The timing of neuronal loss across adolescence in the medial prefrontal cortex of male and female rats
Introduction
Adolescence, often defined as the period between puberty and adulthood, is a time characterized by neuroanatomical changes that coincide with an increased vulnerability to a variety of clinical disorders including depression, anxiety and schizophrenia (Spear, 2000, Steinberg, 2005, Paus et al., 2008). The prefrontal cortex (PFC), a region primarily involved in executive function and emotional regulation, continues to develop across much of the lifespan with adolescence as a critical period of development. In the human PFC, there is significant synaptic pruning during the adolescent period. Between early adolescence and adulthood, there is a decrease in dendritic spine density on PFC pyramidal neurons (Petanjek et al., 2011) and a decrease in synaptic density (Huttenlocher and Dabholkar, 1997). This period also coincides with an overall decrease in PFC volume (Gogtay et al., 2004, Lenroot and Giedd, 2006). Importantly, there is evidence for differences in the timing and trajectory of PFC development in males and females (Lenroot and Giedd, 2006).
Because of the difficulty in quantification and parcellation of the human PFC and given the homology between the rat and primate frontal cortex (Uylings et al., 2003), rat models for PFC development have been reliably used. Similar to primates, the rodent PFC continues to develop into adulthood, as both its volume (Van Eden and Uylings, 1985a) and laminar specificity (Van Eden and Uylings, 1985b) peak during the periadolescent period and then decrease until at least 90 days of age. Synaptic pruning and dendritic organization have been documented in the adolescent rodent medial prefrontal cortex (mPFC) as well. Specifically, between adolescence and adulthood, both male and female rats lose dendritic spines while only female rats lose a significant number of dendrites (Koss et al., 2014). In addition, while the density of PFC neurons projecting to the amygdala decreases between adolescence and adulthood (Cressman et al., 2010), there is an increase in the density of fibers innervating the mPFC from the amygdala (Cunningham et al., 2002).
Evidence from our laboratory indicates that changes in synaptic density and volume could be related to a loss of total neurons in the region. In the rat mPFC, the number of neurons decreases between adolescence and adulthood with neuronal loss being considerably greater in females (Markham et al., 2007). There is further evidence that in females, the actions of hormones secreted after puberty lead to this decrease in mPFC neuron number, as ovariectomy before puberty prevents this loss when neuron number is assessed in adulthood (Koss et al., 2015). This is similar to findings in the primary visual cortex where ovarian hormones after puberty were found to play a role in neuronal pruning (Nuñez et al., 2002). It is currently unclear whether these neuronal losses in the mPFC involve GABAergic interneurons or glutaminergic pyramidal cells, or a combination of both. However, one study showed that ovariectomized adult females have a greater density of parvalbumin-positive GABA neurons in the mPFC than that of intact females (Cholanian et al., 2014), suggesting the neuronal losses in intact females may detectably include GABA cells.
Counter to the pruning of many cellular components of the gray matter is the increase in white matter between the juvenile and adult periods. Myelination is known to continue well into adulthood in the corpus callosum of rats (Nuñez et al., 2000). Electron microscopic analysis of the posterior (splenium) corpus callosum shows that there is an increase in the number of axons that are myelinated between the juvenile period and adulthood, even while axons are pruned (Kim and Juraska, 1997). Furthermore the presence of ovarian hormones from puberty on leads to a lower number of myelinated axons in this region in early adulthood (Yates and Juraska, 2008). Like the posterior corpus callosum, the volume of the white matter under the PFC continues to increase between adolescence and adulthood in both male and female rats. However, a sex difference emerges at P90 with males having a larger white matter volume than females (Markham et al., 2007).
A critical question that remains unanswered is the trajectory of these neuroanatomical changes, specifically whether they occur gradually throughout the adolescent period or within a well-defined temporal window that may coincide with puberty. If puberty is a key component for these changes, there should be differences between males and females due to the difference in pubertal timing and the different hormones involved. Here we quantified neuron and glial cell number, along with the volume of the white matter under the PFC, in male and female rats. In addition to counts of all neurons in the mPFC, we stereologically counted the number of GABA immunoreactive interneurons. Most previous studies have compared two ages: juvenile/early adolescence and adult. Here we examine five ages from the juvenile period to puberty through adulthood in order to delineate when these anatomical changes occur.
Section snippets
Subjects
Subjects were the offspring of Long-Evans hooded rats obtained from Harlan Laboratories (Indianapolis, IN, USA) and bred in the vivarium in the Psychology Department at the University of Illinois. All animals were weaned on postnatal day (P) 24 and housed with same-sex littermates in pairs or triplets until sacrifice. Tissue from both male and female rats was collected at P25, P35, P45, P60 and P90 (n = 10–11 per group) for a total of 105 animals. P25 is during the preadolescent period, P35
Age of puberty
For animals that were not sacrificed prior to pubertal onset, males reached puberty at an average age of 44.9 days with a range of 42–48. The average age for the onset of puberty in females was 34.9 with a range of 32–38.
Neuron number
Because our analysis revealed no differences in neuron counts between cerebral hemispheres, or between cortical layers (2/3, 5/6) in males or females at any age, data presented are pooled from left and right hemispheres from layers 2 to 6. Females lost a significant number of
Discussion
In the present study, there was a decrease in the number of neurons in the female mPFC between P35 and P45, which coincides with the onset of puberty. Furthermore, it appears that these neuronal losses were not predominantly GABAergic interneurons. Male rats showed only a weak trend for neuronal loss across ages. Additionally, while both males and females gained white matter volume under the PFC, this increase was somewhat blunted in female rats after puberty. There was no detectable change in
Acknowledgments
We thank Taehyeon Kim for her assistance and the Beckman Institute Imaging Technology Group for use of the microscopy suite. This work was supported by NIH MH099625 to J.M. Juraska.
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