nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory.

nalyot (nal) is a novel olfactory memory mutant of Drosophila, encoding Adf1, a myb-related transcription factor. Following extended training sessions, Adf1 mutants show normal early memory but defective longterm memory. Adf1 shows widespread spatiotemporal expression, yet mutant alleles reveal no discernible disruptions in gross morphology of the nervous system. Studies at the larval neuromuscular junction, however, reveal a role for Adf1 in the modulation of synaptic growth-in contrast to the role established for dCREB2 in the control of synaptic function (Davis et al., 1996). These findings suggest that Adf1 and dCREB2 regulate distinct transcriptional cascades involved in terminal stages of synapse maturation. More generally, Adf1 provides a novel link between molecular mechanisms of developmental and behavioral plasticity.

Segment-specific retention of a larval neuromuscular system and its role in a new, rhythmic, pupal motor pattern in Manduca sexta.

At pupation in Manduca sexta, accessory planta retractor muscles and their motoneurons degenerate in segment-specific patterns. Accessory planta retractor muscles in abdominal segments 2 and 3 survive in reduced form through the pupal stage and degenerate after adult emergence. Electromyographic and electrophysiological recordings show that these accessory planta retractor muscles participate in a new, rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for extended bouts. Accessory planta retractor muscle contractions are driven by synaptic activation of accessory planta retractor motoneurons and are often accompanied by rhythmic activity in intersegmental muscles and spiracular closer muscles. The pupal motor pattern is influenced by descending neural input although isolated abdominal ganglia can produce a pupal motor pattern-like rhythm. The robust pupal motor pattern first seen after pupal ecdysis weakens during the second half of pupal life. Anemometric recordings indicate that the intersegmental muscle and spiracular closer muscle component of the pupal motor pattern produces ventilation. Accessory planta retractor muscle contractions lift the flexible abdominal floor, to which the developing wings and legs adhere tightly. We hypothesize that, by a bellows-like action, the accessory planta retractor muscle contractions circulate hemolymph in the appendages. Morphometric analysis shows that dendritic regression is similar in accessory planta retractor motoneurons with different pupal fates, and that accessory planta retractor motoneurons begin to participate in the pupal motor pattern while their dendrites are regressed.

Neural mechanisms of behavioral plasticity: metamorphosis and learning in Manduca sexta.

This review summarizes our current understanding of the neural circuit underlying the larval proleg withdrawal reflex (PWR) of Manduca sexta and describes how PWR function changes in two contexts: metamorphosis and learning. The first form of PWR plasticity occurs during the larval-pupal transformation, when the reflex is lost. One mechanism that contributes to this loss is the weakening of monosynaptic excitatory connection from proleg sensory neurons to proleg retractor motor neurons. This change is associated with the hormonally-mediated regression of proleg motor neuron dendrites, which may break synaptic contacts between the sensory and motor neurons. After pupation, some of the proleg motor neurons die in a segment-specific pattern that persists even after individual motor neurons are isolated from the nervous system and exposed to hormones in vitro. The second form of PWR plasticity involves short-term, activity-dependent changes in neural function during the larval stage. The nicotinic cholinergic connections from proleg sensory neurons to motor neurons exhibit several forms of plasticity including facilitation, depression, post-tetanic potentiation and two types of muscarinic modulation. Larval PWR behavior exhibits two simple forms of learning-habituation and dishabituation-which involve alterations in the central PWR circuit. These studies of a simple circuit illustrate neural mechanisms by which behaviors undergo both short- and long-term modifications.

Novel dual innervation of a larval proleg muscle by two similar motoneurons in the tobacco hornworm Manduca sexta.

In Manduca sexta, the accessory planta retractor muscle (APRM), which retracts the larval proleg, is innervated by two excitatory motoneurons, the accessory planta retractor motoneurons (APRs). These muscles and motoneurons have been the focus of a number of developmental and behavioral studies. The present study investigated properties of the pair of APRs that innervate each APRM and determined their pattern of innervation of APRM fibers. Members of APR pairs could not be distinguished by their anatomical or electrical properties (resting membrane potential, input resistance and spike threshold). Spontaneous synaptic inputs to members of APR pairs were highly correlated, whereas spontaneous synaptic inputs to APRs and functionally dissimilar motoneurons were not well correlated. Synaptic inputs from identified mechanosensory neurons and interneurons to the two APRs were qualitatively similar, but the magnitude of the response to sensory stimulation sometimes differed within a pair. Both APRs produced large, rapidly rising excitatory junction potentials in APRM fibers. Within the APRM, some fibers were singly innervated by one or the other APR while the remaining fibers were dually innervated by both APRs. In dually innervated fibers, the motor terminals of the two APRs were spatially segregated. This innervation pattern appears to be unique among insects and shares some properties with the innervation of vertebrate muscle.

Fish oil ameliorates renal injury and hyperlipidemia in the Milan normotensive rat model of focal glomerulosclerosis.

Rats of the Milan normotensive rat strain (MNS) spontaneously develop severe proteinuria and excessive glomerular thromboxane (Tx)A2 production at a young age. These abnormalities are accompanied by podocyte alterations, progressive focal glomerulosclerosis (FGS), and interstitial fibrosis, resembling human FGS. Since it has been shown that pharmacologic Tx-synthase inhibition protects MNS rats from these changes, it was hypothesized that a fish oil (FO) enriched diet, by enhancing TxA3 production instead of TxA2, might afford similar protection, compared with diets enriched in safflower oil (SO) or lard (LD). Rats were pair-fed 11% fat diets from age of 1 to 11 months. Glomerular TxA2 at 11 months was significantly lower in PO-fed rats than in SO- and LD-fed rats (11 +/- 3.0, 69 +/- 3.0, 59 +/- 19.0 nanograms per min/mg, respectively; P < 0.001). At 3 months, urinary albumin excretion was similar among the groups. Over the course of the study, rats fed FO developed significantly less albuminuria than the SO and LD groups (P < 0.001 by analysis of variance for repeated measures), such that the values at 11 months were 25 +/- 5.8, 49 +/- 8.7, and 68 +/- 13.0 mg/24h, respectively. Serum cholesterol and triglycerides were also significantly lower in FO-fed rats than in SO- and LD-fed rats. The extent of FGS was similar in the three groups, but FO-fed rats had less interstitial injury than the other groups. It was observed that a fish-oil diet substantially alleviated albuminuria, normalized nephrotic hyperlipidemia, and reduced interstitial injury, but did not prevent the development of FGS in the MNS model.

Glomerular epithelial and mesangial cells differentially modulate the binding specificities of VLA-1 and VLA-2.

BACKGROUND: Maintenance of glomerular cell interaction with the complex basement membrane is crucial for the normal functioning of the kidney. Because little is known about the receptors utilized by glomerular cells, we examined the attachment of cultured glomerular cells to extracellular matrix proteins. EXPERIMENTAL DESIGN: We produced monoclonal antibodies that inhibited the function of rat VLA-1 and VLA-2 and used these antibodies alone and in combination to explore the attachment of glomerular epithelial cells (GEC) and mesangial cells to extracellular matrix proteins in vitro. RESULTS: Cultured GEC utilize only VLA-2 for attachment to collagen but use it together with VLA-1 for adhesion to laminin. In contrast, mesangial cells use both receptors for their attachment to collagen but utilize only VLA-1 in their interaction with laminin. The use of VLA-1 by GEC and of VLA-2 by mesangial cells was unexpected because the expression of these receptors was barely detectable in an enzyme-linked immunosorbent assay and by immunocytochemistry. CONCLUSIONS: VLA-1, VLA-2, and VLA-3 are integrin receptors with overlapping specificities in that all have the potential to interact with collagen and laminin. Our studies demonstrate that cultured GEC use VLA-1 and VLA-2 almost exclusively for their adhesion to these ligands, and thus VLA-3 appears to play a negligible role in such attachment. Interestingly, GEC and mesangial cells differentially modulate the ligand binding specificities of VLA-1 and VLA-2. In situ, VLA-1 has been localized within the mesangium, whereas VLA-2 has not been detected within the glomerulus leading to the conclusion that GEC do not use VLA-1 or VLA-2 and that mesangial cells fail to utilize VLA-2. However, our studies have shown that, even when such receptors are barely detectable on the surface of cultured cells by sensitive techniques, they can play a functional role. These results suggest either that the levels of expression in situ are too low for the relatively insensitive immunohistochemical techniques employed, and thus the importance of these receptors to glomerular cell attachment in vivo is under appreciated or that such receptors are the result of de novo expression by glomerular cells when they are subjected to in vitro culture conditions. Because it is known that such conditions may mimic pathologic stress, we are presently examining the expression of these receptors by glomerular cells in various disease models.

Cell-mediated immune injury in the kidney: acute nephritis induced in the rat by azobenzenearsonate.

Cell-mediated immune mechanisms have long been suspected of playing an important role in the pathogenesis of various renal diseases. An animal model of active nephritis secondary to an exogenous antigen that requires antigen presentation to immune-competent T cells has not been developed. Consequently, the potential of kidney cells to serve as effective antigen presenting cells after an exposure to a therapeutic, biological, or environmental agent in the intact animal has not been documented. The present experiments were designed to demonstrate the capacity of the kidney to become the target for cell-mediated immune injury. A model system has been developed whereby a chemically reactive form of the hapten azobenzenearsonate is introduced directly into the left kidney of pre-immunized Brown Norway rats. Previous studies have shown that this form of the hapten requires active antigen presentation but no intracellular processing, since the reactive form of the hapten modifies directly surface expressed proteins. Delayed hypersensitivity was demonstrated in the actively immunized animals by standard lymphocyte stimulation index and by in vivo skin testing. Peak foot pad swelling of 220 +/- 13 x 10(-2) mm in response to the hapten was observed between days 11 and 14 as compared to < 10 x 10(-2) mm in the contralateral foot injected with vehicle alone and < 20 x 10(-2) mm in response to azobenzenearsonate injection in animals immunized with adjuvant alone. The exposure of the kidney to the hapten in the primed animal results in an active unilateral granulomatous nephritis with marked destruction of tubules and glomeruli. On average, 71.5 +/- 5.2% of the renal cortex is affected by the inflammatory process in the actively immunized animals, compared to only 8.1 +/- 3.8% in controls. The disease can be reproduced qualitatively by adoptive transfer of T cells but not by passive antibody administration to naive recipients. These studies demonstrate that intrinsic kidney cells can act as effective antigen presenting cells in the intact animal and that the kidney can become the target of a cell-mediated immune injury.

The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions.

Single-strand conformation polymorphism (SSCP) analysis has proven to be a simple and effective technique for the detection of single base substitutions. We have used SSCP to analyze 29 mouse globin mutations, 27 p53 mutations, and 8 rhodopsin mutations contained within different size PCR products. Our results indicate that the type of mutation (transition versus transversion) did not play a major role in determining whether a mutation was detected by SSCP analysis. The position of the base substitution was more important than the precise base substitution in determining whether a mutation was detected. We report that SSCP sensitivity varies dramatically with the size of the DNA fragment being analyzed. The optimal size fragment for sensitive base substitution detection by SSCP is approximately 150 bp. Our results illustrate the need to keep the size of the PCR fragment small when performing SSCP to detect mutations. Larger fragments can be analyzed when screening for polymorphisms when the need to detect every sequence variation is not as critical.

Reidentification of larval interneurons in the pupal stage of the tobacco hornworm, Manduca sexta.

The abdominal prolegs are the primary locomotory appendages of Manduca sexta larvae. After the prolegs are lost at pupation, some of the proleg motoneurons die while the survivors are respecified to carry out different functions in the adult moth. As a first step toward investigating the process of functional respecification at the synaptic level, we searched for larval interneurons that affected the activity of proleg motoneurons, and followed these interneurons into the pupal stage. Interneurons were judged to be individually identifiable based on their effects on proleg motoneuron activity and their anatomical features. Seven larval interneurons were identified and placed in five physiological classes based on their effects on proleg motoneurons: ipsilateral excitors, contralateral excitors, ipsilateral inhibitors, contralateral inhibitors, and bilateral inhibitor-excitors. Four of the larval interneurons produced apparently monosynaptic postsynaptic potentials in proleg motoneuron. Of the five larval interneurons that were reidentified in the early pupal stage, two showed minor but consistent structural modifications from the larval stage. Interneurons that produced unitary postsynaptic potentials in larval motoneurons continued to do so in pupal motoneurons. These studies demonstrate that individually identified interneurons can be followed through the larval-pupal transformation, during the initial stages of motoneuron respecification.