Development of A-type allatostatin immunoreactivity in antennal lobe neurons of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

The antennal lobe (AL) of the sphinx moth Manduca sexta is a well-established model system for studying mechanisms of neuronal development. To understand whether neuropeptides are suited to playing a role during AL development, we have studied the cellular localization and temporal expression pattern of neuropeptides of the A-type allatostatin family. Based on morphology and developmental appearance, we distinguished four types of AST-A-immunoreactive cell types. The majority of the cells were local interneurons of the AL (type Ia) which acquired AST-A immunostaining in a complex pattern consisting of three rising (RI–RIII) and two declining phases (DI, DII). Type Ib neurons consisted of two local neurons with large cell bodies not appearing before 7/8 days after pupal ecdysis (P7/P8). Types II and III neurons accounted for single centrifugal neurons, with type II neurons present in the larva and disappearing in the early pupa. The type III neuron did not appear before P7/P8. RI and RII coincided with the rises of the ecdysteroid hemolymph titer. Artificially shifting the pupal 20-hydroxyecdysone (20E) peak to an earlier developmental time point resulted in the precocious appearance of AST-A immunostaining in types Ia, Ib, and III neurons. This result supports the hypothesis that the pupal rise in 20E plays a role in AST-A expression during AL development. Because of their early appearance in newly forming glomeruli, AST-A-immunoreactive fibers could be involved in glomerulus formation. Diffuse AST-A labeling during early AL development is discussed as a possible signal providing information for ingrowing olfactory receptor neurons.This work was supported by a DFG grant (Scha 678/3-3) to J.S.     

Distribution of neuropeptides in the primary olfactory center of the heliothine moth <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.     

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.     

Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta allatostatin and allatotropin

Juvenile hormone (JH) biosynthesis and the effects of synthetic Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin (Mas-AT) were investigated in isolated corpora allata (CA) of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-phase high-performance liquid chromatography (RP-HPLC) of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA (and other tissues) rather than the lack of JH acid methyltransferase. Mean rates of synthesis (100-200fmol/pr/h) were inhibited ca. 70% by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay (ELISA) to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea.     

Regulation of cyclic GMP elevation in the developing antennal lobe of the Sphinx moth, Manduca sexta.

In the moth, Manduca sexta, 3',5'-guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20-hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity-dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal-lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal-lobe neurons.     

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.     

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target.

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transverse nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell-specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cell bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at approximately 42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Within this region of the nerve, the growth cones of both cell types-exhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Mas-allatotropin/Lom-AG-myotropin I immunostaining in the brain of the locust, <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

Mas-allatotropin (Mas-AT) and Lom-accessory gland-myotropin I (Lom-AG-MTI) are two members of a conserved family of insect neuropeptides, collectively termed allatotropins, which have diverse functions, ranging from stimulation of juvenile hormone secretion to myotropic effects on heart and hindgut. In addition, allatotropins appear to be abundant within the nervous system, suggesting neuroactive roles. To identify neurons in the insect brain suitable for a neurophysiological analysis of the roles of allatotropins, we used antisera against Mas-AT and Lom-AG-MTI to map allatotropin-immunoreactive neurons in the brain of a suitable insect, the locust Schistocerca gregaria. Both antisera revealed basically identical staining patterns throughout the locust brain with more than 12,500 immunostained interneurons per brain hemisphere. Neurosecretory cells were not labeled, and the retrocerebral complex was devoid of immunostaining. Prominent immunoreactive cell types include about 9,600 lamina monopolar neurons, medulla to lobula interneurons, local neurons of the antennal lobe, a giant interneuron of the mushroom body, projection neurons of the glomerular lobe to the mushroom body, and three systems of tangential neurons of the central complex. Several groups of neurons showed colocalization of Mas-AT- and -aminobutyric acid immunostaining. Mass spectrometric analysis identified a peptide with a molecular mass identical to Lom-AG-MTI in all major parts of the locust brain but not in the retrocerebral complex. This study strongly suggests that Lom-AG-MTI is highly abundant in the locust brain, and is likely to play a neuroactive role in many brain circuits including all stages of sensory processing, learning and memory, and higher levels of motor control.This work was supported by DFG grant HO 950/14 to U.H.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transvers nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cells bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at ～42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Withinthis region of the nerve, the growth cones of both cell typesexhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Enhancement by serotonin of the growth in vitro of antennal lobe neurons of the sphinx moth Manduca sexta

Cell culture experiments have been used to examine the effects of serotonin [5-hydroxytryptamine (5-HT)] on the morphological development of antennal lobe (AL) neurons in the brain of the sphinx moth, Manduca sexta. The majority of cells used in this study were from animals at stage 5 of the 18 stages of metamorphic adult development. 5-HT did not affect the survival of M. sexta AL neurons in culture, but did increase the numbers of cells displaying features characteristic of certain cell types. Three morphologically distinct cell types were examined in detail. The principal effect of 5-HT on these neurons was enhancement of cell growth. The magnitude of responses to this amine was cell-type specific. Site-specific responses to 5-HT were apparent also in one cell type. Our results suggest that the effects of 5-HT can change during the course of metamorphic development. These changes coincide temporally with the development of fast, sodium-based action potentials. © 1996 John Wiley & Sons, Inc.     

Effects of 20-hydroxyecdysone and serotonin on neurite growth and survival rate of antennal lobe neurons in pupal stage of the silk moth Bombyx mori in vitro

Effects of 20-hydroxyecdysone and serotonin on the morphological development and the survival of antennal lobe neurons from day-2 pupal brains of the silk moth Bombyx mori were investigated in vitro. Four morphologically distinct neuronal types could be identified in the cultured antennal lobe neurons: unipolar, bipolar, multi-polar and projection neurons. Antennal lobe neurons in culture with 20-hydroxyecdysone and serotonin showed different patterns of the morphological development from those described in Manduca sexta. Projection neurons extend their neurites remarkably by 20-hydroxyecdysone in B. mori, but there is no extension from antennal lobe neurons in M. sexta. Multi-polar neurons conspicuously increase only formation of new branches from their primary neurites by serotonin in B. mori, but there are both extension and branching of the neurites in M. sexta. On day-5, antennal lobe neurons in lower titers of 20-hydroxyecdysone had significantly higher survival rates than those in higher titers. Neurons cultured for 7 days at different levels of 20-hydroxyecdysone generally showed significantly lower survival rates than neurons cultured for 5 days under the same conditions.     

Expression of neuropeptides and their receptors in the developing retina of mammals

The present review examines various aspects of the developmental expression of neuropeptides and of their receptors in mammalian retinas, emphasizing their possible roles in retinal maturation. Different peptidergic systems have been investigated with some detail during retinal development, including substance P (SP), somatostatin (SRIF), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), opioid peptides and corticotrophin-releasing factor (CRF). Overall, the developmental expression of most peptides is characterized by early appearance, transient features and achievement of the mature pattern at the time of eye opening. Concerning possible developmental actions of neuropeptides, recent studies imply a role of SP in the modulation of cholinergic neurotransmission in early postnatal rabbit retinas, when cholinergic cells participate in the retinal spontaneous waves of activity. In addition, the presence of transient SRIF expressing ganglion cells and recent observations in SRIF receptor knock-out mice indicate variegated roles of this peptide in the development of the retina and of retinofugal projections. Furthermore, VIP and PACAP exert protective and growth-promoting actions that may sustain retinal neurons during their development, and opioid peptides may control cell proliferation in the developing retina. Finally, a peak in the expression of certain peptides, including VIP, NPY and CRF, is present around the time of eye opening, when the retina begins the analysis of structured visual information, suggesting important roles of these peptides during this delicate phase of retinal development. In summary, although the physiological actions of peptides during retinal development are far from being clarified, the data reviewed herein indicate promising perspectives in this field of study.     

The development of identified neurosecretory cells in the tobacco hornworm, Manduca sexta

The prothoracicotropic hormone (PTTH) is a principal neuropeptide regulator of insect postembryonic molting and metamorphosis. In the tobacco hornworm, Manduca sexta, PTTH is produced by two neurosecretory cells (NSC) located in each protocerebral lobe of the brain. The development of these neurons, the L-NSC III, has been investigated immunocytologically to establish the time course of their morphological differentiation. PTTH may be one of the earliest neuropeptides expressed in insect embryos. PTTH-immunoreactivity was initially detected in the somata at 24 to 30% of embryonic development. Neurites sprouted shortly thereafter and began to grow medially through the brain anlage. By 42% embryonic development, the neurites had decussated to the contralateral brain lobe. As development progressed, the L-NSC III neurites grew along specific tracts through the contralateral brain lobe reaching the ventrolateral regions of the brain by approximately 60% development. The axons exited the brain through a retrocerebral nerve, the nervi corporis cardiaci I + II. At approximately 63% development, the axons innervated the corpus allatum and began branching to form neurohemal terminals for PTTH release. At 60% development, short collaterals began extending in the protocerebral neuropil. During the remainder of embryogenesis, both the dendritic collaterals and the terminal neurohemal varicosities continued to elongate and arborize. By 85% embryonic development, the basic architecture of the L-NSC III was established.     

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.     

A comparison of the development of neuropeptide and MAP2 immunocytochemical labeling in the macaque visual cortex during pre- and postnatal development

The appearance of Substance P (SP) and Neuropeptide Y (NPY) has been studied using light microscopic immunocytochemical labeling throughout the complete developmental span of Macaca nemestrina monkey striate cortex. In the adult, 80% of the NPY + neurons occur in the white matter (WM) and most of the remainder are medium to large multipolar neurons in layer 2. Fibers occur in all layers except 4C and are very numerous, given the relatively small number of NPY+ cell bodies. NPY+ neurons first were seen at embryonic day (E) 75. Most neurons were in the intermediate zone (IZ), but a few were in the immature cortical plate (CP). An adultlike distribution was present by E125 for neurons and by birth for fibers, but fiber staining intensity and number increased to postnatal year 1 (P1yr). In adult cortex, numerous SP+ nonpyramidal neurons were present in layers 2–6 and WM, but SP+ fibers were surprisingly infrequent. During development, significant numbers of SP+ neurons were not seen in the CP until E113–125. Later prenatal ages had a prominent plexus of SP+ cell bodies and fibers at the layer 5/6 border. This plexus disappeared by P12wk due to either down-regulation of SP or cell death. SP+ neurons in IZ/WM were very sparse until birth after which they increased in number and staining intensity up to P1yr, suggesting a postnatal up-regulation of SP in a preexisting WM subpopulation. Cell densities were determined for SP, NPY, and the neuron-specific marker microtubule-associated protein 2 (MAP2) to clarify the developmental dynamics of IZ/WM neurons. MAP2+ cell densities in WM peaked around birth and then declined 20% in the outer half and 77% in the inner half of WM. SP+ cell density rose 57% from birth to P20wk and then declined 20% into adult hood. NPY+ cell density was fairly constant prenatally and then rose 300% by adulthood. Neuropeptide cell density changes took place predominatly in the outer WM. These data indicate that cell death does occur in the general population of monkey striate cortical WM neurons. In contrast, both SP+ and NPY+ cells are characterized by minimal cell death and a late expression of neuropeptides which causes an increase in neuropeptide+ cell density in postnatal WM. © 1993 John Wiley & Sons, Inc.     

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6-8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.     

Neurotransmitters and neuropeptides in the developing human central nervous system. A review

This is a review on the ontogenesis of major neurotransmitters and neuropeptides in the developing human central nervous system. In general, the molecules under study appeared early in development, usually in the first trimester. Cholinergic neurons were found to be present around the time of neuropeptide formation. The newly formed neuropeptidergic fibers extended towards the cholinergic centers where both might interact. In the major centers of the central nervous system, neuropeptides were also noted to colocalize with various neurotransmitters. For example, in the facial nucleus, enkepahlin and substance P fibers coexisted with cholinergic and catecholaminergic neurons, suggesting complex interactions. In the interpeduncular nucleus, peptidergic neurons acting as interneurons clearly modulated the afferent input to this nucleus. In the hippocampus and in sensory organs such as the retina, there were indications that neuropeptides and gamma-amino butyric acid coexisted. We hypothesize that interactions of neurotransmitters and peptides in neurons and fibers early in development play an indispensable role in the morphogenesis of the human central nervous system.