Effectiveness of a Student-Developed Instructional Video in Learning the Anatomy of the Equine Distal Limb.

The anatomy of the equine distal limb (EDL) is both complex and important to veterinary clinical practice. First-year veterinary students (VM1s) often struggle to adequately understand it. Two third-year veterinary students collaborated with instructors to create an instructional video to facilitate first-year students' comprehension of EDL anatomy. The video was offered to all VM1s. Learning outcomes were assessed via practical exams. Exam scores on EDL structures were compared between students who did (video) and students who did not (no video) watch the video. Students' laboratory experiences and confidence were evaluated with a post-exam survey. The third-year students documented their experiences while producing the video. Eighty percent of VM1s viewed the video; 91% rated the video as very valuable. The video improved student confidence during the practical exam by 9%, and 89% of surveyed students indicated the video positively impacted their exam grade. One item score was significantly improved in the video group (p < .001), as was the score of the five questions combined (p < .001). As expected, overall practical exam scores were not statistically different. Student collaborators indicated that participation reinforced their knowledge while enhancing their professional development. Student collaboration was a beneficial strategy for instructional support development that positively impacted student affect and also generated opportunities for the involved students' professional growth.

The bioimpedance spectroscopy as useful tool for measuring the fluid excess and fluid management in severe polytrauma patients.

Although the fluid therapy plays a fundamental role in the management of polytrauma patients (PP), a tool which could determine it appropriately is still lacking. The aim of this study was to evaluate the application of a bioimpedance spectroscopy (BIS) for body fluids volume and distribution monitoring in these patients. This prospective, observational study was performed on 25 severe PP and 25 healthy subjects. The body fluids composition was repeatedly assessed using BIS between days 3 to 11 of intensive care unit stay while the impact of fluid intake and balance was evaluated. Fluid intake correlated significantly with fluid excess (FE) in edemas, and their values were significantly higher in comparison with the control group. FE was strongly associated with cumulative fluid balance (p<0.0001; r=0.719). Furthermore, this parameter was associated with the entire duration of mechanical ventilation (p=0.001, r=0.791) independently of injury severity score. In conclusion, BIS measured FE could be useful in PP who already achieved negative fluid balance in prevention the risk of repeated hypovolemia through inappropriate fluid restriction. What is more, measured FE has a certain prognostic value. Further studies are required to confirm BIS as a potential instrument for the improvement of PP outcome.

Mu-opioid receptor inhibition decreases voluntary wheel running in a dopamine-dependent manner in rats bred for high voluntary running.

The mesolimbic dopamine and opioid systems are postulated to influence the central control of physical activity motivation. We utilized selectively bred rats for high (HVR) or low (LVR) voluntary running behavior to examine (1) inherent differences in mu-opioid receptor (Oprm1) expression and function in the nucleus accumbens (NAc), (2) if dopamine-related mRNAs, wheel-running, and food intake are differently influenced by intraperitoneal (i.p.) naltrexone injection in HVR and LVR rats, and (3) if dopamine is required for naltrexone-induced changes in running and feeding behavior in HVR rats. Oprm1 mRNA and protein expression were greater in the NAc of HVR rats, and application of the Oprm1 agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) to dissociated NAc neurons produced greater depolarizing responses in neurons from HVR versus LVR rats. Naltrexone injection dose-dependently decreased wheel running and food intake in HVR, but not LVR, rats. Naltrexone (20mg/kg) decreased tyrosine hydroxylase mRNA in the ventral tegmental area and Fos and Drd5 mRNA in NAc shell of HVR, but not LVR, rats. Additionally, lesion of dopaminergic neurons in the NAc with 6-hydroxydopamine (6-OHDA) ablated the decrease in running, but not food intake, in HVR rats following i.p. naltrexone administration. Collectively, these data suggest the higher levels of running observed in HVR rats, compared to LVR rats, are mediated, in part, by increased mesolimbic opioidergic signaling that requires downstream dopaminergic activity to influence voluntary running, but not food intake.

Mosaic structure of the small cryptic plasmid pKST23 from Escherichia coli.

Small plasmid pKST23 was isolated from sheep ruminal Escherichia coli population. Plasmid sequence was determined to be 2,779 bp in length and was found to have an overall 42 % of GC pairs. However, its sequence can be divided into two regions based on genetic composition and the GC content. It was found that the high GC region spanning approximately from nucleotide 1,300 to 2,750 was identical to a group of small Escherichia coli plasmids and encoded a putative replication protein identical to plasmid pKL1 Rep protein. The part with lower GC pairs seemed to be more specific as it showed no similarity to the GenBank database. Computational analysis revealed four open reading frames, two of which showed considerable homology to replication proteins. PCR primers targeting parts of the two different regions of plasmid pKST23 were used to assess the occurrence of related plasmids within ruminal E. coli population.

Estradiol directly attenuates sodium currents and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

The gonadotropin-releasing hormone (GnRH) neuron is the pivotal control center in a tightly regulated reproductive axis. The release of GnRH controls estradiol production by the ovary, and estradiol acts at the hypothalamus to regulate GnRH release. However, the mechanisms of estradiol feedback are just beginning to be understood. We have previously shown that estradiol administered to the female mouse modulates sodium currents in fluorescently-labeled GnRH neurons. In the current studies, estradiol (1 nM) was applied directly, for 16-24h, to hypothalamic cultures from young or aged female ovariectomized mice. The direct application of estradiol modulated a tetrodotoxin-sensitive sodium current in isolated GnRH neurons from both young and aged animals. Estradiol, and the specific estrogen receptor-ß agonist DPN, decreased current amplitude measured in the morning (AM), but had no effect on afternoon currents. These compounds also decreased the rise and decay slope of the current response, increased the width of the current, and increased action potential width in AM recordings. In addition, estradiol decreased the amplitude of the depolarizing afterpotential (DAP); this effect was not time-of-day dependent. The ER-ß agonist DPN did not mimic the effect of estradiol on DAPs, and the modulation of DAPs by estradiol was no longer present in cells from postreproductive animals. These results indicate that estradiol can affect the physiology of GnRH neurons via multiple pathways that are differentially regulated during the transition to reproductive senescence, suggesting that estradiol regulation of GnRH neuronal output is modulated during the aging process.

Flufenamic acid modulates multiple currents in gonadotropin-releasing hormone neurons.

Reproduction in mammals is dependent upon the appropriate neurosecretion of gonadotropin-releasing hormone (GnRH), yet the endogenous generation of activity underlying GnRH secretion remains poorly understood. We have demonstrated that the depolarizing afterpotential (DAP), which modulates bursting activity, is reduced in isolated GnRH neurons from aged animals. Calcium-activated non-specific cation (CAN) channels contribute to the DAP in other vertebrate neurosecretory cells. We used the CAN channel blocker flufenamic acid (FFA) to examine the contribution of CAN channels to the DAP in GnRH neurons during aging. Recordings were performed on isolated fluorescent GnRH neurons from young, middle-aged and aged female mice. Flufenamic acid inhibited spontaneous activity, but significantly increased the DAP in neurons from young and middle-aged animals. Apamin did not significantly potentiate the DAP, but did reduce the effects of FFA, suggesting that the increased DAP is partially due to blockade of apamin-sensitive SK channels. Flufenamic acid increased the current underlying the DAP (I(ADP)) and decreased the preceding fast outward current (I(OUT)) at all ages. These current responses were not affected by apamin, but TEA evoked similar changes. Thus, a potassium current, likely mediated through BK channels, contributes to the fast AHP and appears to offset the DAP; this current is sensitive to FFA, but insensitive to age. The effect of FFA on the DAP, but not I(ADP), is diminished in aged animals, possibly reflecting an age-related modulation of the apamin-sensitive SK channel. Future studies will examine the expression of SK channels during the aging process in GnRH neurons.

Estradiol attenuates multiple tetrodotoxin-sensitive sodium currents in isolated gonadotropin-releasing hormone neurons.

Secretion from gonadotropin-releasing hormone (GnRH) neurons is necessary for the production of gametes and hormones from the gonads. Subsequently, GnRH release is regulated by steroid feedback. However, the mechanisms by which steroids, specifically estradiol, modulate GnRH secretion are poorly understood. We have previously shown that estradiol administered to the female mouse decreases inward currents in fluorescently labeled GnRH neurons. The purpose of this study was to examine the contribution of sodium currents in the negative feedback action of estradiol. Electrophysiology was performed on GnRH neurons dissociated from young, middle-aged, or old female mice. All mice were ovariectomized; half were estradiol replaced. The amplitude of the sodium current underlying the action potential was significantly decreased in GnRH neurons from young estradiol-treated animals. In addition, in vivo estradiol significantly decreased the transient sodium current amplitude, but prolonged the sodium current inactivation time constant. Estradiol decreased the persistent sodium current amplitude, and induced a significant negative shift in peak current potential. In contrast to results obtained from cells from young reproductive animals, estradiol did not significantly attenuate the sodium current underlying the action potential in cells isolated from middle-aged or old mice. Sodium channels can modulate cell threshold, latency of firing, and action potential characteristics. The reduction of sodium current amplitude by estradiol suggests a negative feedback on GnRH neurons, which could lead to a downregulation of cell excitability and hormone release. The attenuation of estradiol regulation in peripostreproductive and postreproductive animals could lead to dysregulated hormone release with advancing age.

The effect of new proteasome inhibitors, belactosin A and C, on protein metabolism in isolated rat skeletal muscle.

The proteasome inhibitors are used as research tools to study of the ATP-dependent ubiquitin-proteasome system. Some of them are at present undergoing clinical trials to be used as therapeutic agents for cancer or inflammation. These diseases are often accompanied by muscle wasting. We herein demonstrate findings about new proteasome inhibitors, belactosin A and C, and their direct effect on protein metabolism in rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus (EDL) were dissected from both legs of male rats (40-60 g) and incubated in a buffer containing belactosin A or C (30 microM) or no inhibitor. The release of amino acids into the medium was estimated using high performance liquid chromatography to calculate total and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasome and cathepsin B, L activity were determined by fluorometric assay. Protein synthesis and leucine oxidation were detected using specific activity of L-[1-14C] leucine added to medium. Inhibited and control muscles from the same rat were compared using paired t-test. The results indicate that after incubation with both belactosin A and C total proteolysis and CTLA of proteasome decreased while cathepsin B, L activity did not change in both SOL and EDL. Leucine oxidation was significantly enhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysis was reduced in both muscles in the presence of belactosin A only. In summary, belactosin A and C affected basic parameters of protein metabolism in rat skeletal muscle. The response was both muscle- and belactosin-type-dependent.

The effect of new proteasome inhibitors, belactosin A and C, on protein metabolism in isolated rat skeletal muscle

The proteasome inhibitors are used as research tools to study of the ATP-dependentubiquitin-proteasome system. Some of them are at present undergoing clinicaltrials to be used as therapeutic agents for cancer or inflammation. These diseases areoften accompanied by muscle wasting. We herein demonstrate findings about newproteasome inhibitors, belactosin A and C, and their direct effect on protein metabolismin rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus(EDL) were dissected from both legs of male rats (40-60g) and incubated in a buffercontaining belactosin A or C (30 ìM) or no inhibitor. The release of amino acids intothe medium was estimated using high performance liquid chromatography to calculatetotal and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasomeand cathepsin B, L activity were determined by fluorometric assay. Proteinsynthesis and leucine oxidation were detected using specific activity of L-[1-14C]leucine added to medium. Inhibited and control muscles from the same rat were comparedusing paired t-test. The results indicate that after incubation with both belactosinA and C total proteolysis and CTLA of proteasome decreased while cathepsinB, L activity did not change in both SOL and EDL. Leucine oxidation was significantlyenhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysiswas reduced in both muscles in the presence of belactosin A only. In summary,belactosin A and C affected basic parameters of protein metabolism in rat skeletalmuscle. The response was both muscle- and belactosin-type-dependent(AU)

Effect of beta-hydroxy-beta-methylbutyrate (HMB) on protein metabolism in whole body and in selected tissues.

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effect. The aim of the study was to examine the role of exogenous HMB on leucine and protein metabolism in whole body and selected tissues. Rats were administered by HMB (0.1 g/kg b.w.) or by saline. The parameters of whole-body protein metabolism were evaluated 24 h later using L-[1-14C]leucine and L-[3,4,5-3H]phenylalanine. Changes in proteasome dependent proteolysis and protein synthesis were determined according the "chymotrypsin-like" enzyme activity and labeled leucine and phenylalanine incorporation into the protein. A decrease in leucine clearance and whole-body protein turnover (i.e., a decrease in whole-body proteolysis and protein synthesis) was observed in HMB treated rats. Proteasome-dependent proteolysis decreased significantly in skeletal muscle, changes in heart, liver, jejunum, colon, kidney, and spleen were insignificant. Decrease in protein synthesis was observed in the heart, colon, kidney, and spleen, while an increase was observed in the liver. There were no significant changes in leucine oxidation. We conclude that protein anabolic effect of HMB in skeletal muscle is related to inhibition of proteolysis in proteasome. Alterations in protein synthesis in visceral tissues may affect several important functions and the metabolic status of the whole body.

Age affects spontaneous activity and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

Neuronal activity underlying the pulsatile secretion of GnRH remains poorly understood, as does the endogenous generation of such activity. It is clear that changes at the level of the hypothalamus are taking place during reproductive aging, yet virtually nothing is known about GnRH neuronal physiology in aging and postreproductive animals. In these studies, we performed cell-attached and whole-cell recordings in GnRH-enhanced green fluorescent protein neurons dissociated from young (3 months), middle-aged (10 months), and old (15-18 months) female mice. All mice were ovariectomized; half were estradiol replaced. Neurons from all ages fired spontaneously, most in a short-burst pattern that is characteristic of GnRH neuronal firing. Membrane characteristics were not affected by age. However, firing frequency was significantly reduced in neurons from old animals, as was spike patterning. The amplitude of the depolarizing afterpotential, evoked by a 200-msec current pulse, was significantly smaller in aged animals. In addition, inward whole-cell currents were reduced in estradiol-treated animals, although they were not significantly affected by age. Because depolarizing afterpotentials have been shown to contribute to prolonged discharges of activity after a very brief excitatory input, a decreased depolarizing afterpotential could lead to attenuated pulses in older animals. In addition, decreases in frequency and pattern generation could lead to improper information coding. Therefore, changes in the GnRH neuron during aging could lead to dysregulated activity, potentially resulting in the attenuated LH pulses observed in the transition to reproductive senescence.

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.

Spike-dependent depolarizing afterpotentials contribute to endogenous bursting in gonadotropin releasing hormone neurons.

Pulsatile secretion of gonadotropin releasing hormone in mammals is thought to depend on repetitive and prolonged bursts of action potentials in specific neuroendocrine cells. We have previously described episodes of electrical activity in isolated gonadotropin releasing hormone neurons, but the intrinsic mechanisms underlying the generation of spike bursts are unknown. In acutely isolated gonadotropin releasing hormone neurons, which had been genetically targeted to express enhanced green fluorescent protein, current pulses generated spike-mediated depolarizing afterpotentials in 69% of cells. Spike-dependent depolarizing afterpotentials could evoke bursts of action potentials that lasted for tens of seconds. Brief pulses of glutamate (as short as 1 ms), which simulated excitatory postsynaptic potentials, also triggered spike-mediated depolarizing afterpotentials and episodic activity. These data indicate that spike-dependent depolarizing afterpotentials, an endogenous mechanism in gonadotropin releasing hormone neurons, likely contribute to the episodic firing thought to underlie pulsatile secretion of gonadotropin releasing hormone. Furthermore, fast excitatory postsynaptic potentials mediated by glutamate can activate this intrinsic mechanism.

Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging.

The present study was designed to determine if functional age differences in the NMDA epsilon2 (NR2B) subunit were detectable at the level of individual cortical neurons. Neurons were acutely dissociated from the frontal and prefrontal cortices of young adult, middle-aged, or old mice, using a combination of proteinase K and trypsin followed by manual trituration. After overnight culture, patch-clamp electrophysiology and rapid perfusion were used to obtain whole-cell responses to 300 microM NMDA, with or without the potent NR2B antagonist ifenprodil. Healthy, phase-bright cortical neurons were isolated from animals of all ages. Cell diameter and capacitance was consistent between ages. We were able to perform kinetic analyses of the NMDA-evoked response, and demonstrated a significant increase in the rate of deactivation with increasing age. In addition, we observed a significant effect of high-concentration ifenprodil on the NMDA-evoked response in old animals. Thus, this method is ideal for the dissociation of neurons from the brain of both young and old animals, and offers a powerful tool for functional analysis at the level of the individual cell.

Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein.

The gonadotropin-releasing hormone (GnRH) system, considered to be the final common pathway for the control of reproduction, has been difficult to study because of a lack of distinguishing characteristics and the scattered distribution of neurons. The development of a transgenic mouse in which the GnRH promoter drives expression of enhanced green fluorescent protein (EGFP) has provided the opportunity to perform electrophysiological studies of GnRH neurons. In this study, neurons were dissociated from brain slices prepared from prepubertal female GnRH-EGFP mice. Both current- and voltage-clamp recordings were obtained from acutely dissociated GnRH neurons identified on the basis of EGFP expression. Most isolated GnRH-EGFP neurons fired spontaneous action potentials (recorded in cell-attached or whole-cell mode) that typically consisted of brief bursts (2-20 Hz) separated by 1-10 sec. At more negative resting potentials, GnRH-EGFP neurons exhibited oscillations in membrane potential, which could lead to bursting episodes lasting from seconds to minutes. These bursting episodes were often separated by minutes of inactivity. Rapid application of glutamate or NMDA increased firing activity in all neurons and usually generated small inward currents (<15 pA), although larger currents were evoked in the remaining neurons. Both AMPA and NMDA receptors mediated the glutamate-evoked inward currents. These results suggest that isolated GnRH-EGFP neurons from juvenile mice can generate episodes of repetitive burst discharges that may underlie the pulsatile secretion of GnRH, and glutamatergic inputs may contribute to the activation of endogenous bursts.

Electrophysiological analysis of NMDA receptor subunit changes in the aging mouse cortex.

NMDA receptors play an important role in memory processes and plasticity in the brain. We have previously demonstrated a significant decrease in NMDARepsilon2 subunit mRNA and protein with increasing age in the C57Bl/6 mouse frontal cortex. In the present study, two-electrode voltage clamp electrophysiology on Xenopus oocytes injected with total RNA harvested from the frontal cortex of young and old C57Bl mice was used to detect changes in receptor composition during aging. Ifenprodil concentration-response curves, magnesium current-voltage curves, and single channel conductances were determined for native receptors. In addition, ifenprodil and magnesium curves were generated for recombinant NMDA receptors of varying subunit ratios. Ifenprodil dose-response curves for all receptors were biphasic. The low affinity component of the curve increased slightly with age, while the high affinity population decreased, mimicking recombinant receptors with decreasing levels of epsilon2. A decrease in maximal current was also observed in aged animals with decreased levels of epsilon2, although single channel conductances were identical between young and old mice. In addition, an increase in sensitivity to magnesium was observed for receptors from older animals. Results are consistent with the interpretation that the epsilon2 subunit is reduced in older mouse frontal cortex. A change in NMDA receptor subunit composition could influence memory processes during aging.

Development of the facial and hypoglossal motor nuclei in the neonatal Brazilian opossum brain.

The development of the facial and hypoglossal motor nuclei were examined in the neonatal Brazilian opossum (Monodelphis domestica), a marsupial in which postnatal central nervous system development has been well characterized. In this study, we utilized postnatal injection of the retrograde tracer cholera toxin subunit B (CtB) to characterize the formation of the facial and hypoglossal motor nuclei in the developing neonatal opossum brainstem. Injections of CtB were made into the cheek/lip region or tongue of opossum pups to retrogradely label the facial or hypoglossal motor nuclei, respectively. Following a 2 h survival time, facial motoneurons in newborn opossum pups (1 PN) exhibited CtB labeling, with their cell bodies localized near the developing cranial abducens nucleus. At 3 and 5 PN, following a 48 h survival time, CtB-labeled facial motoneurons were observed in and migrating to the region of the adult facial motor nucleus in the rostral medulla. Between 7 and 10 PN, almost all facial motoneurons had migrated to their destination within the facial motor nucleus. Hypoglossal motoneurons also exhibited CtB labeling from 1 PN; however, their cell bodies were localized within the hypoglossal motor nucleus at the earliest age examined. Double label studies, to examine guidance of facial motoneurons during migration, demonstrated that CtB-labeled facial motoneurons are in close proximity to vimentin-like immunostained radial glial fibers during migration. These results suggest: (1) migration of facial motoneurons to the facial motor nucleus is a postnatal event, (2) efferent projections from facial and hypoglossal motoneurons project into the peripheral region of their target muscles from the day of birth, and (3) facial motoneurons migrate to their destination in the brainstem thereafter, in close association with radial glial fibers.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

Characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei of the Brazilian opossum.

The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker tau and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that, during the first 10 postnatal days (1-10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, tau, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and tau, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, tau, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period.

Ontogeny of cholecystokinin binding sites in the hindbrain of the laboratory rat.

Studies in our laboratory have revealed a robust, transient expression of cholecystokinin binding sites in the facial motor nucleus during development in the Brazilian opossum, Monodelphis domestica. To investigate the ubiquity of this phenomenon, we have performed receptor autoradiography on the hindbrains of embryonic and neonatal rat pups. Cholecystokinin binding sites are present at very low levels in the embryonic day-16 rat hindbrain, but binding sites are abundant prior to birth. The greatest increase in labelled nuclei occurs prior to 5 days of postnatal age. Binding levels are heavy in the nucleus of the solitary tract, medial vestibular nucleus, posterior dorsal tegmental nucleus, area postrema, and caudal spinal trigeminal nucleus by 30 days postnatal. Both A-type and B-type receptors are present in the neonatal brainstem, although most labelled areas appear to be B-type. A-type binding sites are present in the ventral cochlear nucleus, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, the area postrema, the spinal nucleus of the trigeminal, and the cuneate and gracile nuclei by 5 days postnatal. As reported for the Brazilian opossum, cholecystokinin binding sites are expressed in the facial motor nucleus of neonatal rats and are transient. In this study of the brainstem in laboratory rats, a transient expression is also observed in the rubrospinal tract, parvocellular reticular nucleus, raphe obscurus, cuneate and gracile nuclei, and the ventral median fissure of the spinal cord. As vasopressin binding sites and estrogen receptors have also been shown to be expressed transiently in the laboratory rat facial motor nucleus, the physiological and developmental significance of transient binding site expression remains to be elucidated.