Intranasal Inhibition of Tropomyosin Receptor Kinase B Signaling during Early Postnatal Development Disrupts Growth and Sensorimotor Maturation in Neonatal Rodents

Animals and Treatments

All procedures were approved by the Institutional Animal Care and Use Committee at the College of Charleston (Protocol #2024-007). Sprague Dawley neonatal rodents were housed with their biological dams in a temperature-controlled vivarium (12 h light/dark cycle; ad libitum access to food and water). Litters ranged from 10 to 13 pups, and pups were randomly assigned to treatment groups to minimize litter effects. Each experimental group contained n = 10 pups per group derived from multiple litters to minimize litter-specific effects. Pups received either saline vehicle (n = 12), ANA-12 at 0.5 mg/kg (n = 10), or ANA-12 at 1 mg/kg (n = 13) delivered intranasally once daily from postnatal day (PND) 8–14. This developmental window corresponds to a critical period in rodent cortical maturation characterized by rapid synaptogenesis and peak BDNF/TrkB signaling, making it highly sensitive to trophic manipulation.

Intranasal Delivery

The ANA-12 doses were selected based on previously published in vivo studies demonstrating effective TrkB inhibition following systemic administration. Although intranasal drug delivery can sometimes require lower doses than systemic administration, neonatal nasal absorption and distribution differ substantially from adult models due to differences in mucosal surface area, enzymatic metabolism, and cerebrospinal fluid dynamics. Therefore, doses consistent with previously validated in vivo TrkB inhibition studies were used to ensure sufficient CNS exposure during this developmental period. Intranasal administration was performed on awake pups using a standardized neonatal delivery protocol. ANA-12 (a selective, noncompetitive TrkB antagonist) was purchased from Sigma Aldrich and prepared according to the manufacturer’s specifications. Stock solutions were dissolved in dimethyl sulfoxide and diluted in sterile saline to achieve final working concentrations. Doses of 0.5 mg/kg and 1 mg/kg were selected based on prior in vivo studies demonstrating effective TrkB inhibition within the central nervous system [8]. Solutions were freshly prepared and administered intranasally once daily. Pups were gently held in a semi-supine position, and doses were delivered in 2 µL droplets alternated between nostrils with 2-min intervals to prevent aspiration and optimize absorption through olfactory and trigeminal pathways. Total administration volumes did not exceed 16 µL per pup per day. ANA-12 doses were adjusted daily to each animal’s body weight.

Developmental Assessments

Body weights were recorded at PND7, PND9, PND12, and PND14 to assess the impact of chronic TrkB inhibition on physical growth. Early-life growth rate is highly dependent on neurotrophin-regulated metabolic and neuromuscular maturation, making weight an important developmental indicator.

Sensorimotor Reflex Testing

Behavioral assessments were conducted at PND7 and PND14 to evaluate the developmental progression of core reflexes, which depend on brainstem, cerebellar, and cortical maturation. Each test was performed in three trials, and the mean latency was used for analysis. These tasks were chosen because they capture hallmark developmental milestones known to depend on intact trophic signaling during PND7–P14.

ELISA Quantification

At PND14, pups were deeply anesthetized with isoflurane inhalation anesthesia, and brains were rapidly harvested. Cortical and hippocampal tissue samples were microdissected, flash frozen on dry ice, and stored at –80°C until processing. Tissue homogenates were prepared in lysis buffer with protease and phosphatase inhibitors to preserve phosphorylation states. Protein assays and a series of ELISAs were performed in both experiments to assess protein levels and neuroinflammation markers, including TNF-α and IL-6, as well as ELISA analysis of total and phosphorylated TrkB. ELISA assays were used to quantify total TrkB and phosphorylated TrkB at Tyr816, a phosphorylation site associated with PLCγ signaling downstream of TrkB activation. Samples from the cortex and hippocampus were harvested and utilized. Quantitative assessments were gathered via Tukey’s post hoc test and one-way ANOVA analyses. Values were normalized to total protein (pg/mg) to account for differences in tissue mass and homogenate concentration.

Statistics

All data are presented as mean ± SEM. Behavioral and longitudinal growth data were analyzed using two-way ANOVAs with treatment and age as factors. Neurochemical data (ELISA-based measurements) were analyzed using one-way ANOVAs within each brain region. When appropriate, Tukey’s multiple comparisons test was used for post hoc pairwise comparisons. Assumptions of normality and homogeneity of variance were evaluated using Brown-Forsythe and Bartlett’s tests. Statistical significance was defined as p < 0.05, with thresholds denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Intranasal ANA-12 Reduced Postnatal Growth

Postnatal body weight serves as a key indicator of developmental progress, enabling the assessment of how trkB inhibition affects early growth trajectories. Intranasal administration of ANA-12 produced a dose-dependent reduction in postnatal body weight at PND14 (Fig. 1). While all groups showed the expected age-related increases in mass, pups receiving either 0.5 mg/kg or 1 mg/kg ANA-12 displayed attenuated growth trajectories relative to saline-treated controls. By PND14, both ANA-12 groups exhibited significantly lower body weights compared to saline (p < 0.001), with the 1 mg/kg group showing the most pronounced reduction.

TrkB Inhibition Delayed Sensorimotor Reflex Maturation

A two-way ANOVA for negative geotaxis (Fig. 2a) revealed a significant interaction between time and treatment (F(2,21) = 16.10, p < 0.001). At PND7, post hoc analyses indicated significantly longer geotaxis latencies in both the ANA-12 1 mg/kg (p < 0.0001) and 0.5 mg/kg groups (p = 0.0003) compared to saline controls. By PND14, the high-dose ANA-12 group continued to exhibit elevated latencies compared to the saline group (p = 0.0048). Saline-treated pups exhibited a significant developmental improvement between postnatal day 7 (PND7) and postnatal day 14 (PND14) (p < 0.0001). In contrast, the ANA-12 1 mg/kg group demonstrated minimal change across this interval, suggesting a marked disruption in the normal maturation of this reflex.

For cliff avoidance, a two-way ANOVA likewise demonstrated a significant time × treatment interaction (F(2,21) = 41.20, p < 0.0001) (Fig. 2b). There were significant main effects of treatment (F(2,21) = 31.35, p < 0.001) and age (F(1,21) = 27.30, p < 0.0001). ANA-12-treated pups exhibited longer avoidance latencies at PND14 compared to PND7, while saline-treated pups showed the expected developmental decrease in latency over time.

A two-way ANOVA for righting reflex (Fig. 2c) performance revealed a significant effect of time (F(1,21) = 10.76, p = 0.0036). All groups exhibited reduced righting latencies from PND7 to PND14, with a repeated-measures analysis confirming a significant improvement, specifically within the saline group (p = 0.0015). By PND14, righting performance did not differ significantly among treatment groups, indicating partial developmental recovery of this reflex despite early delays associated with ANA-12 exposure.

Cortical TrkB Expression Increased after ANA-12 Exposure

Cortical TrkB expression was altered following ANA-12 exposure (Fig. 3). Total TrkB and phosphorylated TrkB levels were analyzed separately across treatment groups. One-way ANOVA revealed a significant increase in total TrkB expression in ANA-12-treated animals relative to saline controls (F(2,31) = 7.928, p = 0.0017). Post hoc Tukey’s multiple comparisons test confirmed that both the 0.5 mg/kg (p = 0.0016) and 1 mg/kg (p = 0.0151) ANA-12 groups exhibited significantly higher total TrkB levels compared to saline controls, with no difference between ANA-12 dose groups (p = 0.6389) (Fig. 3b).

In contrast, phosphorylated TrkB levels did not differ significantly among treatment groups (F(2,23) = 1.317, p = 0.2875) (Fig. 3a). To further assess receptor activation, the ratio of phosphorylated TrkB to total TrkB (pTrkB/pan-TrkB) was calculated as an index of receptor activity (Fig. 3c). One-way ANOVA revealed a significant effect of treatment on this ratio (F(2,22) = 5.482, p = 0.0117). Post hoc analysis indicated that both ANA-12 treatment groups showed significantly reduced pTrkB/pan-TrkB ratios compared to saline controls (0.5 mg/kg: p = 0.0135; 1 mg/kg: p = 0.0351), with no difference between doses (p = 0.9487). Together, these findings indicate that ANA-12 treatment increased total TrkB expression while reducing relative receptor activation, consistent with compensatory upregulation of TrkB in response to pharmacological inhibition without a corresponding increase in receptor phosphorylation (Fig. 3).

ANA-12 Reduced Cortical and Hippocampal Inflammation

ANA-12 differentially modulated inflammatory signaling in the cortex and hippocampus. To assess neuroinflammatory responses, TNF-α and IL-6 levels were quantified separately in cortical and hippocampal tissue using ELISA and analyzed by one-way ANOVA (Fig. 4).

In the cortex, TNF-α levels differed significantly across treatment groups (F(2,22) = 4.501, p = 0.0230) (Fig. 4c). Post hoc Tukey’s comparisons revealed a significant reduction in TNF-α levels in the ANA-12 1 mg/kg group compared to saline controls (p = 0.0342), whereas the 0.5 mg/kg group did not differ significantly from saline (p = 0.0697). No difference was observed between ANA-12 dose groups (p = 0.9117). In contrast, cortical IL-6 levels did not differ significantly across treatment groups (F(2,28) = 1.209, p = 0.3137) (Fig. 4a).

In the hippocampus, both cytokines were significantly affected by treatment. TNF-α levels differed across groups (F(2,25) = 3.743, p = 0.0378) (Fig. 4d), with post hoc analysis indicating a significant reduction in the ANA-12 1 mg/kg group relative to saline controls (p = 0.0472), but not in the 0.5 mg/kg group (p = 0.1189). Similarly, IL-6 levels were significantly reduced in the hippocampus following ANA-12 treatment (F(2,26) = 38.27, p < 0.0001) (Fig. 4b), with both the 0.5 mg/kg and 1 mg/kg groups showing significantly lower IL-6 levels compared to saline controls (both p < 0.0001), and no difference between ANA-12 doses (p = 0.9776). Together, these findings indicate that ANA-12 treatment reduces pro-inflammatory cytokine expression in a region- and cytokine-specific manner, with more robust and consistent effects observed in the hippocampus compared to the cortex (Fig. 4).

Functional Consequences of TrkB Inhibition on Neonatal Development

Across all behavioral assays, pups receiving either 0.5 mg/kg or 1 mg/kg ANA-12 consistently showed delayed maturation compared to saline controls. This was evident in cliff avoidance latencies and slower negative geotaxis responses in ANA-12-treated animals. These findings are consistent with the known roles of BDNF in sensorimotor development [18, 19]. The relative absence of developmental improvement between PND7 and PND14 in the higher ANA-12 dose group suggests that continuous TrkB suppression disrupts the normal progression of sensorimotor reflexes. This pattern parallels findings that reducing BDNF activity alters synaptic plasticity, decreases cortical neuronal responsiveness, and increases susceptibility to developmental perturbations [20, 21].

Neurochemical Effects of ANA-12: A Paradoxical Shift in Cytokines and TrkB

ANA-12 administration significantly reduced IL-6 in the hippocampus. In contrast, cortical IL-6 levels were not significantly altered, and TNF-alpha levels showed a selective reduction in the cortex at the higher dose, with more consistent reductions observed in the hippocampus. Although BDNF is widely recognized for its role in neuroprotection, developmental neuroimmune interactions differ markedly from adult responses. BDNF influences microglial maturation, cytokine responsiveness, and astrocyte-neuron communication during early life [22, 23]. Inhibiting TrkB may suppress activity-dependent cytokine release or alter neuron-glial communication, resulting in a net decrease in pro-inflammatory markers. This interpretation aligns with prior findings showing that TrkB modulation can influence immune tone at different developmental stages [24].

A second key observation was the significant increase in total TrkB expression following ANA-12 exposure, while phosphorylated TrkB levels were not significantly altered. As a result, the ratio of phosphorylated to total TrkB was reduced, indicating decreased relative receptor activation. At face value, increased receptor expression during pharmacological inhibition appears contradictory. However, this pattern reflects a well-characterized homeostatic response in which neurons compensate for reduced signaling by upregulating receptor expression. When downstream TrkB signaling pathways such as ERK, Akt, and PLCγ1 are suppressed by ANA-12, neurons may increase transcription and translation of TrkB receptors in an attempt to restore trophic signaling [25, 26].

Although phosphorylation of TrkB was not significantly altered, phosphorylation alone does not necessarily indicate functional signaling. ANA-12 is a noncompetitive allosteric antagonist that binds the TrkB extracellular domain and prevents the conformational changes required for full receptor activation [8]. In this context, basal or heterologous phosphorylation may still occur even in the absence of effective downstream signaling. Heterologous kinases, including Src family kinases, GPCR-associated kinases, and calcium-dependent enzymes, can phosphorylate TrkB residues independent of canonical ligand-driven activation [27, 28]. The resulting pool of phosphorylated but functionally limited receptors may contribute to the observed dissociation between receptor expression and behavioral outcomes.

Developmental Vulnerability and the Importance of Timing

The neonatal brain exhibits heightened sensitivity to trophic modulation due to its rapid rate of development and maturation. BDNF levels rise sharply during the first two postnatal weeks, supporting synaptic refinement, dendritic stabilization, and the integration of cortical circuits [29]. Interrupting this process with ANA-12 is likely to disrupt critical periods of plasticity. Negative geotaxis and cliff avoidance are sensitive to early deficits in cerebellar development, vestibular integration, and corticospinal maturation, all of which rely on TrkB signaling [30].

The observed weight differences further reflect systemic developmental consequences. BDNF is known to influence hypothalamic regulation of metabolism, neuromuscular development, and overall growth rate. Reduced trophic signaling may impair energy utilization, feeding behavior, or growth-related cellular processes, contributing to slower weight gain [31].

Broader Significance for Neurodevelopmental Disorders

Disrupted TrkB signaling has been implicated in autism spectrum disorders, intellectual disability, motor coordination disorders, and early-life stress exposure. The developmental delays observed here may reflect mechanisms relevant to these conditions. Reduced BDNF/TrkB activity during critical developmental windows may alter synaptic connectivity, weaken sensorimotor integration, and impair cortical maturation [32–34]. This study provides a model for understanding how early-life reductions in trophic signaling influence neurodevelopmental trajectories.

Limitations and Future Directions

Several limitations warrant consideration. First, ELISA-based quantification cannot distinguish between phosphorylation at different TrkB residues, which may provide more mechanistic insight into the nature of compensatory phosphorylation. Second, long-term behavioral or structural outcomes beyond PND14 were not assessed; future studies should examine whether ANA-12-induced delays persist into adolescence or recover spontaneously.

Importantly, a baseline difference in negative geotaxis performance was observed between groups at PND7 prior to treatment initiation. Although pups were randomly assigned to treatment groups to minimize litter effects, behavioral stratification based on baseline performance was not performed. This variability likely reflects natural differences in early sensorimotor development between litters and may influence interpretation of developmental trajectories. Future studies incorporating baseline-matched group allocation or larger sample sizes may help reduce this variability and strengthen interpretation of treatment effects. Future work should also investigate whether compensatory increases in TrkB expression enhance responsiveness to subsequent BDNF treatment or influence recovery following injury.