Physiology and morphology of projection neurons in the antennal lobe of the male mothManduca sexta

1. We have used intracellular recording and staining, followed by reconstruction from serial sections, to characterize the responses and structure of projection neurons (PNs) that link the antennal lobe (AL) to other regions of the brain of the male sphinx moth Manduca sexta. 2. Dendritic arborizations of the AL PNs were usually restricted either to ordinary glomeruli or to the male-specific macroglomerular complex (MGC) within the AL neuropil. Dendritic fields in the MGC appeared to belong to distinct partitions within the MGC. PNs innervating the ordinary glomeruli had arborizations in a single glomerulus (uniglomerular) or in more than one ordinary glomerulus of one AL (multiglomerular) or in one case, in single glomeruli in both ALs (bilateral-uniglomerular). One PN innervated the MGC and many or all ordinary glomeruli of the AL. 3. PNs with dendritic arborizations in the ordinary glomeruli and PNs associated with the MGC typically projected both to the calyces of the ipsilateral mushroom body and to the lateral protocerebrum, but some differences in the patterns of termination in those regions have been noted for the two classes of PNs. One PN conspicuously lacked branches in the calyces but did project to the lateral protocerebrum. The PN innervating the MGC and many ordinary glomeruli projected to the calyces of the ipsilateral mushroom body and the superior protocerebrum. 4. Crude sex-pheromone extracts excited all neurons with arborizations in the MGC, although some were inhibited by other odors. One P(MGC) was excited by crude sex-pheromone extract and by a mimic of one component of the pheromone blend but was inhibited by another component of the blend. 5. PNs with dendritic arborizations in ordinary glomeruli were excited or inhibited by certain non-pheromonal odors. Some of these PNs also responded to mechanosensory stimulation of the antennae. 6. The PN with dendritic arborizations in the MGC and many ordinary glomeruli was excited by crude sex-pheromone extracts and non-pheromonal odors and also responded to mechanosensory stimulation of the antenna.     

The Neuroanatomical Organization of Projection Neurons Associated with Different Olfactory Bulb Pathways in the Sea Lamprey,Petromyzon marinus

Although there is abundant evidence for segregated processing in the olfactory system across vertebrate taxa, the spatial relationship between the second order projection neurons (PNs) of olfactory subsystems connecting sensory input to higher brain structures is less clear. In the sea lamprey, there is tight coupling between olfaction and locomotion via PNs extending to the posterior tuberculum from the medial region of the olfactory bulb. This medial region receives peripheral input predominantly from the accessory olfactory organ. However, the axons from olfactory sensory neurons residing in the main olfactory epithelium extend to non-medial regions of the olfactory bulb, and the non-medial bulbar PNs extend their axons to the lateral pallium. It is not known if the receptive fields of the PNs in the two output pathways overlap; nor has the morphology of these PNs been investigated. In this study, retrograde labelling was utilized to investigate the PNs belonging to medial and non-medial projections. The dendrites and somata of the medial PNs were confined to medial glomerular neuropil, and dendrites of non-medial PNs did not enter this territory. The cell bodies and dendrites of the non-medial PNs were predominantly located below the glomeruli (frequently deeper in the olfactory bulb). While PNs in both locations contained single or multiple primary dendrites, the somal size was greater for medial than for non-medial PNs. When considered with the evidence-to-date, this study shows different neuroanatomical organization for medial olfactory bulb PNs extending to locomotor control centers and non-medial PNs extending to the lateral pallium in this vertebrate.     

Serotonin-like immunoreactivity in the optic lobes of three insect species

The cellular localization of 5-HT in the optic lobes of three insect species was assayed with the use of antibodies raised against 5-HT. In Schistocerca, Periplaneta, and Calliphora all neuropil regions of the optic lobe, the lamina, medulla and lobula, contain 5-HT-immunoreactive varicose fibres in different patterns, like columns and layers. Such fibres also connect the lobula to neuropil in the lateral protocerebrum. In Calliphora also 5-HT-positive fibres of the medulla and lobula plate have projections to the lateral protocerebrum, whereas the origin of the lamina fibres is not certain. In all species the processes displaying 5-HT-like immunoreactivity appear to be derived from a relatively small number of cell bodies, each neuron thus having processes over a large volume of the neuropil of the optic lobe in different layers.     

Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage

The antennal lobe (AL) is the primary structure in the Drosophila brain that relays odor information from the antennae to higher brain centers. The characterization of uniglomerular projection neurons (PNs) and some local interneurons has facilitated our understanding of olfaction; however, many other AL neurons remain unidentified. Because neuron types are mostly specified by lineage and temporal origins, we use the MARCM techniques with a set of enhancer-trap GAL4 lines to perform systematical lineage analysis to characterize neuron morphologies, lineage origin and birth timing in the three AL neuron lineages that contain GAL4-GH146-positive PNs: anterodorsal, lateral and ventral lineages. The results show that the anterodorsal lineage is composed of pure uniglomerular PNs that project through the inner antennocerebral tract. The ventral lineage produces uniglomerular and multiglomerular PNs that project through the middle antennocerebral tract. The lateral lineage generates multiple types of neurons, including uniglomeurlar PNs, diverse atypical PNs, various types of AL local interneurons and the neurons that make no connection within the ALs. Specific neuron types in all three lineages are produced in specific time windows, although multiple neuron types in the lateral lineage are made simultaneously. These systematic cell lineage analyses have not only filled gaps in the olfactory map, but have also exemplified additional strategies used in the brain to increase neuronal diversity.     

Structure of the visual system of the larva of the tiger beetle (Cicindela chinensis)

The visual system of the larval tiger beetle (Cicindela chinensis) consists of six (two large, two mediumsized, and two small) stemmata on either side of the head, and an underlying neuropil mass. Each stemma exhibits a corneal lens and an underlying rhabdom layer. Retinular cells extend single proximal axons into the neuropil mass. The neuropil mass has a flattened heart-shape, and consists of two juxtaposed identical structures, each being a neuropil complex of each of the two large stemmata. The complex consists of lamina and medulla neuropils. Most retinular axons terminate in the lamina neuropil. Axons of two types of lamina monopolar neurons descend parallel to each other into the lamina neuropil. Moreover, each lamina neuropil contains a single giant monopolar neuron. Possible centrifugal processes and tangential neurons also occur. Lamina monopolar axons descend straight into the medulla neuropil. Medulla neurons spread fan-shaped dendrites distally in the medulla neuropil and send single axons toward the protocerebrum. These data are discussed with respecct to the unique visual behavior of this larva and in comparison with other insect visual systems.     

Immunocytochemical identification of neuroactive substances in the antennal lobe of the male silkworm moth Bombyx mori

As a first step towards understanding the functional role of neuroactive substances in the first olfactory center of the male silkworm moth Bombyx mori, we carried out an immunocytochemical identification of antennal lobe neurons. Antibodies against gamma-aminobutyric acid (GABA), FMRFamide, serotonin, tyramine and histamine were applied to detect their existence in the antennal lobe. In the present immunocytochemical study, we clarified four antenno-cerebral tracts from their origin and projection pathways to the protocerebrum, and revealed the following immunoreactive cellular organization in the antennal lobe. 1) Local interneurons with cell bodies in the lateral cell cluster showed GABA, FMRFamide and tyramine immunoreactivity. 2) Projection neurons passing through the middle antenno-cerebral tract with cell bodies in the lateral cell cluster showed GABA and FMRFamide immunoreactivity. Projection neurons passing through the outer antenno-cerebral tract with cell bodies in the lateral cell cluster showed FMRFamide immunoreactivity. 3) Centrifugal neurons passing through the inner antenno-cerebral tract b with cell bodies located outside the antennal lobe showed serotonin and tyramine immunoreactivity. Our results revealed basic distribution patterns of neuroactive substances in the antennal lobe and indicated that each projection pathway from the antennal lobe to the protocerebrum contains specific combination of neuroactive substances.     

Functional organization of olfactory processing in the accessory lobe of the spiny lobster

An isolated brain preparation was used to characterize neurons innervating the accessory lobe (AL) of the spiny lobster (Panulirus argus). Four distinct classes of neurons responded to electrical stimulation of the olfactory (antennular) nerve. These cells responded to electrical stimulation with a long and variable latency; they also responded to odor stimulation in a nose-brain preparation. Neurons connecting the AL with the olfactory lobe branched in the central AL layer and selectively innervated olfactory lobe glomeruli. These cells had response latencies which were significantly shorter than those of other AL neurons. Intrinsic AL interneurons were heterogeneous as a population, and most arborized in irregular but circumscribed regions of either the lateral or medial layers. The final class of neurons branched ipsilaterally in the deutocerebral neuropil and bilaterally innervated only a few AL glomeruli. The physiology and morphology of these four classes of neurons confirm an olfactory function for the AL and identify the input and output regions of the lobe. Based on these findings, we propose that the AL processes odor information in the context of higher order multimodal input.Abbreviations AL accessory lobe - DCN deutocerebral neuropil - OGT olfactory-globular tract - OGTN olfactory-globular tract neuropil - OL olfactory lobe     

The maxillary palp sensory pathway of Orthoptera

Primary sensory projections and arborisations of higher-order neurons associated with the maxillary palps were examined in Tettigoniidae, Gryllidae, Tetrigidae and Acrididae representing the two sub-orders of Orthoptera, Ensifera and Caelifera. Anterograde filling and Golgi impregnation of maxillary receptor neurons revealed two patterns of innervation, the ensiferous and the caeliferous type. In both ensiferans and caeliferans, receptor neurons arborised within the tritocerebrum, the antennal motor- and mechano-sensory centre and the lobus glomerulatus. In ensiferans, additional areas of innervation were found in the lobus glomerulatus and in a previously undescribed neuropil, here referred to as the accessory lobus glomerulatus. In relation to the anatomical data a putative functional segregation of the neuropil into gustatory-, olfactory- and mechano-sensory centres is implied.     

Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta

Summary Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta were individually reconstructed. Serotonin immunoreactivity was detected in 19–20 bilaterally symmetrical pairs of interneurons in the midbrain and 10 pairs in the suboesophageal ganglion. These neurons were also immunoreactive with antisera against DOPA decarboxylase. All major neuropil regions except the protocerebral bridge are innervated by these neurons. In addition, efferent cells are serotonin-immunoreactive in the frontal ganglion (5 neurons) and the suboesophageal ganglion (2 pairs of neurons). The latter cells probably give rise to an extensive network of immunoreactive terminals on the surface of the suboesophageal ganglion and suboesophageal nerves. Most of the serotonin-immunoreactive neurons show a gradient in the intensity of immunoreactive staining, suggesting low levels of serotonin in cell bodies and dendritic arbors and highest concentrations in axonal terminals. Serotonin-immunoreactive cells often occur in pairs with similar morphological features. With one exception, all serotonin-immunoreactive neurons have bilateral projections with at least some arborizations in identical neuropil areas in both hemispheres. The morphology of several neurons suggests that they are part of neuronal feedback circuits. The similarity in the arborization patterns of serotonin-immunoreactive neurons raises the possibility that their outgrowing neurites experienced similar forces during embryonic development. The morphological similarities further suggest that serotonin-immunoreactive interneurons in the midbrain and suboesophageal ganglion share physiological characteristics.Abbreviations CNS central nervous system - DDC DOPA decarboxylase - LAL lateral accessory lobe - SLI serotonin-like immunoreactivity - SOG suboesophageal ganglion - VLP ventro-lateral protocerebrum     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Active dendritic conductances dynamically regulate GABA release from thalamic interneurons

Inhibitory interneurons in the dorsal lateral geniculate nucleus (dLGN) process visual information by precisely controlling spike timing and by refining the receptive fields of thalamocortical (TC) neurons. Previous studies indicate that dLGN interneurons inhibit TC neurons by releasing GABA from both axons and dendrites. However, the mechanisms controlling GABA release are poorly understood. Here, using simultaneous whole-cell recordings from interneurons and TC neurons and two-photon calcium imaging, we find that synchronous activation of multiple retinal ganglion cells (RGCs) triggers sodium spikes that propagate throughout interneuron axons and dendrites, and calcium spikes that invade dendrites but not axons. These distinct modes of interneuron firing can trigger both a rapid and a sustained component of inhibition onto TC neurons. Our studies suggest that active conductances make LGN interneurons flexible circuit-elements that can shift their spatial and temporal properties of GABA release in response to coincident activation of functionally related subsets of RGCs.     

Localization of catecholamine-containing nerve fibres in the branchial heart and cardiac ganglion of the common cuttlefish Sepia officinalis L. (cephalopoda)

The innervation of the branchial heart of Sepia officinalis was examined using TEM and glyoxylic acid induced fluorescence. In the cardiac ganglion and in cardiac nerves bluish-green fluorophores were seen associated with perikarya and varicose nerve fibres. Microspectrofluorometric analysis provided clear evidence that monoaminergic neurons in the branchial heart contain only catecholamines. Considering pharmacological data, it is more than likely that 5-hydroxytryptamine (serotonin) is not present in this system.     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Functional divergence of spatially conserved olfactory glomeruli in two related moth species

In different moth species, the number and spatial arrangement of olfactory glomeruli in the antennal lobe (AL) vary widely, but the spatial map within a species is thought to be invariant, making it possible to identify single glomeruli across individuals. We investigated the relationship between the physiological tuning of pheromone-selective interneurons and their association with specific, identified glomeruli in the macroglomerular complex (MGC) of the noctuid moth, Heliothis subflexa. Three odorants that are required for pheromone-source location in this species were tested individually and in blends. Recordings from 27 pheromone-specific projection neurons (PNs) indicated that the majority (48%) were selectively activated by the major pheromone component of this species, Z-11-hexadecenal (Z11-16:Ald), with 33% primarily tuned to Z-9-hexadecenal and 19% to Z-11-hexadecenol. Intracellular staining revealed that the dendrites of PNs tuned to Z11-16:Ald always branched within the largest glomerulus of the MGC, the cumulus. Similarly, each of the other two classes of PN was associated with a different 'satellite' glomerulus in the MGC. The spatial configuration of the four-glomerulus H. subflexa MGC was indistinguishable from that previously reported in the closely related species, Heliothis virescens. Hence, as these two species diverged, changes in the association of satellite MGC glomeruli with particular odorants have occurred without a measurable change in the anatomical arrangement of the glomerular array.     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

Neuronal connectivity patterns in the compound eyes of Artemia salina and Daphnia magna (Crustacea: Branchiopoda)

The neuronal types and patterns in the visual system of the species Artemia salina and Daphina magna have been studied with the Golgi method and electron microscopy. The lamina contains five classes of neurons: photoreceptor axons, monopolar, centrifugal, tangential and amacrine neurons. The terminals of the receptor axons are distributed in two (A. salina) or three (D. magna) layers. The dilated terminals have an extensive and wide array of fine branches. One axon from each ommatidium bypasses the lamina and terminates in the medula in A. salina. A. salina has four types of monopolar neurons, two of which are stratified, whereas in D. magna only two types are found, one of which is bistratified. Tangential T-neurons connect the lamina with the protocerebrum. D. magna has in addition one tangential T-neuron connecting both the lamina and the medulla with the protocerebrum. In both species monopolar-type centrifugal neurons connect the medulla and the lamina, whereas that of A. salina has a wide laminar distribution. Both species also have amacrine cells in the lamina. The medulla contains, besides those shared with the lamina, transmedullary neurons (two types in A. salina), amacrine cells and neurons originating in the protocerebrum. "Cartridge"-type synaptic compartments are lacking in the investigated species, although a periodic arrangement is discernible in the distal portion of the lamina of A. salina. The receptors from three types of specialized contacts in Artemia, one of which involves a dyad. D. magna has only one-to-one synapses. Neurosecretory fibres are absent in A. salina.     

Serotonin-immunoreactive neurons in the antennal sensory system of the brain in the carpenter ant, Camponotus japonicus

Social Hymenoptera such as ants or honeybees are known for their extensive behavioral repertories and plasticity. Neurons containing biogenic amines appear to play a major role in controlling behavioral plasticity in these insects. Here we describe the morphology of prominent serotonin-immunoreactive neurons of the antennal sensory system in the brain of an ant, Camponotus japonicus. Immunoreactive fibers were distributed throughout the brain and the subesophageal ganglion (SOG). The complete profile of a calycal input neuron was identified. The soma and dendritic elements are contralaterally located in the lateral protocerebrum. The neuron supplies varicose axon terminals in the lip regions of the calyces of the mushroom body, axon collaterals in the basal ring but not in the collar region, and other axon terminals ipsilaterally in the lateral protocerebrum. A giant neuron innervating the antennal lobe has varicose axon terminals in most of 300 glomeruli in the ventral region of the antennal lobe (AL) and a thick neurite that spans the entire SOG and continues towards the thoracic ganglia. However, neither a soma nor a dendritic element of this neuron was found in the brain or the SOG. A deutocerebral projection neuron has a soma in the lateral cell-body group of the AL, neuronal branches at most of the 12 glomeruli in the dorsocentral region of the ipsilateral AL, and varicose terminal arborizations in both hemispheres of the protocerebrum. Based on the present results, tentative subdivisions in neuropils related to the antennal sensory system of the ant brain are discussed.     

Pulses, patterns and paths: neurobiology of acoustic behaviour in crickets

Crickets use acoustic communication for pair formation. Males sing with rhythmical movements of their wings and the mute females approach the singing males by phonotaxis. Females walking on a trackball rapidly steer towards single sound pulses when exposed to split-song paradigms. Their walking path emerges from consecutive reactive steering responses, which show no temporal selectivity. Temporal pattern recognition is tuned to the species-specific syllable rate and gradually changes the gain of auditory steering. If pattern recognition is based on instantaneous discharge rate coding, then the tuning to the species-specific song pattern may already be present at the level of thoracic interneurons. During the processing of song patterns, changes in cytosolic Ca²⁺ concentrations occur in phase with the chirp rhythm in the local auditory interneurone. Male singing behaviour is controlled by command neurons descending from the brain. The neuropil controlling singing behaviour is located in the anterior protocerebrum next to the mushroom bodies. Singing behaviour is released by injection of cholinergic agonists and inhibited by γ-butyric acid (GABA). During singing, the sensitivity of the peripheral auditory system remains unchanged but a corollary discharge inhibits auditory processing in afferents and interneurons within the prothoracic auditory neuropil and prevents the auditory neurons from desensitisation.     

Spiking patterns and their functional implications in the antennal lobe of the tobacco hornworm Manduca sexta

Bursting as well as tonic firing patterns have been described in various sensory systems. In the olfactory system, spontaneous bursts have been observed in neurons distributed across several synaptic levels, from the periphery, to the olfactory bulb (OB) and to the olfactory cortex. Several in vitro studies indicate that spontaneous firing patterns may be viewed as "fingerprints" of different types of neurons that exhibit distinct functions in the OB. It is still not known, however, if and how neuronal burstiness is correlated with the coding of natural olfactory stimuli. We thus conducted an in vivo study to probe this question in the OB equivalent structure of insects, the antennal lobe (AL) of the tobacco hornworm Manduca sexta. We found that in the moth's AL, both projection (output) neurons (PNs) and local interneurons (LNs) are spontaneously active, but PNs tend to produce spike bursts while LNs fire more regularly. In addition, we found that the burstiness of PNs is correlated with the strength of their responses to odor stimulation--the more bursting the stronger their responses to odors. Moreover, the burstiness of PNs was also positively correlated with the spontaneous firing rate of these neurons, and pharmacological reduction of bursting resulted in a decrease of the neurons' responsiveness. These results suggest that neuronal burstiness reflects a physiological state of these neurons that is directly linked to their response characteristics.     

Immunocytochemistry of GABA in the brain and suboesophageal ganglion ofManduca sexta

Summary We have used specific antisera against protein-conjugated-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. About 20000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers.