Terminal field volume of the glossopharyngeal nerve in adult rats reverts to prepruning size following microglia depletion with PLX5622.

Programmed reduction of synapses is a hallmark of the developing brain, with sensory systems emerging as useful models with which to study this pruning. The central projections (terminal field) of the gustatory glossopharyngeal nerve (GL) of the rat are a prime example of developmental pruning, undergoing an approximate 66% reduction in volume from postnatal day 15 (P15) to P25. Later in adulthood, developmental GL pruning can be experimentally reversed, expanding to preweaning volumes, suggesting mature volumes may be actively maintained throughout the life span. Microglia are central nervous system glia cells that perform pruning and maintenance functions in other sensory systems, including other gustatory nerves. To determine their role in GL pruning, we depleted microglia from Sprague-Dawley rat brains from P1 to P40 using daily intraperitoneal injections of the colony-stimulating factor 1 receptor inhibitor PLX5622. This prevented GL developmental pruning, resulting in preweaning terminal field volumes and innervation patterns persisting through P40, 2 weeks after pruning is normally completed. These findings show microglia are necessary for developmental GL pruning. Ceasing PLX5622 treatments at P40 allowed microglia repopulation, and within 4 weeks the GL terminal field had reduced to control volumes, indicating that pruning can occur outside of the typical developmental period. Conversely, when microglia were depleted in adult rats, GL terminal fields expanded, reverting to sizes comparable to the neonatal rat. These data indicate that microglia are required for GL pruning and may continue to maintain the GL terminal field at a reduced size into adulthood.

Astrocytic response to neural injury is larger during development than in adulthood and is not predicated upon the presence of microglia.

While contributions of microglia and astrocytes are regularly studied in various injury models, how these contributions differ across development remains less clear. We previously demonstrated developmental differences in microglial profiles across development in an injury model of the gustatory system. Nerves of the rat gustatory system have limited capacity to regenerate if injured during neonatal ages but show robust recovery if the injury occurs in adulthood. Using this developmentally disparate model of regenerative capacity, we quantified microglia and astrocytes in the rostral nucleus of the solitary tract (rNTS) following transection of the gustatory chorda tympani nerve (CTX) of neonatal and adult rats. We found that neonatal CTX induced an attenuated microglia response but a larger astrocyte response compared to adult CTX. To elucidate the interplay between the microglia and astrocyte responses in the CTX model, we used our novel intraperitoneal injection protocol for the colony-stimulating factor 1 receptor inhibitor PLX5622 to deplete microglia in the neonatal and adult rat brain prior to and after CTX. PLX5622 depleted microglia by 80-90% within 3 days of treatment, which increased to > 90% by 7 days. After 14 days of PLX5622 treatment, microglia were depleted by > 96% in both neonates and adults while preserving baseline astrocyte quantity. Microglia depletion eliminated the adult astrocyte response to CTX, while the neonatal astrocyte response after injury remained robust. Our results show injecting PLX5622 is a viable means to deplete microglia in neonatal and adult rats and suggest developmentally distinct mechanisms for astrogliosis following neural injury.

Microglia density decreases in the rat rostral nucleus of the solitary tract across development and increases in an age-dependent manner following denervation.

Microglia are critical for developmental pruning and immune response to injury, and are implicated in facilitating neural plasticity. The rodent gustatory system is highly plastic, particularly during development, and outcomes following nerve injury are more severe in developing animals. The mechanisms underlying developmental plasticity in the taste system are largely unknown, making microglia an attractive candidate. To better elucidate microglia's role in the taste system, we examined these cells in the rostral nucleus of the solitary tract (rNTS) during normal development and following transection of the chorda tympani taste nerve (CTX). Rats aged 5, 10, 25, or 50days received unilateral CTX or no surgery and were sacrificed four days later. Brain tissue was stained for Iba1 or CD68, and both the density and morphology of microglia were assessed on the intact and transected sides of the rNTS. We found that the intact rNTS of neonatal rats (9-14days) shows a high density of microglia, most of which appear reactive. By 29days of age, microglia density significantly decreased to levels not significantly different from adults and microglia morphology had matured, with most cells appearing ramified. CD68-negative microglia density increased following CTX and was most pronounced for juvenile and adult rats. Our results show that microglia density is highest during times of normal gustatory afferent pruning. Furthermore, the quantity of the microglia response is higher in the mature system than in neonates. These findings link increased microglia presence with instances of normal developmental and injury induced alterations in the rNTS.