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.

Taste activity in the parabrachial region in adult rats following neonatal chorda tympani transection.

The chorda tympani is a gustatory nerve that fails to regenerate if sectioned in rats 10 days of age or younger. This early denervation causes an abnormally high preference for NH4Cl in adult rats, but the impact of neonatal chorda tympani transection on the development of the gustatory hindbrain is unclear. Here, we tested the effect of neonatal chorda tympani transection (CTX) on gustatory responses in the parabrachial nucleus (PbN). We recorded in vivo extracellular spikes in single PbN units of urethane-anesthetized adult rats following CTX at P5 (chronic CTX group) or immediately prior to recording (acute CTX group). Thus, all sampled PbN neurons received indirect input from taste nerves other than the CT. Compared to acute CTX rats, chronic CTX animals had significantly higher responses to stimulation with 0.1 and 0.5 M NH4Cl, 0.1 and 0.5 M NaCl, and 0.01 M citric acid. Activity to 0.5 M sucrose and 0.01 M quinine stimulation was not significantly different between groups. Neurons from chronic CTX animals also had larger interstimulus correlations and significantly higher entropy, suggesting that neurons in this group were more likely to be activated by stimulation with multiple tastants. Although neural responses were higher in the PbN of chronic CTX rats compared to acute-sectioned controls, taste-evoked activity was much lower than observed in previous reports, suggesting permanent deficits in taste signaling. These findings demonstrate that the developing gustatory hindbrain exhibits high functional plasticity following early nerve injury.NEW & NOTEWORTHY Early and chronic loss of taste input from the chorda tympani is associated with abnormal taste behaviors. We found that compared to when the chorda tympani is sectioned acutely, chronic nerve loss leads to amplification of spared inputs in the gustatory pons, with higher response to salty and sour stimuli. Findings point to plasticity that may compensate for sensory loss, but permanent deficits in taste signaling also occur following early denervation.

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.

Regenerative Failure Following Rat Neonatal Chorda Tympani Transection is Associated with Geniculate Ganglion Cell Loss and Terminal Field Plasticity in the Nucleus of the Solitary Tract.

Neural insult during development results in recovery outcomes that vary dependent upon the system under investigation. Nerve regeneration does not occur if the rat gustatory chorda tympani nerve is sectioned (CTX) during neonatal (≤P10) development. It is unclear how chorda tympani soma and terminal fields are affected after neonatal CTX. The current study determined the impact of neonatal CTX on chorda tympani neurons and brainstem gustatory terminal fields. To assess terminal field volume in the nucleus of the solitary tract (NTS), rats received CTX at P5 or P10 followed by chorda tympani label, or glossopharyngeal (GL) and greater superficial petrosal (GSP) label as adults. In another group of animals, terminal field volumes and numbers of chorda tympani neurons in the geniculate ganglion (GG) were determined by labeling the chorda tympani with DiI at the time of CTX in neonatal (P5) and adult (P50) rats. There was a greater loss of chorda tympani neurons following P5 CTX compared to adult denervation. Chorda tympani terminal field volume was dramatically reduced 50 days after P5 or P10 CTX. Lack of nerve regeneration after neonatal CTX is not caused by ganglion cell death alone, as approximately 30% of chorda tympani neurons survived into adulthood. Although the total field volume of intact gustatory nerves was not altered, the GSP volume and GSP-GL overlap increased in the dorsal NTS after CTX at P5, but not P10, demonstrating age-dependent plasticity. Our findings indicate that the developing gustatory system is highly plastic and simultaneously vulnerable to 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.

Contributory role of sex differences in the variations of gustatory function.

Remarkable variability between males and females occurs for an array of taste-guided behaviors in both rodents and humans. Sex differences have been noted for taste preference, detection thresholds, and reactivity to taste stimuli. Manipulating sex hormones during early postnatal development or altering the amount of circulating estrogen in adulthood can dramatically alter the pattern of these behaviors. Receptors for sex hormones appear to be prominent in several nuclei associated with central gustatory pathways, indicating that steroid hormones may modulate central taste processing. Electrophysiological recordings from the rat brainstem suggest that taste-elicited activity to sweet stimuli is organized by hormones during early development, and activity during bitter stimulation is altered by circulating ovarian hormones. Sex differences in gustatory function appear to emerge at the level of the taste bud. Among ovariectomized rats, estradiol treatment decreases activity in the chorda tympani nerve during NaCl stimulation. Although there is no evidence that chorda tympani responses to NaCl differ between intact male and female rats, glossopharyngeal nerve responses are lower in intact females for both NaCl and sodium acetate. Responses in the glossopharyngeal nerve to citric acid stimulation are also higher in female rats relative to males. These findings suggest that, in addition to differential central modulation of taste input based on sex, taste information from the periphery varies between males and females. Although the extent of sex differences in taste processing and the underlying causal mechanisms require further clarification, it is clear that studying one sex alone provides an incomplete picture of gustatory function. © 2016 Wiley Periodicals, Inc.

Chronic Oral Capsaicin Exposure During Development Leads to Adult Rats with Reduced Taste Bud Volumes.

INTRODUCTION: Cross-sensory interaction between gustatory and trigeminal nerves occurs in the anterior tongue. Surgical manipulations have demonstrated that the strength of this relationship varies across development. Capsaicin is a neurotoxin that affects fibers of the somatosensory lingual nerve surrounding taste buds, but not fibers of the gustatory chorda tympani nerve which synapse with taste receptor cells. Since capsaicin is commonly consumed by many species, including humans, experimental use of this neurotoxin provides a naturalistic perturbation of the lingual trigeminal system. Neonatal or adults rats consumed oral capsaicin for 40 days and we examined the cross-sensory effect on the morphology of taste buds across development. METHODS: Rats received moderate doses of oral capsaicin, with chronic treatments occurring either before or after taste system maturation. Tongue morphology was examined either 2 or 50 days after treatment cessation. Edema, which has been previously suggested as a cause of changes in capsaicin-related gustatory function, was also assessed. RESULTS: Reductions in taste bud volume occurred 50 days, but not 2 days post-treatment for rats treated as neonates. Adult rats at either time post-treatment were unaffected. Edema was not found to occur with the 5 ppm concentration of capsaicin we used. CONCLUSIONS: Results further elucidate the cooperative relationship between these discrete sensory systems and highlight the developmentally mediated aspect of this interaction. IMPLICATIONS: Chronic exposure to even moderate levels of noxious stimuli during development has the ability to impact the orosensory environment, and these changes may not be evident until long after exposure has ceased.

Developmental time course of peripheral cross-modal sensory interaction of the trigeminal and gustatory systems.

Few sensory modalities appear to engage in cross-modal interactions within the peripheral nervous system, making the integrated relationship between the peripheral gustatory and trigeminal systems an ideal model for investigating cross-sensory support. The present study examined taste system anatomy following unilateral transection of the trigeminal lingual nerve (LX) while leaving the gustatory chorda tympani intact. At 10, 25, or 65 days of age, rats underwent LX with outcomes assessed following various survival times. Fungiform papillae were classified by morphological feature using surface analysis. Taste bud volumes were calculated from histological sections of the anterior tongue. Differences in papillae morphology were evident by 2 days post-transection of P10 rats and by 8 days post in P25 rats. When transected at P65, animals never exhibited statistically significant morphological changes. After LX at P10, fewer taste buds were present on the transected side following 16 and 24 days survival time and remaining taste buds were smaller than on the intact side. In P25 and P65 animals, taste bud volumes were reduced on the denervated side by 8 and 16 days postsurgery, respectively. By 50 days post-transection, taste buds of P10 animals had not recovered in size; however, all observed changes in papillae morphology and taste buds subsided in P25 and P65 rats. Results indicate that LX impacts taste receptor cells and alters epithelial morphology of fungiform papillae, particularly during early development. These findings highlight dual roles for the lingual nerve in the maintenance of both gustatory and non-gustatory tissues on the anterior tongue.

Long-term alterations in peripheral taste responses to NaCl in adult rats following neonatal chorda tympani transection.

The peripheral taste system of the adult rodent is highly resilient against damage, with morphological, behavioral, and functional recovery evident after regeneration of a transected nerve. If chorda tympani transection (CTX) occurs at early postnatal ages however, the nerve fails to regenerate and effects on tongue morphology and behavior are more severe and longer-lasting compared to adult denervation. To examine whether neonatal CTX induces functional changes in intact nerves, whole-nerve electrophysiology was performed on the glossopharyngeal (GL) and chorda tympani (CT) nerves of adult rats that received CTX at P10. Attenuation of NaCl-elicited GL responses were observed in CTX rats 2 months after surgery, with bilateral denervation causing the largest decreases in responses. When assessed 1 year after neonatal CTX, amiloride-sensitive responses to NaCl in the contralateral CT increased while amiloride-insensitive responses decreased. Responses to other tastants were consistent with control animals. This is the first evidence of long-term functional changes to the peripheral taste system after injury in rats fed a normal diet. This study further characterizes the developing peripheral taste system as highly susceptible to change following neural injury.

Chorda tympani nerve transection at different developmental ages produces differential effects on taste bud volume and papillae morphology in the rat.

Chorda tympani nerve transection (CTX) results in morphological changes to fungiform papillae and associated taste buds. When transection occurs during neonatal development in the rat, the effects on fungiform taste bud and papillae structure are markedly more severe than observed following a comparable surgery in the adult rat. The present study examined the potential "sensitive period" for morphological modifications to tongue epithelium following CTX. Rats received unilateral transection at 65, 30, 25, 20, 15, 10, or 5 days of age. With each descending age at the time of transection, the effects on the structural integrity of fungiform papillae were more severe. Significant losses in total number of taste buds and filiform-like papillae were observed when transection occurred 5-30 days of age. Significant reduction in the number of taste pores was indicated at every age of transection. Another group of rats received chorda tympani transection at 10, 25, or 65 days of age to determine if the time course of taste bud degeneration differed depending on the age of the rat at the time of transection. Taste bud volumes differed significantly from intact sides of the tongue at 2, 8, and 50 days post-transection after CTX at 65 days of age. Volume measurements did not differ 2 days post-transection after CTX at 10 or 25 days of age, but were significantly reduced at the other time points. Findings demonstrate a transitional period throughout development wherein fungiform papillae are highly dependent upon the chorda tympani for maintenance of morphological integrity.

In vivo recordings from rat geniculate ganglia: taste response properties of individual greater superficial petrosal and chorda tympani neurones.

Coding of gustatory information is complex and unique among sensory systems; information is received by multiple receptor populations located throughout the oral cavity and carried to a single central relay by four separate nerves. The geniculate ganglion is the location of the somata of two of these nerves, the greater superficial petrosal (GSP) and the chorda tympani (CT). The GSP innervates taste buds on the palate and the CT innervates taste buds on the anterior tongue. To obtain requisite taste response profiles of GSP neurones, we recorded neurophysiological responses to taste stimuli of individual geniculate ganglion neurones in vivo in the rat and compared them to those from the CT. GSP neurones had a distinct pattern of responding compared to CT neurones. For example, a small subset of GSP neurones had high response frequencies to sucrose stimulation, whereas no CT neurones had high response frequencies to sucrose. In contrast, NaCl elicited high response frequencies in a small subset of CT neurones and elicited moderate response frequencies in a relatively large proportion of GSP neurones. The robust whole-nerve response to sucrose in the GSP may be attributable to relatively few, narrowly tuned neurones, whereas the response to NaCl in the GSP may relate to proportionately more, widely tuned neurones. These results demonstrate the diversity in the initial stages of sensory coding for two separate gustatory nerves involved in the ingestion or rejection of taste solutions, and may have implications for central coding of gustatory quality and concentration as well as coding of information used in controlling energy, fluid and electrolyte homeostasis.

Each sensory nerve arising from the geniculate ganglion expresses a unique fingerprint of neurotrophin and neurotrophin receptor genes.

Neurons in the geniculate ganglion, like those in other sensory ganglia, are dependent on neurotrophins for survival. Most geniculate ganglion neurons innervate taste buds in two regions of the tongue and two regions of the palate; the rest are cutaneous nerves to the skin of the ear. We investigated the expression of four neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4, and five neurotrophin receptors, trkA, trkB, trkC, p75, and truncated trkB (Trn-B) in single sensory neurons of the adult rat geniculate ganglion associated with the five innervation fields. For fungiform papillae, a glass pipette containing biotinylated dextran was placed over the target papilla and the tracer was iontophoresed into the target papilla. For the other target fields, Fluoro-Gold was microinjected. After 3 days, geniculate ganglia were harvested, sectioned, and treated histochemically (for biotinylated dextran) or immunohistochemically (for Fluoro-Gold) to reveal the neurons containing the tracer. Single labeled neurons were harvested from the slides and subjected to RNA amplification and RT-PCR to reveal the neurotrophin or neurotrophin receptor genes that were expressed. Neurons projecting from the geniculate ganglion to each of the five target fields had a unique expression profile of neurotrophin and neurotrophic receptor genes. Several individual neurons expressed more than one neurotrophin receptor or more than one neurotrophin gene. Although BDNF is significantly expressed in taste buds, its primary high affinity receptor, trkB, was not prominently expressed in the neurons. The results are consistent with the interpretation that at least some, perhaps most, of the trophic influence on the sensory neurons is derived from the neuronal somata, and the trophic effect is paracrine or autocrine, rather than target derived. The BDNF in the taste bud may also act in a paracrine or autocrine manner on the trkB expressed in taste buds, as shown by others.

Injury-induced functional plasticity in the peripheral gustatory system.

Combining unilateral denervation of anterior tongue taste buds with a low-sodium diet in rats results in a rapid, dramatic, and selective attenuation of neurophysiological sodium taste responses from the intact side of the tongue. The transduction pathway responsible for the attenuated response is through the epithelial sodium channel (Hill and Phillips, 1994). Current experiments extend these findings by detailing the effects of experimentally induced injury on taste responses from anterior tongue taste receptors in sodium-restricted rats. Experiments focused on functional salt taste responses from the intact chorda tympani nerve in sodium-restricted rats in which a gustatory nerve was sectioned that innervates the anterior tongue (chorda tympani), the posterior tongue (glossopharyngeal), or palatal taste receptors (greater superficial petrosal) or in which a nongustatory nerve was sectioned that also has its target in the anterior tongue (trigeminal). An additional group was studied that received thermal injury to the anteroventral tongue. Substantial and selective suppression of sodium salt responses occurred in a graded manner generally related to the distance from the target field of the injury to anterior tongue taste buds. The order of effectiveness was: chorda tympani section > trigeminal section > thermal injury = glossopharyngeal section > greater superficial petrosal section. These results support the hypothesis that local, diffusible factors liberated from immune-derived cells as a result of neural and/or epithelial damage are involved in regulating the transduction pathway responsible for sodium salt sensation, and that these factors may become evident through dietary sodium restriction.

Time course of morphological alterations of fungiform papillae and taste buds following chorda tympani transection in neonatal rats.

The time course of structural changes in fungiform papillae was analyzed in rats that received unilateral chorda tympani nerve transection at 10 days of age. Morphological differences between intact and denervated sides of the tongue were first observed at 8 days postsection, with an increase in the number of fungiform papillae that did not have a pore. In addition, the first papilla with a filiform-like appearance was noted on the denervated side at 8 days postsectioning. By 11 days after surgery, the total number of papillae and the number of papillae with a pore were significantly lower on the transected side of the tongue as compared to the intact side. At 50 days postsection, there was an average of 70.5 fungiform papillae on the intact side and a mean of only 20.8 fungiform papillae the denervated side. Of those few remaining papillae on the cut side, an average of 13.5 papillae were categorized as filiform-like, while no filiform-like papillae occurred on the intact side. Significant reduction in taste bud volume was noted at 4 days posttransection and further decrements in taste bud volume were noted at 8 and 30 days postsection. Electron microscopy of the lingual branch of the trigeminal nerve from adult rats that received neonatal chorda tympani transection showed normal numbers of both myelinated and unmyelinated fibers. Thus, in addition to the well-characterized dependence of taste bud maintenance on the chorda tympani nerve, the present study shows an additional role of the chorda tympani nerve in papilla maintenance during early postnatal development.