogen-response element (ERE) half-site located within the TPH2 5'-UTR. Deletion and site-directed mutation of this site abolished ligand-induced TPH2-luc activity. These results support the concept that there is a direct and functional interaction between E2:ERß and the ERE half-site of the TPH2 promoter to regulate Tph2 expression. We illustrate a direct regulation of the TPH2 transcription by estradiol and ERß via a newly identified ERE half-site within the TPH2 promoter: (i) Estradiol- or an ERß agonist-induced TPH2 transcription was blocked by an ER antagonist, while (ii) membrane impermeable form of estradiol did not induce transcription. (iii) Deletion or mutation of the ERE half-site abolished ligand-induced TPH2 transcription.

The androgen metabolite, 5α-androstane-3ß,17ß-diol (3ß-diol), activates the oxytocin promoter through an estrogen receptor-ß pathway.

Testosterone has been shown to suppress the acute stress-induced activation of the hypothalamic-pituitary-adrenal axis; however, the mechanisms underlying this response remain unclear. The hypothalamic-pituitary-adrenal axis is regulated by a neuroendocrine subpopulation of medial parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). These neurons are devoid of androgen receptors (ARs). Therefore, a possibility is that the PVN target neurons respond to a metabolite in the testosterone catabolic pathway via an AR-independent mechanism. The dihydrotestosterone metabolite, 5α-androstane-3ß,17ß-diol (3ß-diol), binds and activates estrogen receptor-ß (ER-ß), the predominant ER in the PVN. In the PVN, ER-ß is coexpressed with oxytocin (OT). Therefore, we tested the hypothesis that 3ß-diol regulates OT expression through ER-ß activation. Treatment of ovariectomized rats with estradiol benzoate or 3ß-diol for 4 days increased OT mRNA selectively in the midcaudal, but not rostral PVN compared with vehicle-treated controls. 3ß-Diol treatment also increased OT mRNA in the hypothalamic N38 cell line in vitro. The functional interactions between 3ß-diol and ER-ß with the human OT promoter were examined using an OT promoter-luciferase reporter construct (OT-luc). In a dose-dependent manner, 3ß-diol treatment increased OT-luc activity when cells were cotransfected with ER-ß, but not ER-α. The 3ß-diol-induced OT-luc activity was reduced by deletion of the promoter region containing the composite hormone response element (cHRE). Point mutations of the cHRE also prevented OT-luc activation by 3ß-diol. These results indicate that 3ß-diol induces OT promoter activity via ER-ß-cHRE interactions.

Perinatal dexamethasone-induced alterations in apoptosis within the hippocampus and paraventricular nucleus of the hypothalamus are influenced by age and sex.

Exposure to high levels of glucocorticoids (GCs) during development leads to long-term changes in hypothalamic-pituitary-adrenal (HPA) axis regulation, although little is known about the neural mechanisms that underlie these alterations. In this study, we investigated the effects of late gestational (days 18-22) or postnatal (days 4-6) administration of the GC receptor agonist dexamethasone (DEX) on an apoptosis marker in two brain regions critical to HPA axis regulation, the hippocampus and the hypothalamic paraventricular nucleus (PVN). One day after the final DEX injection, male and female rats were sacrificed, and brains were processed for immunohistochemical detection of cleaved caspase-3, an apoptotic cell death indicator. DEX increased cleaved caspase-3 immunoreactivity in the CA1 hippocampal region of both sexes following prenatal but not postnatal treatment. Prenatal DEX also increased caspase-3 immunoreactivity in the CA3 region, an elevation that tended to be greater in females. In contrast, postnatal DEX resulted in a much smaller, albeit significant, induction in CA3 caspase-3 compared with prenatal treatment. Quantitative real-time PCR analysis revealed that prenatal but not postnatal DEX-induced hippocampal cleaved caspase-3 correlated with elevated mRNA of the proapoptotic gene Bad. Few caspase-3-ir cells were identified within the PVN regardless of treatment age, although postnatal but not prenatal DEX increased this number. However, the region immediately surrounding the PVN (peri-PVN) showed significant increases in caspase-3-ir cells following pre- and postnatal DEX. Together these findings indicate that developmental GC exposure increases apoptosis in HPAaxis-associated brain regions in an age- and sex-dependent manner.

Prenatal dexamethasone exposure potentiates diet-induced hepatosteatosis and decreases plasma IGF-I in a sex-specific fashion.

The clinical use of synthetic glucocorticoids in preterm infants to promote lung development has received considerable attention due to the potential for increased risk of developing metabolic disease in adulthood after such treatment. In this study, we examined the hypothesis that exposure to the synthetic glucocorticoid, dexamethasone (DEX), during late gestation in the rat results in the development of nonalcoholic fatty liver disease in adult offspring. Pregnant Sprague Dawley dams were treated with 0.4 mg/kg DEX beginning on gestational d 18 until parturition (gestational d 23). At postnatal d 21, offspring were weaned onto either a standard chow or high-fat (60% fat-derived calories) diet. In adulthood (postnatal d 60-65), hepatic tissue was harvested and examined for pathology. Liver steatosis, or fat accumulation, was found to be more severe in the DEX-exposed female offspring that were weaned onto the high-fat diet. This finding corresponded with decreased plasma IGF-I concentrations, as well as decreased hypothalamic expression of GHRH mRNA. Morphological measurements on body and long bone length further implicate a GH signaling deficit after fetal DEX exposure. Collectively, these data indicate suppression of GH axis function in the female DEX/high-fat cohort but not in the male offspring. Because deficits in the GH signaling can be linked to the development of nonalcoholic fatty liver disease, our results suggest that the prominent liver injury noted in female offspring exposed to DEX during late gestation may stem from abnormal development of the GH axis at the hypothalamic level.

Serotonin 1B autoreceptors originating in the caudal dorsal raphe nucleus reduce expression of fear and depression-like behavior.

BACKGROUND: Serotonin 1B (5-HT(1B)) autoreceptors regulate release of serotonin from terminals of dorsal raphe nucleus (DRN) projections. Expression of 5-HT(1B) in the DRN inversely correlates with behavioral measures of emotion, and viral-mediated overexpression of 5-HT(1B) receptors in the middle DRN inversely reduces measures of fear and anxiety in unstressed rats. Because the caudal subregion of the DRN is important in translating stress into emotional dysregulation, we explored behavioral functions of 5-HT(1B) autoreceptors in the caudal DRN. METHODS: We manipulated 5-HT(1B) autoreceptor function in rats using either viral-mediated gene transfer into the caudal DRN or systemic injections of the 5-HT(1B) agonist 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP-94253). Rats were tested in forced swim test, open field test, and contextual fear conditioning. RESULTS: Overexpression of 5-HT(1B) in the caudal DRN increased swimming in the forced swim test. It did not alter locomotion or thigmotaxis in the open field test but did reduce conditioned freezing. Freezing was reduced when 5-HT(1B) overexpression was present only during testing but not training. The CP-94253 exerted an inverted U-shaped dose response curve on conditioned freezing, with most pronounced effects seen at 1 mg/kg. At this dose, CP-94253 administered before a fear retention test reduced freezing both during that session and in subsequent drug-free testing, but only when drug was paired with re-exposure to the fear context. CONCLUSIONS: The 5-HT(1B) autoreceptors originating in the caudal DRN regulate behavioral expression of helplessness and fear. Because systemic pharmacologic treatment with a 5-HT(1B) agonist facilitates reductions in fear, 5-HT(1B) receptors may be a target for the treatment of certain anxiety disorders.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of TV-methyl-D-aspartate receptors

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of A'-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of N-methyl-D-aspartate receptors.

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of N-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

Estrogen selectively increases tryptophan hydroxylase-2 mRNA expression in distinct subregions of rat midbrain raphe nucleus: association between gene expression and anxiety behavior in the open field.

BACKGROUND: Ovarian steroids modulate anxiety behavior, perhaps by regulating the serotonergic neurons in the midbrain raphe nucleus. The regulation of the brain-specific isoform of rat tryptophan hydroxylase (TPH2) by ovarian hormones has not yet been investigated. Therefore, we examined the effects of estrogen and progesterone on TPH2 mRNA in the rat dorsal and median raphe nuclei (DRN and MRN, respectively) and whether TPH2 mRNA levels correlated with anxiety behavior. METHODS: Ovariectomized rats were treated for two weeks with placebo, estrogen or estrogen plus progesterone, exposed to the open field test, and TPH2 mRNA was quantified by in situ hybridization histochemistry. RESULTS: Estrogen increased TPH2 mRNA in the mid-ventromedial and caudal subregions of the DRN and the caudal MRN. Combined estrogen and progesterone treatment did not change TPH2 mRNA relative to ovariectomized controls. TPH2 mRNA in caudal DRN was associated with lower anxiety-like behavior, whereas TPH2 mRNA in rostral dorsomedial DRN was associated with increased anxiety-like behavior. CONCLUSIONS: These results suggest that estrogen may increase the capacity for serotonin synthesis in discrete subgroups of raphe neurons, and reinforce previous observations that different subregions of DRN contribute to distinct components of anxiety behavior.

Differential effects of ovarian steroids on anxiety versus fear as measured by open field test and fear-potentiated startle.

The ovarian steroids, estrogen (E) and progesterone (P), have been shown to affect anxiety and fear in humans and animals, although with inconsistent results. These ambiguous findings may be due to differential actions of ovarian steroids on anxiety versus fear. To investigate such a role, we used the open field test (OFT) and fear-potentiated startle (FPS). We examined these behaviors between cycling female rats in proestrus (high E and rising P) or diestrus (low E and P), as well as between ovariectomized rats treated for 2 weeks with placebo, E, or E plus P (OVX, OVX/E, OVX/EP, respectively). We found no differences in anxiety-like or fear behaviors in OFT or FPS between proestrus and diestrus rats, perhaps due to the opposing effects of E and P. In contrast, we found that the OVX/E rats spent more time in the center of the OFT compared to the OVX and OVX/EP rats with no difference in overall activity level, suggesting that E reduced anxiety and this was opposed by P. With FPS, the OVX/E rats showed increased startle in the first third of the testing session, followed by a rapid decline in startle magnitude in subsequent trials. The addition of P to E treatment counteracted this effect. In conclusion, E may have differential effects on specific components of anxiety and fear; E may decrease anxiety in a naturalistic environment, but intensify both fear learning and extinction processes. P antagonizes these E effects on anxiety and fear.

Retinal influences specify cortico-cortical maps by postnatal day six in rats and mice.

Studies of callosal projections in striate cortex show that the retina is involved in the development of topographical connections. In normal animals callosal fibers connect retinotopically corresponding, nonmirror-symmetric cortical loci, whereas in animals bilaterally enucleated at birth, callosal fibers connect topographically mismatched, mirror-symmetric loci. Moreover, in rodents the overall pattern of visual callosal connections is adult-like by postnatal day 12 (P12). In this study we delayed the onset of retinal deafferentation in rats and mice in order to determine the period when retinal influences are critically needed for the development of retinotopically matched callosal linkages. Callosal maps were revealed by placing small injections of retrogradely and anterogradely transported tracers into different loci of lateral striate cortex. We found that the patterns of callosal linkages in rats enucleated at P12, P8, and P6 were nonmirror-symmetric, as in normally reared rats. In contrast, the patterns of linkages in rats enucleated at P4 closely resembled the mirror-symmetric pattern seen in rats enucleated at birth (P0). A similar reversal in topography (from symmetric to nonsymmetric) occurred in mice when enucleation was delayed from P4 to P6. These findings indicate that retinal input prior to P6, but not prior to P4, is sufficient for specifying normal callosal topography. Moreover, they suggest that development of retinotopically matched callosal linkages depends critically on retinal influences during a brief period between P4 and P6, when callosal connections are still very immature.