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GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system.

We used an antiserum directed against gamma-aminobutyric acid (GABA) fixed with glutaraldehyde (Hoskins et al., Cell Tissue Res. 244:243-252, '86) to label neurons with GABA-like immunoreactivity (GLI) in wholemounts of the stomatogastric ganglion and each segmental ganglion of crayfish, except the brain. Each abdominal ganglion had an average of 63 labeled neurons, or 10% of all their neurons. Each peripheral nerve of each abdominal ganglion except the last contained labeled axons. Within each segment, the first peripheral nerve, N1, had five axons; the second peripheral nerve, N2, had at most four; and the third peripheral nerve, N3, had two. In the last ganglion, N2 had one labeled axon, N3 had two and N6 had two; the other nerves contained no labeled axons. A tabulation of the identified inhibitory neurons in the abdominal ganglia revealed that 40% of these GABA-ergic neurons have been identified. The subesophageal ganglion had many labeled neurons in clusters that formed a repeating pattern; it also had labeled neurons near its dorsal midline. The thoracic ganglia contained more labeled neurons than did the abdominals, but their patterns of labeling were similar. The commissural ganglia contained three clusters of labeled neurons and sent labeled axons to the esophageal ganglion. The esophageal ganglion contained four labeled neurons and many labeled axons. The stomatogastric ganglion contained labeled axon terminals but not labeled neurons.

Animals↗

Substance P antibody reveals homologous neurons with axon terminals among somata in the crayfish and crab brain.

In the search for particular neurons that stain selectively and can be identified, the cerebral ganglia (brains) of the crayfish Cherax destructor and the crab Leptograpsus variegatus were immunocytochemically treated with a monoclonal antibody raised against substance P. Four large neurons in the cerebral ganglion of the crayfish and crab label selectively with a monoclonal antibody raised against substance P. Two of the large neurons have their cell bodies in the protocerebrum and two in the deutocerebrum in both animals. Each protocerebral cell in both animals projects through the ipsilateral and contralateral olfactory lobes to end among the lateral cell somata of the olfactory lobe and not in the neuropile. Electron micrographs show the presence of synapses within the cell somata area and on the cell somata themselves. Each deutocerebral cell in both animals projects only ipsilaterally and ends within the neuropile of the olfactory lobes. The immunoreactivity to substance P antibody and the shapes and the unique projections of the four cells suggest that they are homologous in the two species. Synaptic connections between axons and cell somata are rare in the arthropods but have been found on the Kenyon cells of the mushroom bodies of Limulus. This raises questions about homologies between the crustacean olfactory lobe and the mushroom bodies of Limulus and insects.

Animals↗

Nontopographic projection of olfactory sensory neurons in the crayfish brain.

We have used tritiated leucine to trace the input projection pattern of olfactory sensory neurons in crayfishes. The olfactory neurons are associated with cuticular sensilla on the external antennular filaments. Each sensillum, or aesthetasc, harbors the distal dendritic segments of about 175 bipolar sensory neurons, the cell bodies for which are grouped in a subcuticular ensemble or ganglion. About 150-175 individual ganglia may be found on each antennule in an adult crayfish. When an aesthetasc is exposed to tritated leucine, the tracer is taken up by the associated olfactory sensory neurons and is transported along the axons to their central terminations within the glomeruli of the ipsilateral olfactory lobe. We tested the possibility that the sensory neurons from specific aesthetascs project to specific glomeruli. By restricting access of the leucine to small groups of aesthetascs, we exposed less than 2% of the olfactory sensory neurons to the tracer. Nonetheless, all glomeruli were labeled following such treatment. We conclude that the sensory neurons are generally distributed to the olfactory glomeruli. If each neuron terminates in a single glomerulus, these data support a divergent pattern of sensory projection from individual ganglia to all regions of the olfactory lobe.

Animals↗

Effect of temperature on long-term survival of anucleate giant axons in crayfish and goldfish.

The effect of temperature on the electrophysiology and morphology of anucleate axons was examined following severance of crayfish medial giant axons and goldfish Mauthner axons from their respective cell bodies. Although anucleate segments of each giant axon exhibited long-term survival for weeks to months at 5-25 degrees C in crayfish and 10-30 degrees C in goldfish, the two axons differed in their survival characteristics. All measures of long-term survival in crayfish medial giant axons were independent of animal holding temperature, whereas all measures in Mauthner axons were dependent on holding temperature. Medial giant axons survived for at least 90 days in crayfish maintained at 5-25 degrees C in this and previous studies. Mauthner axons survived for over 5 months in goldfish maintained at 10 degrees C but survived for 1 month at 30 degrees C. Postoperative time had different effects on many single measures of long-term survival (axonal diameter, amplitude of action or resting potentials) in medial giant axons compared to Mauthner axons. For example, resting and action potentials in crayfish medial giant axons remained remarkably constant at all holding temperatures for 0-90 postoperative days. In contrast, resting and action potentials in goldfish Mauthner axons declined abruptly in the first 10-20 postoperative days followed by a slower decline at each holding temperature. We suggest that the mechanism of long-term survival is not necessarily the same in all anucleate axons.

Animals↗

Ultrastructure of the electrotonic and chemical components of the lateral-to-motor and medial-to-motor synapses in crayfish nerve cord.

Lateral-to-motor and medial-to-motor synapses in crayfish nerve cords are composed of an electrical and a chemical component. The presynaptic terminals showed localized clusters of synaptic vesicles, electron-dense areas, coated pits, and coated vesicles. In thin sections, active zones were defined by electron-dense regions where synaptic vesicles attached and, in freeze-fracture replicas, by clusters of intramembrane particles localized in bands with vesicle openings on the sides of these bands. The cytoplasmic surface of the postsynaptic membrane opposite the active zones was coated with electron-dense material that in freeze-fracture replicas was seen as an increase in intramembrane particles located in the external leaflet (EF-face). This specialization of the postsynaptic membrane may correspond to the neurochemical receptor. Also, pre- and postsynaptic membranes were separated by a wider extracellular gap than those of adjacent nonsynaptic regions and electrical synapses or gap junctions. Synaptic vesicles were located exclusively at the synaptic regions by means of a cytoskeleton that was different for the electrical and the chemical components. The vesicles associated with the electrical component were anchored to a cytoskeleton composed of a beaded layer of densities located parallel to the membrane. This cytoskeleton maintained the synaptic vesicles separated from the presynaptic membrane by a distance of 13 +/- 2 nm. The synaptic vesicles associated with the chemical component were anchored to electron-dense regions formed by filaments arranged in bundles, anchored to the presynaptic membrane. Vesicles lined both sides positioned to discharge their contents into the extracellular space and to replace the discharged vesicles.

Animals↗

A crustacean neuronal cytoskeletal protein with characteristics of neurofilaments and microtubule-associated proteins.

The purpose of this study was to characterize further a unique protein that is a component of the cytoskeleton of crayfish neurons. This protein, referred to as P600, is unique because it is unusually large (Mr greater than 600 kD), and because it has characteristics in common with both mammalian microtubule-associated proteins and neurofilaments. Immunohistochemical techniques have shown that P600 colocalizes with microtubules and is a component of the fibrous side-arms that extend from microtubules (Weaver and Viancour, Brain Res. 544:49, 1991). We have developed a method for obtaining purified P600 by using gel filtration techniques. When viewed by negative staining electron microscopy, P600 obtained by that method produced 11 nm-wide beaded filaments. The number of filaments was strictly related to the P600 concentration in a column fraction. A small amount of P600 consistently copurified with taxol-stabilized microtubules. The proportion copurifying with microtubules was increased by using apyrase to deplete ATP, or by using a nonhydrolyzable ATP analogue to compete with ATP. Immunogold labeling localized P600 near the ends of a subset of the fibrous side-arms extending from endogenous axonal microtubules. Several polyclonal antibodies against mammalian microtubule-associated proteins were tested for P600 labeling on immunoblots, and positive labeling was obtained with an antiserum directed against a region of microtubule-associated protein 1B that has microtubule binding activity. Epitope homology between P600, mammalian microtubule-associated protein 1B, and the mammalian mid-molecular weight neurofilament subunit is discussed in the context of possible evolutionary relationships among these cytoskeletal proteins.

Animals↗

Anatomy and fine structure of neurons in the deutocerebral projection pathway of the crayfish olfactory system.

Golgi impregnation and neurobiotin injection were used to examine details of the neural pathways in the olfactory system of the freshwater crayfish, Procambarus clarkii. Deutocerebral projection neurons (globuli cells) were directly injected with neurobiotin. These neurons have dendritic arborizations in the ipsilateral olfactory and accessory lobes, and they project axons to the lateral protocerebrum, where they terminate in microglomeruli of the hemi-ellipsoid body. The axons of the deutocerebral projection neurons are readily impregnated by Golgi procedures, and they terminate as an expanded membranous knot about 5 microns in diameter. Electron microscopy on Golgi-stained terminals has revealed that each knot makes several hundred synapses with small spine-like or shaft-like processes of postsynaptic neurons. Injection of neurobiotin into local interneurons of the hemi-ellipsoid body and subsequent examination of stained preparations with the electron microscope reveals that these cells are a major postsynaptic target of the deutocerebral projection neurons. Furthermore, the local interneurons make extensive efferent synaptic connections with unidentified neurons in the terminal medulla.

Animals↗

Two classes of vesicles are present and change in relative proportion during post-embryonic development of rectifying electrical synapses in the crayfish.

The size and shape of vesicles at junctional appositions of the rectifying electrical synapses between the medial giant fibre and motor giant neurone of the crayfish were measured during the first 2 months after hatching. Summed data over this period reveal a bimodal distribution in vesicle diameter. From the day of hatching until about 7 days of age, small vesicles (circa 25 nm diameter) predominate. From day 7 onwards, larger vesicles (circa 55 nm diameter) occur in increasing numbers, until at day 56 they constitute about 85% of the population at any one junctional apposition. At intermediate ages (day 7-28) individual junctional appositions may show the same bimodal distribution in size as does the age group as a whole, indicating that large and small vesicles occur together at the same junction. The larger vesicles are mainly circular, while the small vesicles are pleomorphic, with shapes ranging from almost circular down to a shape factor of about 0.6.

Aging↗

Functional degeneration of isolated central stumps of crayfish sensory axons.

In the crayfish, Procambarus clarkii, nerve 5 carries primarily sensory axons from the tail fan to the 6th abdominal ganglion where they synaptically activate interneuron A. Since the sensory neurons have their somata located at the periphery, transection of nerve 5 part way to the ganglion allowed us to examine the fate of their soma-less central stumps. Up to 3 weeks postlesion the response to stimulation of nerve 5 consisted of a brief latency spike in interneuron A, similar to that in control animals and to stimulation of the intact nerve 4. Stimulation of the lesioned nerve 5 beyond 3 weeks failed to fire interneuron A. This loss of function was correlated to loss of axons in nerve 5 deduced by comparing the numbers in the lesioned nerve 5 to its contralateral intact counterpart. The numbers are about equal in the paired nerves but rapidly decline on the lesioned side to 50% within 1 week, 20% within 3 weeks, and less than 10% in subsequent weeks. This loss affects all size classes of axons. However, in the 3 week lesioned nerve large glial infoldings subdivided some of the larger axons and single nuclei were seen in a few of the medium-sized axons. Possibly subdivision of large axons by glial infolding may introduce glial nuclei into axons.

Afferent Pathways↗

Ultrastructure of the circuit providing input to the crayfish lateral giant neurons.

Labeled or otherwise identified neurons of the crayfish lateral giant escape reaction circuit were examined electron microscopically and the findings compared to expectations from physiology. Terminals of primary afferents contained clear, approximately 45 nm, irregularly round synaptic vesicles, while sensory interneuron terminals had slightly larger, 50 nm, more strictly round vesicles, permitting tentative classification based on anatomical criteria. Excitatory synapses on the lateral giants, believed from physiology to be electrical, generally had some gap junctions, but these were almost invariably paralleled by more prominent chemical junctional regions of unknown function. There may also be a class of interneurons making purely chemical synapses on the lateral giants. Synapses from primary afferents to sensory interneurons, believed from physiology to be cholinergic, had purely chemical morphology. Synapses with narrow elongated vesicles, similar to GABAergic vesicles seen in other neurons, frequently occurred on terminals of primary afferents. These synapses provide a basis for known presynaptic inhibition of afferent input. Consistent with physiology, such inhibitors sometimes also contacted the postsynaptic targets of the primary afferents and sometimes received input from other primary afferents. Afferent terminals also received some input from profiles rich in large dense cored vesicles. Presumptive inhibitory input found on proximal dendrites of lateral giants provides a basis for known recurrent inhibition. However, similar inhibitory synapses that sometimes received local input from excitors of the lateral giants were also found distally mixed with excitatory inputs. These provide a basis for recently discovered distal inhibitory input following excitation and for tonic inhibition.

Afferent Pathways↗

Intersegmental ascending interneurons controlling uropod movements of the crayfish Procambarus clarkii.

The premotor effects of intersegmental ascending interneurons upon uropod motor neurones in the crayfish Procambarus clarkii (Girard) are examined with intracellular recording and staining techniques. We show that many ascending interneurones can affect the activity of the antagonistic opener and closer motor neurones in the terminal ganglion. Based upon soma position, ascending interneurones are divided into three groups of rostral, medial, and caudal interneurones. Twenty-four ascending interneurones are characterized physiologically according to their inputs from the tailfan and their output effects on the uropod motor neurones of both sides. Each interneurone is identifiable as a unique individual by means of overall shape, soma position, number of main branches, the commissure in which primary neurites cross the midline, axon position in the 5th-6th abdominal connective and physiological responses. They are classified into six classes; coactivating, coinhibiting, reciprocally closing, reciprocally opening, variably effective, and not effective interneurones, according to their premotor effects on the uropod motor neurones. These ascending interneurones seem to act as multifunctional units conveying sensory information from the tailfan to the anterior abdominal ganglia and, at the same time, influencing the uropod motor pattern in the terminal abdominal ganglion.

Animals↗

Comparative brain ontogeny of the crayfish and clawed lobster: implications of direct and larval development.

The freshwater crayfish Cherax destructor and the lobster Homarus americanus have many similarities including life style, body form, and neural organization. However, the ontogenic history is very different in the two species. The development of Cherax is short and direct whereas the development of Homarus comprises three pelagic larval stages and takes more than twice as long from extrusion to benthic stages at constant temperature. In order to determine the progression of maturation of the nervous system in each species and the potential implications of pelagic forms on brain structure, the timing of appearance of 22 general and neural developmental events clearly identifiable in both species was compared. The onset of serotonin antigenicity in the different parts of the brain was chosen as one marker of neural development. During the first month of embryogenesis the timing of morphological, physiological, and neural events is similar in the two species. Morphological development is then accelerated in the crayfish near hatching time and over the two postembryonic stages before the advent of the independent benthic stage. Such heterochronic processes can at least partly account for the different developmental patterns in the two decapods. Among the characters showing similar timing in the two species is the formation of glomeruli (presumptive zones of synaptic contact) in the olfactory lobes of the deutocerebrum, although this event is embryonic in Homarus but postembryonic in Cherax. In contrast, glomerular formation in the accessory lobes is heterochronic: in both species, the glomeruli of the accessory lobes are acquired postembryonically, that is, 3 to 4 months earlier in Cherax than in Homarus. These data suggest that the development of the glomeruli in the olfactory lobes may depend primarily on internal developmental signals, whereas the triggering of glomerular formation in the accessory lobes may depend on external cues. The fact that, in Homarus, only the postlarval stages show mature accessory glomeruli may be a reflection of the functional requirements of benthic life.

Animals↗

The inhibitory motoneurons of crayfish thoracic limbs: identification, structures, and homology with insect common inhibitors.

The three inhibitory motoneurons supplying crayfish thoracic limbs were identified, stained, and compared structurally. The inhibitors to the walking leg muscles (in Orconectes) were identified anatomically by a combination of immunocytochemical staining for gamma-aminobutyric acid (GABA) or glutamate decarboxylase and differential backfill staining with nickel and cobalt ions. The cheliped inhibitors were identified intracellularly and injected with Lucifer Yellow (Pacifastacus) or cobalt (Procambarus). The common inhibitor (CI) in each thoracic segment has a medial or slightly contralateral soma near the ganglion's posterior boundary, a gently curving primary neurite, an extensive ipsilateral dendritic tree, and an axon emerging through the anterior root. The stretcher-closer inhibitor (SI) has a soma slightly anterior and ipsilteral to the CI's a sharply bent proximal neurite, a smaller dendritic tree, and an axon in the posterior root. The opener inhibitor (OI) lies more laterally and often posterior to the CI; its diagnoally directed neurite enters the posterior root. The inhibitors' structures were related to major neuroanatomical landmarks within the ganglion, to soma positions of excitatory motoneurons revealed by backfilling, and to soma locations of inhibitory interneurons revealed by GABA-like immunoreactivity. In their peripheral distributions to the leg muscles and in their central structures, these crayfish limb inhibitors show striking similarities with those of the locust. Crayfish and locust thoracic ganglia also show more general neuroanatomical similarities. These observations suggest that the crayfish CI, SI, and OI are, respectively, homologous with the locust CI1, CI2, and CI3. The implications of such a homology for arthropod phylogeny are discussed.

Animals↗

Physiology and morphology of spiking local interneurons in the terminal abdominal ganglion of the crayfish.

Not only nonspiking local interneurons but also spiking local interneurons in the terminal abdominal ganglion of the crayfish (Procambarus clarkii Girard) contribute to form the motor output of the uropod. In this study, 14 spiking local interneurons are identified by their gross morphology and physiological properties including the sensory inputs from the uropod and premotor output onto the uropod motor neurons on both sides. Morphologically, they are divided into three groups based on the position of their somata: anterior, medial, and posterior. The main branches of interneurons in each group are usually extended on the side contralateral to the soma, or they have profuse bilateral branches. Physiologically, all of them receive excitatory inputs from the afferents innervating the exopodite on the side ipsilateral their main branches. They are usually silent, and spike only in response to sensory stimulation. The majority of them (11 out of 14 neurons) have output effects on the uropod motor neurons that elicit reciprocally closing pattern. They increased the activity of the closer motor neurons and decreased the activity of the opener motor neurons. Only one interneuron inhibits the closer motor neurons. The remaining two interneurons coactivate both the antagonistic motor neurons. Since the reciprocal closing pattern of the uropod motor neurons is elicited in response to sensory stimulation of the uropod, spiking local interneurons may initiate this local reflex circuit. They detect the initiation of the stimulus and convey signals from the sensory to the motor side.

Animals↗

Electrical responses and synaptic connections of giant serotonin-immunoreactive neurons in crayfish olfactory and accessory lobes.

Five pairs of identified 5HT-IR cells in the deutocerebrum of the crayfish Cherax are known to have their synaptic endings in the accessory and olfactory lobes. Two of these cells, one on each side of the brain, are significantly larger than the others. Dye fills of these "giant" cells reveal each to be an interneuron with its branches confined to, but distributed throughout, the olfactory and accessory lobes on the side of the brain ipsilateral to its cell body and with no branches to the contralateral side. Intracellular recordings from the giant cells were made while stimulating the olfactory afferents and tracts within the brain in an attempt to discover the inputs and outputs to the cells. Electrical stimulation of chemoreceptor sensilla on the outer branch of the antennule does not excite the giant 5HT neurons. Focal extracellular electrical stimulation of the olfactory globular tract containing the axons of projection neurons from the olfactory and accessory lobes produces excitatory synaptic potentials and action potentials in the giant cells. Focal extracellular electrical stimulation of the deutocerebral commissure, the axons of which terminate in the glomeruli of the accessory lobes, also results in excitation of the giant cells. We conclude that the input to the giant cells is via axons in the deutocerebral commissure and collaterals from the projection neurons, ending in the glomeruli of the accessory lobes. The output of the giant cells is to the olfactory lobes, where it may serve to modulate olfactory signals.

Action Potentials↗

Biotin staining in the giant fiber systems of the lobster.

The avidin-biotin-complex method is a popular immunocytochemical technique. This method labels consistently a group of neurons in the lobster ventral nerve cord in the absence of primary antibodies. The specific staining is due to a relatively high level of endogenous biotin (or biocytin) in these neurons. These biotin-positive neurons are located in the supraesophageal, thoracic, and abdominal ganglia. Intraaxonal injection of Lucifer yellow followed by Texas red-conjugated streptavidin staining reveals that the neurons are members of the medial giant (MG) and lateral giant (LG) systems, which are important in mediating rapid tail flipping during escape maneuvers. In neuronal somata, staining is restricted to the cytoplasm. Within MG axons, staining appears as punctate, subaxolemmal structures. Preincubating nerve cords in biocytin or direct intraaxonal injection of biocytin enhances staining of these punctate organelles. In LG axons, staining is localized to fragments of braided filamentous structures that also appear to be associated with the axolemma. Preincubation of ventral nerve cords in various concentrations of biocytin results in the appearance of additional groups of stained neurons, suggesting that there are subsets of neurons with specific biocytin-uptake or -retention mechanisms. In the crayfish, biotin-positive staining is confined to the MG neurons; the LG neurons are not stained. In the earthworm, no staining is observed in the MG and LG axon escape systems. In the goldfish, no biotin-staining is seen in the Mauthner neurons and their axons. The significance of specific localization of biotin or biocytin to subsets of neurons is unclear. It may reflect the presence of high levels of biocytin moieties on biotin-dependent enzymes. Biotin is an important cofactor in the catalytic functions of several decarboxylases crucial in energy production and lipogenesis. Axons of the giant fiber systems in lobsters and crayfish may have high energy and fatty acid synthesis requirements. Increased levels of biotin accumulation may also be related to other functions of the giant axon systems, such as the formation of electrical synapses among themselves and with phasic motoneurons.

Animals↗

Proctolin and excitation of the crayfish swimmeret system.

The ventral nerve cord of crayfish contains axons of five pairs of excitatory interneurons, each of which can activate the swimmeret system. Perfusion of the ventral nerve cord with the neuropeptide proctolin also activates the swimmeret system. The experiments reported here were conducted to test the hypothesis that one or more of these excitatory interneurons uses proctolin as a transmitter. Each of the five excitatory axons was located and stimulated separately in an individual crayfish, and similar motor activity was elicited by stimulating each of them. Quantitative comparison of spontaneous swimmeret motor patterns with activity caused by stimulating one of these excitatory axons, EC, or by perfusing with proctolin solutions showed that the motor patterns produced under these three conditions were not significantly different (P > 0.05). By using a new, affinity-purified proctolin antiserum, we labeled axons in the connective tissue between the last thoracic and first abdominal ganglion and compared the positions of labeled axons with the previously described positions of the excitatory axons. About 0.3% of the axons in these connective tissues showed proctolin-like immunoreactivity, but heavily labeled pairs of axons did occur bilaterally in the regions of excitatory swimmeret axons. The projections of these labeled axons into the abdominal ganglia were traced in serial plastic sections. Labeled processes were abundant in the lateral neuropils, the loci of the swimmeret pattern-generating circuitry. From this evidence, we propose that three of these excitatory swimmeret interneurons use proctolin as a transmitter, but that a fourth does not. The evidence for the fifth axon is ambiguous.

Amino Acid Sequence↗

Crustacean cardioactive peptide-immunoreactive neurons in the ventral nervous system of crayfish.

Crustacean cardioactive peptide-immunoreactive neurons have been mapped in whole-mount preparations and sections of the ventral nervous system of the crayfish Astacus astacus and Orconectes limosus. Based on their morphology, projection patterns, and staining characteristics, two types of contralaterally projecting neurons are individually identifiable. In both species, these neurons occur in all neuromers as apparent serial homologs. In adult specimens, one type of cell has a small, densely stained dorsal lateral perikaryon, and a descending axon, and appears to be an interneuron. Each neuromer contains a single pair of these cells. Only in maxillary ganglia, these cells may have an additional ascending projection. The other type, a neurosecretory cell, has a larger, weakly stained perikaryon and a projection to the segmental third root of the next anterior neuromer. All neuromers contain a single pair of these neurons adjacent to the interneurons except for the abdominal neuromers, which contain two pairs of the neurosecretory cells. Central arborizations and varicose processes toward the surface of the third roots and within the perineural sheath of the ventral nerve cord arise from these neurons. Electron microscopy of granule-containing terminals substantiated that these newly discovered extensive neurohemal areas are release sites for the peptide. In young immature specimens, the perikarya of both neuron types do not differ in size. Additional weakly stained small perikarya occur in all neuromers of Astacus astacus. These two types of crayfish neurons and other comparable aminergic and peptidergic neurons of crayfish and lobster are differentially distributed in the ventral cord. Furthermore, comparison of similar neuron types in crab, locust, meal worm, and moth species indicates intra- and interphyletic structural homologies.

Animals↗