A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation.

Upon contacting its postsynaptic target, a neuronal growth cone transforms into a presynaptic terminal. A membrane component on the growth cone that facilitates synapse formation was identified by means of a complementary DNA-based screen followed by genetic analysis. The late bloomer (lbl) gene in Drosophila encodes a member of the tetraspanin family of cell surface proteins. LBL protein is transiently expressed on motor axons, growth cones, and terminal arbors. In lbl mutant embryos, the growth cone of the RP3 motoneuron contacts its target muscles, but synapse formation is delayed and neighboring motoneurons display an increase in ectopic sprouting.

Genetic analysis of the mechanisms controlling target selection: target-derived Fasciclin II regulates the pattern of synapse formation.

In Drosophila, motoneuron growth cones initially probe many potential muscle targets but later withdraw most of these contacts to form stereotypic synapses with only one or a few muscles. Prior to synapse formation, Fasciclin II (Fas II) is expressed at low levels on muscle. During synapse formation, Fas II concentrates at the synapse and disappears from the rest of the muscle. We previously showed that Fas II is required both pre- and postsynaptically for synaptic stabilization. Here, we show that the differential expression of target-derived Fas II has a profound influence on the patterning of synapse formation. A transient increase in muscle Fas II stabilizes growth cone contacts and leads to novel synapses that are functional and stable; targets that normally receive two inputs can now receive up to six inputs. Changing the relative levels of Fas II on neighboring muscles leads to dramatic shifts in target selection.

Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity.

Increased cAMP (in dunce mutants) leads to an increase in the structure and function of the Drosophila neuromuscular junction. Synaptic Fasciclin II (Fas II) controls this structural plasticity, but does not alter synaptic function. Here, we show that CREB, the cAMP response element-binding protein, acts in parallel with Fas II to cause an increase in synaptic strength. Expression of the CREB repressor (dCREB2-b) in the dunce mutant blocks functional but not structural plasticity. Expression of the CREB activator (dCREB2-a) increases synaptic strength only in FasII mutants that increase bouton number. This CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a FasII mutant background genetically reconstitutes this cAMP-dependent plasticity. Thus, cAMP initiates parallel changes in CREB and Fas II to achieve long-term synaptic enhancement.

Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.

Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth.

We present evidence that Futsch, a novel protein with MAP1B homology, controls synaptic growth at the Drosophila neuromuscularjunction through the regulation of the synaptic microtubule cytoskeleton. Futsch colocalizes with microtubules and identifies cytoskeletal loops that traverse the lateral margin of select synaptic boutons. An apparent rearrangement of microtubule loop architecture occurs during bouton division, and a genetic analysis indicates that Futsch is necessary for this process. futsch mutations disrupt synaptic microtubule organization, reduce bouton number, and increase bouton size. These deficits can be partially rescued by neuronal overexpression of a futsch MAP1B homology domain. Finally, genetic manipulations that increase nerve-terminal branching correlate with increased synaptic microtubule loop formation, and both processes require normal Futsch function. These data suggest a common microtubule-based growth mechanism at the synapse and growth cone.

Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.

The glutamatergic neuromuscular synapse in Drosophila forms and differentiates into distinct boutons in the embryo and grows by sprouting new boutons throughout larval life. We demonstrate that two axons form approximately 18 boutons on muscles 7 and 6 by hatching and grow to approximately 180 boutons by third instar. We further show that, after synapse formation, the homophilic cell adhesion molecule Fasciclin II (Fas II) is localized both pre- and postsynaptically where it controls synapse stabilization. In FasII null mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and resulting lethality are rescued by transgenes that drive Fas II expression both pre- and postsynaptically; driving Fas II expression on either side alone is insufficient. Fas II can also control synaptic growth; various FasII alleles lead to either an increase or decrease in sprouting, depending upon the level of Fas II.

Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity.

Increased neuronal activity (eag Shaker mutants) and cAMP concentration (dunce mutants) lead to increased synaptic structure and function at the Drosophila neuromuscular junction. Here, we show that the increase in synaptic growth is accompanied by an approximately 50% decrease in synaptic levels of the cell adhesion molecule Fasciclin II (Fas II). This decrease in Fas II is both necessary and sufficient for presynaptic sprouting; FasII mutants that decrease Fas II levels by approximately 50% lead to sprouting similar to eag Shaker and dunce, while transgenes that maintain synaptic Fas II levels suppress sprouting in eag Shaker and dunce. However, FasII mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons.

Postsynaptic PKA controls quantal size and reveals a retrograde signal that regulates presynaptic transmitter release in Drosophila.

Two distinct mechanisms regulate synaptic efficacy at the Drosophila neuromuscular junction (NMJ): a PKA-dependent modulation of quantal size and a retrograde regulation of presynaptic release. Postsynaptic expression of a constitutively active PKA catalytic subunit decreases quantal size, whereas overexpression of a mutant PKA regulatory subunit (inhibiting PKA activity) increases quantal size. Increased PKA activity also decreases the response to direct iontophoresis of glutamate onto postsynaptic receptors. The PKA-dependent modulation of quantal size requires the presence of the muscle-specific glutamate receptor DGluRIIA, since PKA-dependent modulation of quantal size is lost in homozygous viable DGluRIIA- mutants. Furthermore, elevated postsynaptic PKA reduces the quantal amplitude and the time constant of miniature excitatory junctional potential (mEJP) decay to values that are nearly identical to those observed in DGluRIIA mutants. The PKA-dependent reduction in quantal size is accompanied developmentally by an increase in presynaptic quantal content, indicating the presence of a retrograde signal that regulates presynaptic release.

A genetic analysis of synaptic development: pre- and postsynaptic dCBP control transmitter release at the Drosophila NMJ.

Postsynaptic dCBP (Drosophila homolog of the CREB binding protein) is required for presynaptic functional development. Viable, hypomorphic dCBP mutations have a approximately 50% reduction in presynaptic transmitter release without altering the Ca2+ cooperativity of release or synaptic ultrastructure (total bouton number is increased by 25%-30%). Exogenous expression of dCBP in muscle rescues impaired presynaptic release in the dCBP mutant background, while presynaptic dCBP expression does not. In addition, overexpression experiments indicate that elevated dCBP can also inhibit presynaptic functional development in a manner distinct from the effects of dCBP loss of function. Pre- or postsynaptic overexpression of dCBP (in wild type) reduces presynaptic release. However, we do not observe an increase in bouton number, and presynaptic overexpression impairs short-term facilitation. These data suggest that dCBP participates in a postsynaptic regulatory system that controls functional synaptic development.

Long-term regulation of short-term transmitter release properties: retrograde signaling and synaptic development.

The dynamics of presynaptic transmitter release are often matched to the physiological properties and functions of the postsynaptic cell. In organisms ranging from cats to crickets, evidence suggests that retrograde signaling is essential for matching these presynaptic release properties to individual postsynaptic partners. Retrograde interactions appear to control the development of presynaptic, short-term facilitation and homosynaptic depression through local, retrograde signaling at the synapse.

Genetic analysis of synaptic development and plasticity: homeostatic regulation of synaptic efficacy.

When experimentally challenged with perturbations in synaptic structure and function, neurons have the remarkable ability to regulate their synaptic efficacy back to the normal range. Recent genetic analysis has provided insights into the mechanisms controlling this form of synaptic homeostasis, with implications for our understanding of synaptic development and plasticity.

Effect of Bacillus Calmette-Guérin on the development of primary lung cancer in Syrian golden hamsters.

The effect of a single systemic or intratracheal administration of viable Mycobacterium bovis strain Bacillus Calmette-Guérin on the development of primary lung cancer in Syrian golden hamsters was assessed. M. bovis strain Bacillus Calmette-Guérin administered i.v. prior to, during, or after carcinogen treatment did not alter the incidence of lung tumors. Animals receiving intratracheal M. bovis strain Bacillus Calmette-Guérin had an increased incidence of lung tumors. Both i.v. and intratracheal routes accelerated the appearance of tumors.

Experimental oncornavirus vaccines in the cat.

An experimental approach to the immunoprophylatic control of feline oncornavirus-mediated diseases has included induction of antivirus immunity and antibodies to the feline oncornavirus-associated membrane (tumor) antigens. A suitable model for exploring the effectiveness of killed oncornavirus vaccines in the cat has been provided by the use of feline sarcoma virus. Immunization of seven pregnant queens over a 6-week period with ultraviolet light-inactivated Gardner-Arnstein feline sarcoma virus resulted in significant protection among 12 kittens challenged with a tumor-forming Dose 90 at 7 days of age. This immunity was not present in kittens challenged at 35 days of age. Among 12 kittens born of queens immunized during pregnancy with ultraviolet light-inactivated Kawakami-Theilen feline leukemia virus and challenged with the same live virus at 4 days of age, significant protection was noted, ranging from prolongation of survival time to complete protection in 3 kittens. In general, the higher the antibody titer in the mother, the more effective the protection afforded the kittens. Immunization of 43 kittens during their first 5 weeks of life with the same vaccines used in adult cats did not immunize sufficiently to protect against feline sarcoma virus challenge at 5 weeks of age. Neutralizing antibody responses in these kittens were significantly lower than in pregnant queens. That kittens of this age are immunologically responsive was established, since complete protection of 9 kittens to feline sarcoma virus was obtained by immunization with a crude tumor extract inactivated with 5 to 7 megarads of gamma-irradiation. All these kittens developed feline oncornavirus-associated membrane antibodies while 3 developed demonstrable levels of virus-neutralizing antibodies. The results of these studies are believed indicative that killed virus vaccines and tumor vaccines can be effective immunoprophylatic measures in the control of RNA tumor virus oncogenesis in the cat. Developments in this model system should be relevant to any consideration given similar vaccines in humans.

Long-term regulation of short-term plasticity: a postsynaptic influence on presynaptic transmitter release.

The dynamics of presynaptic transmitter release are often matched to the physiological properties and function of the postsynaptic cell. Evidence in organisms as diverse as the cricket central nervous system and the cat spinal cord suggests that retrograde signaling is essential for matching presynaptic release properties to the postsynaptic cell. The cricket central nervous system is favorably organized for analysis of synaptic function in the central nervous system. Several lines of independent evidence suggest that it is possible to reliably estimate the size of single quantal release events at the sensory to interneuron synapses of the cricket. A quantal analysis suggests that a retrograde influence on the probability of presynaptic release is responsible for matching presynaptic dynamic properties to postsynaptic targets. This retrograde interaction is hypothesized to be a long-term modification on the basal probability of presynaptic release.

Conditioning of relative frequency of sniffing by rabbits to odors.

The sniff was identified by a brief episode of increased respiratory rate, usually with a well-defined time of onset. It was detected against the background of respiratory activity in rabbits simply, reliably, and noninvasively by statistical evaluation of digitized pneumograph records. The basal rate of exploratory sniffing was controlled by familiarization. Upon conditioning to olfactory cues, the rate of sniffing for CS+ increased sharply above the basal rate during the first 10 trials and was maintained at high levels by continued reinforcement. During extinction with discrimination between olfactory cues, the rate for CS- fell sharply at first and then more slowly toward the basal rate. With pseudoconditioning, the rabbits responded to an unpaired odor after several sessions; the rates of response acquisition and extinction and the maintained level of responding were lower than with a paired odor in classical delayed conditioning, and the response was not discriminative in respect to another novel odor given during extinction. The sniff displayed a prominent sensory bias for olfactory cues. The relative frequencies of sniffing and respiratory slowing were measured as conditioned responses by screening procedures with a small computer.

Automated percutaneous discectomy.

Automated percutaneous discectomy is a new, safe procedure for treating herniated lumbar discs still contained by the annulus or posterior longitudinal ligament. In 1985, one of the authors reported a percutaneous nucleus aspiration technique using a 2-mm aspiration probe. This small probe produced minimal tissue damage, allowing the procedure to be done on an outpatient. In this series, 518 patients were treated using this technique for an overall success rate of 85%. Compensation patients, elderly patients, and patients with previous surgery were treated successfully using percutaneous discectomy on an outpatient basis. No intraoperative or postoperative complications occurred.

Pre-clamp cardioprotection by protein kinase C (PKC) inhibitor improves left ventricular function following canine normothermic arrest.

Since protein kinase C (PKC) has been proven to be a mediator of neutrophil activation and of intracellular calcium homeostasis, its inhibition could protect the myocardium from the deleterious effects of ischemic/reperfusion inury (IRI). The principal objective of this study was to evaluate the efficacy of the PK inhibitor SPC-100270 (2S,3S)-2-amino, 3-octadecanediol in a canine model of IRI. A double-blind study was conducted in which 19 coonhound dogs received either SPC-100270 or a vehicle before going on cardiopulmonary bypass (CPB). After 60 minutes of global normothermic (37 degree C) cardiac arrest (cross-clamp time 65-81 minutes for SPC-100270 and 65-72 minutes for control) and discontinuation of CBP, an epicardial short axis view echocardiogram was performed and reviewed by a double-blinded observer to determine the ejection fraction (EF). EF value exceeded 20% in 5 out of 9 SPC-100270 animals (27%-44%) and in 0 of 10 controls (0%-16%). These data show that SPC-10027 significantly (p=0.01 by Fisher's Exact Test) increased the probability that the animals would exhibit an EF greater than 20%.

Effects of infused glucose, sodium and potassium chlorides and polyphosphates on palatability of hot-boned pork.

Longissimus muscle sections were excised from eight pork carcasses 1 h postmortem and sectioned into six .5-kg roasts to determine the effects of glucose, salt and polyphosphates (aqueous solution to 110% of fresh weight) on palatability of hot-boned pork. Treatments were hot-boned control (HB) with no infusion or infusions of 2% KCl and 3% of a 1:1 mixture of sodium hexametaphosphate and sodium pyrophosphate (PP) plus either 8% NaCl; 2% glucose (G) plus 6% NaCl; 6% G plus 2% NaCl; or 8% G. Another muscle section was chilled at 0 degrees C for 24 h on each carcass as a cold-processed control (CP). The roasts were frozen until cooked and evaluated by a sensory panel. The infused groups were more tender, juicy and salty and higher in moisture and ash but lower in protein content than either the CP or HB controls (P less than .05). The fat content of the infused groups was lower than of the HB control but was not different from that of the CP control. Either 2% NaCl plus 6% G or equal amounts (4%) of NaCl and G produced the most tender and juicy product. The substitution of 4% glucose for NaCl not only reduced the NaCl content of the infusion solution, but also improved the palatability of the meat. This substitution allows production of a hot-boned, lower-sodium precooked pork that is tender and juicy.

Cholesterol concentration of longissimus muscle, subcutaneous fat and serum of two beef cattle breed types.

The cholesterol concentration of uncooked and cooked longissimus muscle, uncooked subcutaneous fat and serum, as affected by days on feed, breed type and sex class in a factorial arrangement, and the relationship between serum cholesterol and tissue cholesterol were studied. Days on feed, breed type and sex class had no effect (P greater than .05) on tissue cholesterol concentrations. Overall least-squares means for uncooked and cooked longissimus muscle and subcutaneous fat were 63.32, 80.27 and 98.90 mg of cholesterol/100 g of tissue, respectively. Cholesterol concentration was 26.8% higher in cooked vs uncooked muscle and 56.2% higher in uncooked subcutaneous fat compared with uncooked muscle. Serum cholesterol concentration was lower (P less than .05) for fullblood Chianinas and steers (122.6 and 133.9 mg/dl) compared with crossbreds and heifers (162.3 and 151.0 mg/dl), respectively. Serum cholesterol concentration showed a cubic response (P less than .05) over days on feed; it was lowest at 0 d (79.6 mg/dl), increased up to 77 d (156.3 mg/dl), leveled off (154.7 mg/dl), then increased again (179.3 mg/dl). Serum cholesterol had low and insignificant correlation coefficients with all tissue cholesterol concentrations (r = .08 to .22; P greater than .05). Results indicate that muscle and fat tissue cholesterol concentrations do not vary in response to the differences in breed type, sex class or time on feed represented in the present study and thus may be an inherent characteristic dependent upon quantitative needs for cellular membrane functions. However, serum cholesterol concentration was affected by treatments evaluated in this study and may be related to factors affecting lipid metabolism.

Estimates of beef carcass intermuscular fat.

Three experienced persons evaluated 158 carcasses 24 h postmortem for USDA yield grade (YG) and quality grade factors, nine subcutaneous (SC) fat indicators, and four intermuscular (IM) fat indicators. Forty sides (YG 1.1 to 3.8) were selected for determination of chemical composition, two measures of cutability, and total IM fat from the round, loin, rib, and chuck. The IM fat estimates at the 12th rib, rib-plate juncture, and 5th rib were correlated with percentage of chemical fat (r = -.72, -.70, and -.55, respectively). Simultaneous consideration of YG factors accounted for 61% of the variation in chemical fat. Substituting the IM fat estimate at the 12th rib for adjusted fat thickness (AFT) in the equation explained 60% of the variation in percentage of chemical fat. An equation containing two IM fat estimates, marbling score and longissimus muscle area explained 68% of the variation in chemical fat. Simultaneous consideration of the YG factors accounted for 59% of the variation in boneless, closely trimmed (6 mm SC fat and no IM fat) retail cuts from the round, loin, rib, and chuck. Substituting the IM fat estimate at the 12th rib for AFT in the equation accounted for 65% of the variation. These data from a fairly uniform set of steer carcasses show that percentage of chemical fat and cutability can be reliably predicted from IM fat estimates and other traits that can be visually estimated on hot-fat trimmed carcasses.