PHM is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila.

To understand the roles of secretory peptides in developmental signaling, we have studied Drosophila mutant for the gene peptidylglycine alpha-hydroxylating monooxygenase (PHM). PHM is the rate-limiting enzyme for C-terminal alpha-amidation, a specific and necessary modification of secretory peptides. In insects, more than 90% of known or predicted neuropeptides are amidated. PHM mutants lack PHM protein and enzyme activity; most null animals die as late embryos with few morphological defects. Natural and synthetic PHM hypomorphs revealed phenotypes that resembled those of animals with mutations in genes of the ecdysone-inducible regulatory circuit. Animals bearing a strong hypomorphic allele contain no detectable PHM enzymatic activity or protein; approximately 50% hatch and initially display normal behavior, then die as young larvae, often while attempting to molt. PHM mutants were rescued with daily induction of a PHM transgene and complete rescue was seen with induction limited to the first 4 days after egg-laying. The rescued mutant adults produced progeny which survived to various stages up through metamorphosis (synthetic hypomorphs) and displayed prepupal and pupal phenotypes resembling those of ecdysone-response gene mutations. Examination of neuropeptide biosynthesis in PHM mutants revealed specific disruptions: Amidated peptides were largely absent in strong hypomorphs, but peptide precursors, a nonamidated neuropeptide, nonpeptide transmitters, and other peptide biosynthetic enzymes were readily detected. Mutant adults that were produced by a minimal rescue schedule had lowered PHM enzyme levels and reproducibly altered patterns of amidated neuropeptides in the CNS. These deficits were partially reversed within 24 h by a single PHM induction in the adult stage. These genetic results support the hypothesis that secretory peptide signaling is critical for transitions between developmental stages, without strongly affecting morphogenetic events within a stage. Further, they show that PHM is required for peptide alpha-amidating activity throughout the life of Drosophila. Finally, they define novel methods to study neural and endocrine peptide biosynthesis and functions in vivo.

Maturation of locomotor and Fos responses to the NMDA antagonists, PCP and MK-801.

Antagonists at the N-methyl-D-aspartate (NMDA)-type glutamate receptor, such as phencyclidine (PCP) and dizocilpine (MK-801), are well-known to evoke increases in locomotor activity in adult rats and mice. However, little is known about the effects of NMDA antagonists on locomotor activity as a function of development. The present study examined locomotor responses to PCP or MK-801 in male rats of varying ages and found that prepubertal rats were more sensitive to the locomotor-elevating effects of PCP (1.5 mg/kg and 3. 0 mg/kg, s.c.) than were adults. Locomotor responses to MK-801 (0.1 and 0.2 mg/kg, s.c.) were not dependent on age. The age-dependent response to PCP may be related to developmental events in the motor cortex, since more Fos-immunoreactive neurons were observed in the motor cortex of prepubertal animals after PCP administration relative to adult animals. An opposite pattern of age-dependent Fos responses was observed in the posterior retrosplenial cortex. The results suggest that locomotor responses to NMDA antagonists can be influenced in an age- and drug-dependent manner and that maturational events in the motor cortex may modify responses to PCP.

Metamorphosis of tangential visual system neurons in Drosophila.

To learn about construction of the adult nervous system, we studied the differentiation of imaginal neurons in the Drosophila visual system. OL2-A and OL3 are tangential neurons that display dFMRFa neuropeptide gene expression in adults but not in larvae. The two large OL2-A neurons are generated near the end of the embryonic period and already show morphological differentiation at the start of metamorphosis. The numerous small OL3 neurons are generated postembryonically and first detected later in metamorphosis. The onset of dFMRFa transcription coincides with that of neuropeptide accumulation in OL2-A neurons, but it precedes peptide accumulation in the OL3 neurons by days. Altering each of the five conserved sequences within the minimal 256-bp OL dFMRFa enhancer affected in vivo OL transcriptional activity in two cases: alteration of a TAAT element greatly diminished and alteration of a 9-bp tandem repeat completely abolished OL2-A/OL3 reporter activity. A 46-bp concatamer containing the TAAT element, tested separately, was not active in OL neurons. We propose a model of neuronal differentiation at metamorphosis that features developmental differences between classes of imaginal neurons.

Kainic acid lesions enhance locomotor responses to novelty, saline, amphetamine, and MK-801.

Intracerebroventricular (i.c.v.) administration of kainic acid (KA) to rats produces neuronal loss in the hippocampus and other areas of the limbic system. The present study demonstrates that i.c.v. KA enhances the locomotor response to novelty and saline injection, as well as to amphetamine and MK-801. Sixteen to 18 days after i.c.v. administration of KA or vehicle, lesioned and control rats were placed in a novel cage, and locomotor activity and grooming were recorded for 30 min prior to and 60 min following a subcutaneous injection of saline, D-amphetamine, or MK-801. In response to the novel cage and after each injection, KA rats exhibited increased locomotor activity relative to controls. Grooming behavior was found to be elevated in the KA rats when compared to controls, but only in response to the novel cage and saline injection. The possibility that damage to the limbic system disrupts dopaminergic regulation of locomotor behavior is discussed, as well as implications for neuropathology in schizophrenia.