Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf.

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.

Electrophysiological and morphological characteristics of GABAergic respiratory neurons in the mouse pre-Bötzinger complex.

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.

Development of GABAergic neurons from the ventricular zone in the superior colliculus of the mouse.

The superior colliculus (SC) is a layered structure in the midbrain and is particularly rich in gamma-aminobutyric acid (GABA). The present investigation aimed to determine whether the development of GABAergic neurons in the SC is common to that of the neocortex in which they are produced in a distinct area called the ganglionic eminence and are transported by tangential migration. A green fluorescent protein (GFP) knock-in mouse was used in which a GFP gene was introduced into the gene for glutamic acid decarboxylase (GAD) 67 and all GABAergic neurons were fluorescent. At embryonic day (E) 11-14, GFP-positive cells increased strikingly. They were spindle-shaped with processes at both poles and oriented radially between the ventricular and pial surface, together with other GFP-negative cells. After the cutting of the embryonic SC, GFP-positive cells accumulated on one side of the injury as expected from their radial but not tangential migration. In the living slice preparations GFP-positive cells migrated radially during the observation. These results indicate that tangential migration of GABAergic neurons as observed in the neocortex is not applicable and that radial migration from the underlying ventricular zone is predominant in the SC. At E12-13, bundles of commissural GFP-positive fibers which appeared to originate outside the SC were distributed at the superficial layer. These superficial fibers were no longer observed at the later stages.

Development of spontaneous mouth/tongue movement and related neural activity, and their repression in fetal mice lacking glutamate decarboxylase 67.

Spontaneous body movement starts at early fetal stage, at embryonic day (E) 12-15 in mice. In the present study, the movement of the head region was studied in E13-14 mice by in utero ultrasound imaging, together with the in vitro recording of underlying neural activities in the hypoglossal nerve and the ventral root of the upper cervical cord of an isolated brainstem-spinal cord preparation. The role of gamma-aminobutyric acid (GABA) in the generation of fetal movement was assessed using mice lacking GABA-synthesizing glutamate decarboxylase 67 (GAD67). At E14, mouth opening and tongue withdrawal were observed independently at frequency of 14/h each. This movement was rarely observed in the GAD67-deficient mouse. The intraventricular administration of picrotoxin or 3-mercaptopropionic acid abolished mouth opening in the wild-type mice. In a brainstem-spinal cord preparation, three types of neural discharge were recorded: mouth/tongue-moving burst, respiratory burst and irregular activity on the basis of their waveform, regularity in occurrence and concomitant muscle activity. In the GAD67-deficient mice, the occurrence of mouth/tongue-moving burst and irregular activity was inhibited to about 15 and 40% of those in the wild-type mice, respectively. Respiratory burst was slightly inhibited but the difference was not significant. Picrotoxin greatly reduced the frequency of mouth/tongue-moving burst. These results indicate that GABA is involved in rhythm generation in movement of the head region and support the hypothesis that cleft palate in the GAD67-deficient mouse is due to the impairment of mouth or tongue movement that assists palate formation.

Cleft palate by picrotoxin or 3-MP and palatal shelf elevation in GABA-deficient mice.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system. Gene targeting of GABA-synthetic glutamic acid decarboxylase (GAD) 67 and GABAA receptor beta(3) subunit induces cleft palate in the mouse. These findings appear to contradict previous pharmacological investigations using benzodiazepines and GABA itself, which indicate that GABA suppresses palatogenesis. Therefore, the effects of picrotoxin and 3-mercaptopropionic acid (3-MP) on palate formation were investigated in the present study. Picrotoxin and 3-MP impair GABA functions by blocking the GABA receptor and synthesis, respectively. Pregnant mice in the critical period [Embryonic Day (E) 11-15] of palatogenesis were administered these substances by subcutaneous injection or continuous infusion at subconvulsive doses, and their fetuses at E17-18 were investigated. A complete cleft in the secondary palate was observed in 15% of 333 embryos in 28 litters. In the remaining fetuses, a complete cleft palate was not observed, but microscopic examination of serial sections revealed partial defects of the palate. Furthermore, rescue from cleft palate in GAD67-deficient mice was attempted by GABA infusion. Horizontal elevation of palatal shelves, which is not observed in nontreated mice, did occur after the infusion in all 14 GABA-infused GAD67-deficient fetuses, although cleft palate still persisted. These results indicate that GABA is required for palatogenesis and is consistent with findings in gene knockout mice.

Inhibited longitudinal growth of bones in young male rats by clenbuterol.

PURPOSE: Clenbuterol is one of the beta-2 adrenergic receptor agonists with potent anabolic properties in muscles, yet the concomitant effects on muscle and bone in young animals remain to be resolved. Therefore, the purpose of this study was to determine the effects of clenbuterol administration on muscles and bones of young rats. METHODS: Twelve male Sprague-Dawley rats (9-wk-old) were randomly assigned to either a control (CON, N = 6) or clenbuterol group (CLE, N = 6). Clenbuterol of 2 mg x kg body wt x d(-1) was administered subcutaneously for 4 wk. After treatment, the soleus (SOL), extensor digitorum longus (EDL), and ventricle (VENT) muscles and the femurs (FE) and tibiae (TI) bones were excised and analyzed. The bone mineral content (BMC), area, and bone mineral density (BMD) of FE and TI were measured by dual-energy x-ray absorptiometry (DXA). The longitudinal lengths of bones were measured with the Vernier calipers. RESULTS: CLE showed smaller body weight than CON (P < 0.05) after the treatment. The muscle wet weights in CLE tended (P = 0.08) to be higher than CON in SOL (9%) and EDL (12%), but the ratio of muscle wet weight-to-body weight were higher (SOL: P < 0.05, EDL: P < 0.01) than CON. VENT of CLE showed increases in both the wet weight and the ratio (P < 0.01). FEs in CLE showed smaller values in BMC (P < 0.01), area (P < 0.01), and length (P < 0.05) than CON but not in BMD. TIs showed significant decreases (P < 0.01) in BMC, area, and length but not in BMD. CONCLUSION: These results indicated that clenbuterol induced the muscular hypertrophy but inhibited the longitudinal growth of bones in young male rats, which may be a serious concern in any ergogenic use.