Jack E. Turman

NMDA receptor NR1 and NR2A/B subunit expression in trigeminal neurons during early postnatal development

Trigeminal motoneurons (Mo5), mesencephalic trigeminal neurons (Me5), and supratrigeminal (Su5) and intertrigeminal (I5) neurons are important constituents of the neural circuitry responsible for jaw movements observed during ingestive behaviors. In addition, in adult animals, N‐methyl‐D‐aspartate (NMDA) receptors are a critical component of the brainstem circuitry responsible for reflex‐ and centrally activated jaw movements. However, little is known about the expression of this receptor in circuitry used to produce neonatal jaw movements. Receptor immunohistochemistry was used to describe changes in the expression of NMDA NR1 and NR2A/B receptor subunits in Mo5, Me5, Su5, and I5 neurons during postnatal development. Rats at postnatal days (P) 1, 3, 8, 15–16, 21–24, and 28–35 were used. An affinity‐purified polyclonal antibody against the NR1 subunit and an affinity‐purified polyclonal antibody that recognizes both NR2A and 2B subunits were used to depict the expression of these subunits. In Mo5, immunoreactivity was noted for both antibodies throughout the time frame sampled. NR1 expression in Me5 neurons emerged at P1. NR2A/B expression emerged at P3 in caudal and middle regions of Me5 and at P8 for rostral regions of the nucleus. NR1 immunoreactivity was present at P1 for neurons in I5 and at P3 for neurons in the Su5 region. NR2A/B subunit expression in Su5 and I5 neurons emerged at P8. These results provide evidence for NMDA receptor subunits in neonatal trigeminal neurons used in oral–motor circuitry and suggest a role for the NMDA receptor in synaptogenesis associated with these neurons during postnatal development. J. Comp. Neurol. 409:237–249, 1999.

Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4- and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20(+/-) mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20(+/-) splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis.

AMPA receptor subunit expression in trigeminal neurons during postnatal development.

Trigeminal motoneurons (Mo5) and mesencephalic trigeminal neurons (Me5) are important constituents of the neural circuitry responsible for jaw movements. Non–N–methyl–D–aspartate (NMDA) glutamate receptors are a critical component of the brainstem circuitry responsible for reflex and centrally activated jaw movements; however, little is known about the expression of these receptors in neonatal oral–motor circuitry. Receptor immunohistochemistry using affinity–purified polyclonal antibodies directed against GluR1, GluR2/3/4c, and GluR4, respectively, and a monoclonal antibody directed against the GluR2 subunit, were used in rats at postnatal day (P)1, P3, P5, P10, P19–21, P32–35, and P60 to describe the expression of the alpha–amino–d–3–hydroxy–5–methyl–4–isoxazolepropionic acid (AMPA) receptor in Mo5 and Me5 neurons. In Mo5, immunoreactivity was noted for all antibodies throughout the time frame sampled. Neurons in caudal portions of Me5 displayed immunoreactivity to each antibody except at P60 when GluR2 immunoreactivity was absent. Neurons located in rostral Me5 displayed GluR2/3/4c and GluR4 immunoreactivity throughout the time frame, GluR1 immunoreactivity emerged at P3 and a transient expression of GluR2 expression was observed between P10 and P32–35. The lack of labeling of some neurons in both regions, coupled with differences in temporal expression, suggests that there are differences in the AMPA receptor phenotype within and between Mo5 and Me5 during postnatal development. Differences in AMPA subunit composition suggest a complex role for AMPA–mediated glutamatergic neurotransmission in brainstem circuits controlling jaw movements. J. Comp. Neurol. 427:109–123, 2000.

Growth deficits in a postnatal day 3 rat model of hypoxic-ischemic brain injury.

The postnatal day (P) 3 rat model of hypoxic-ischemic (HI) brain injury provides valuable information regarding the cellular response to HI injury in a very immature brain. Our present study is the first to examine growth, metabolic, and behavioral outcomes following a P3 HI brain injury. Rats were injured by cauterizing the right common carotid, and exposure to 8% oxygen for 1.5h. Control rats received sham surgery and exposure to 1.5h of room air. One cohort of rats was examined for growth patterns through P33, evaluated using a battery of tests focused on early postnatal feeding behaviors, and studied using the open field paradigm during the early postnatal and postweaning periods. Another cohort of rats was used to examine metabolic parameters using indirect calorimetry. Significant growth deficits emerged in injured rats during the second postnatal week. No significant differences between groups were noted in the expression of feeding-related behaviors or in metabolic parameters between groups. However, we did observe significant associations between feeding-related behaviors and P14 growth parameters in injured rats. In the open field assessment, HI rats showed increased circling and supination behaviors only during the early postnatal period. Our data reveal that P3 HI brain injury results in generalized growth deficits that persist through postweaning. Analyses suggest that alterations in feeding-related behaviors contribute to growth deficits following a P3 HI brain injury.

The ontogeny of Krox-20 expression in brainstem and cerebellar neurons.

The central expression of Krox-20, a C(2)H(2)-type zinc-finger transcription factor and immediate early gene, is primarily studied in the young embryo, where it contributes to rhombomere (r) r3 and r5 development. Data regarding the cellular localization and developmental regulation of Krox-20 protein expression in brainstem neurons are lacking. Our interest in brainstem development, coupled with findings from our lab and others that demonstrate a profound impact of a Krox-20 null mutation on brainstem-mediated behaviors, led us to investigate the spatiotemporal expression of Krox-20 protein in brainstem and cerebellar neurons to gain insight into potential cellular targets of the mutation. Understanding the cellular localization of Krox-20 is important in light of studies showing the impact of immediate early gene expression on neuronal function. Krox-20 immunohistochemistry experiments were conducted on animals at embryonic days (E) 17.0 and 18.5 and postnatal days (P) 0-1, 3-4, 7, 14, 22, and adulthood. Krox-20 expression is developmentally regulated in motoneurons, somatosensory-related neurons, Purkinje cells, and components of auditory circuitry. Neurons in the ventral cochlear nucleus and inferior colliculus show a sustained Krox-20 expression. Ultrastructural data demonstrate Krox-20 expression in somata and dendrites of central neurons. Our identification of Krox-20 expressing neurons provides us a better understanding of the behavioral consequences of the mutation. Furthermore, our results suggest that Krox-20 protein has a role in the maturation of particular brainstem and cerebellar neurons and fluctuations in Krox-20 protein expression coincide with the development of circuitry underlying brainstem-mediated behaviors.

Novel Indirect Calorimetry Technology to Analyze Metabolism in Individual Neonatal Rodent Pups

Background The ability to characterize the development of metabolic function in neonatal rodents has been limited due to technological constraints. Low respiratory volumes and flows at rest pose unique problems, making it difficult to reliably measure O2 consumption, CO2 production, respiratory quotient (RQ), and energy expenditure (EE). Our aim was to develop and validate a commercial-grade indirect calorimetry system capable of characterizing the metabolic phenotype of individual neonatal rodents. Methodology/Principal Findings To address this research need, we developed a novel, highly sensitive open-circuit indirect calorimetry system capable of analyzing respiratory gas exchange in a single neonatal rodent pup. Additionally, we derived an equation from known metabolic relationships to estimate inlet flow rates, improving the efficiency of data collection. To validate the neonatal rodent indirect calorimetry system and evaluate the applicability of the derived equation for predicting appropriate flow rates, we conducted a series of experiments evaluating the impact of sex, litter size, time of day (during the light phase), and ambient temperature on neonatal rat metabolic parameters. Data revealed that the only metabolic parameter influenced by litter size is a neonatal rats RQ, with rat pups reared in a small litter (5 pups) having lower RQs than rat pups reared in either medium (8 pups) or large (11 pups) litters. Furthermore, data showed that ambient temperature affected all metabolic parameters measured, with colder temperatures being associated with higher CO2 production, higher O2 consumption, and higher energy expenditure. Conclusion/Significance The results of this study demonstrate that the modified Panlab Oxylet system reliably assesses early postnatal metabolism in individual neonatal rodents. This system will be of paramount importance to further our understanding of processes associated with the developmental origins of adult metabolic disease.

GABAA receptor β2/β3 subunit and GAD67 immunoreactivity in the trigeminal motor nucleus during early postnatal development

GABA neurotransmission plays a role in brainstem circuitry responsible for jaw movements. We investigated the developmental relationship between terminals expressing GAD67 and GABA(A) receptor beta(2)/beta(3) subunit expression within the trigeminal motor nucleus. GAD67 immunoreactivity was intense throughout development. Neuropilar beta(2)/beta(3) immunoreactivity emerged during the 2nd postnatal week. Our data provide anatomical evidence for a GABAergic innervation of neonatal trigeminal motoneurons and suggest that beta(2)/beta(3) subunit expression is developmentally regulated in trigeminal motoneurons.