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Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6, and Dlx2 expression, in vitro differentiation into neurons expressing GABA, and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies. SIGNIFICANCE STATEMENT Here we demonstrate that porcine medial ganglionic eminence, like rodents, exhibit a distinct transcriptional and interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic interneuron origins in the pig; and because a rich neurodevelopmental literature on embryonic mouse medial ganglionic eminence exists (with some additional characterizations in other species, e.g., monkey and human), our work allows direct neurodevelopmental comparisons with this literature., (Copyright © 2021 the authors.)

The Drosophila giant fiber (GF) escape circuit is an extensively studied model for neuron connectivity and function. Researchers have long taken advantage of the simple linear neuronal pathway, which begins at peripheral sensory modalities, travels through the central GF interneuron (GFI) to motor neurons, and terminates on wing/leg muscles. This circuit is more complex than it seems, however, as there exists a complex web of coupled neurons connected to the GFI that widely innervates the thoracic ganglion. Here, we define four new neuron clusters dye coupled to the central GFI, which we name GF coupled (GFC) 1-4. We identify new transgenic Gal4 drivers that express specifically in these neurons, and map both neuronal architecture and synaptic polarity. GFC1-4 share a central site of GFI connectivity, the inframedial bridge, where the neurons each form electrical synapses. Targeted apoptotic ablation of GFC1 reveals a key role for the proper development of the GF circuit, including the maintenance of GFI connectivity with upstream and downstream synaptic partners. GFC1 ablation frequently results in the loss of one GFI, which is always compensated for by contralateral innervation from a branch of the persisting GFI axon. Overall, this work reveals extensively coupled interconnectivity within the GF circuit, and the requirement of coupled neurons for circuit development. Identification of this large population of electrically coupled neurons in this classic model, and the ability to genetically manipulate these electrically synapsed neurons, expands the GF system capabilities for the nuanced, sophisticated circuit dissection necessary for deeper investigations into brain formation.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1 + , Dlx2 + , and Sox2 + cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1 + progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2 + cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1 + and Dlx2 + cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants. SIGNIFICANCE STATEMENT Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants., (Copyright © 2018 the authors 0270-6474/18/381100-14$15.00/0.)

Taste perception begins in the oral cavity by interactions of taste stimuli with specific receptors. Specific subsets of taste receptor cells (TRCs) are activated upon tastant stimulation and transmit taste signals to afferent nerve fibers and ultimately to the brain. How specific TRCs impinge on the innervating nerves and how the activation of a subset of TRCs leads to the discrimination of tastants of different qualities and intensities is incompletely understood. To investigate the organization of taste circuits, we used gene targeting to express the transsynaptic tracer barley lectin (BL) in the gustatory system of mice. Because TRCs are not synaptically connected with the afferent nerve fibers, we first analyzed tracer production and transfer within the taste buds (TBs). Surprisingly, we found that BL is laterally transferred across all cell types in TBs of mice expressing the tracer under control of the endogenous Tas1r1 and Tas2r131 promotor, respectively. Furthermore, although we detected the BL tracer in both ganglia and brain, we also found local low-level Tas1r1 and Tas2r131 gene, and thus tracer expression in these tissues. Finally, we identified the Tas1r1 and Tas2r131-expressing cells in the peripheral and CNS using a binary genetic approach. Together, our data demonstrate that genetic transsynaptic tracing from bitter and umami receptor cells does not selectively label taste-specific neuronal circuits and reveal local taste receptor gene expression in the gustatory ganglia and the brain., Significance Statement: Previous papers described the organization of taste pathways in mice expressing a transsynaptic tracer from transgenes in bitter or sweet/umami-sensing taste receptor cells. However, reported results differ dramatically regarding the numbers of synapses crossed and the reduction of signal intensity after each transfer step. Nevertheless, all groups claimed this approach appropriate for quality-specific visualization of taste pathways. In the present study, we demonstrate that genetic transsynaptic tracing originating from umami and bitter taste receptor cells does not selectively label taste quality-specific neuronal circuits due to lateral transfer of the tracer in the taste bud and taste receptor expression in sensory ganglia and brain. Moreover, we visualized for the first time taste receptor-expressing cells in the PNS and CNS., (Copyright © 2015 the authors 0270-6474/15/359717-13$15.00/0.)

Disrupted-in-Schizophrenia 1 (DISC1) is a prominent susceptibility gene for major psychiatric disorders. Previous work indicated that DISC1 plays an important role during neuronal proliferation and differentiation in the cerebral cortex and that it affects the positioning of radial migrating pyramidal neurons. Here we show that in mice, DISC1 is necessary for the migration of the cortical interneurons generated in the medial ganglionic eminence (MGE). RT-PCR, in situ hybridizations, and immunocytochemical data revealed expression of DISC1 transcripts and protein in MGE-derived cells. To study the possible functional role of DISC1 during tangential migration, we performed in utero and ex utero electroporation to suppress DISC1 in the MGE in vivo and in vitro. Results indicate that after DISC1 knockdown, the proportion of tangentially migrating MGE neurons that reached their cortical target was strongly reduced. In addition, there were profound alterations in the morphology of DISC1-deficient neurons, which exhibited longer and less branched leading processes than control cells. These findings provide a possible link between clinical studies reporting alterations of cortical interneurons in schizophrenic patients and the current notion of schizophrenia as a neurodevelopmental disorder.

In the present study, we investigated the role of Neuroligin 2 (NL2) in synaptic transmission and network function using the mouse retina as a model circuit. We show that NL2 is preferentially located at GABAergic rather than glycinergic or glutamatergic postsynapses. The absence of NL2 from the retina resulted in a severe reduction of GABA(A) receptor clustering, and in subtle alterations of the retinal circuitry. Light processing was impaired accordingly, and retinal ganglion cells, the output neurons of the retina, showed increased basal activity and altered coding of visual information. Together, our data indicate that NL2 is essential for the functional integrity of GABAergic signaling and as a consequence, for information processing in the retina.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Rhythmic oscillations of up to 600 Hz in grouped neurons frequently occur in the brains of animals. These high-frequency oscillations can be sustained in calcium-free conditions and may be blocked by gap junction blockers, implying a key role for electrical synapses in oscillation generation. Mathematical theories have been developed to demonstrate oscillations mediated by electrical synapses without chemical modulation; however, these models have not been verified in animals. Here we report that oscillations of up to 686 Hz are induced by paired spikes of short spike intervals (SIs) in a junction-coupled network. To initiate oscillations, it was essential that the second spike was elicited during the relative refractory period. The second spike suffered from slow propagation speed and failure to transmit through a low-conductance junction. Thus, at the spike initiation site, paired spikes of short SIs triggered one transjunctional spike in the postsynaptic neuron. At distant synaptic sites, two transjunctional spikes were produced as the SI increased during spike propagation. Consequently, spike collision of these asymmetrical transjunctional spikes occurred in the interconnected network. The remaining single spike reverberated in a network serving as an oscillator center. Paired-spike-induced oscillations were modeled by computer simulation and verified electrophysiologically in a network that mediates the tail-flip escape response of crayfish.

Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of approximately 100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction.

In the leech, the command-like neuron called cell Tr2 is known to stop swimming, but the connections from cell Tr2 to the swim central pattern generator have not been identified. We used fluorescence resonance energy transfer voltage-sensitive dyes to identify three neurons that are synaptic targets of cell Tr2. We then used electrophysiological techniques to show that these connections are monosynaptic, chemical, and excitatory. Two of the novel targets, cell 256 and cell 54, terminate swimming when stimulated. These neurons are likely to mediate swim cessation caused by cell Tr2 activity, and thus play the role of intermediate control cells in the leech CNS.

Dopamine is a neuromodulator the functions of which in the regulation of complex behaviors such as mood, motivation, and attention are well known. Dopamine appears in the brain early in the embryonic period when none of those behaviors is robust, raising the possibility that dopamine may influence brain development. The effects of dopamine on specific developmental processes such as neurogenesis are not fully characterized. The neostriatum is a dopamine-rich region of the developing and mature brain. If dopamine influenced neurogenesis, the effects would likely be pronounced in the neostriatum. Therefore, we examined whether dopamine influenced neostriatal neurogenesis by influencing the cell cycle of progenitor cells in the lateral ganglionic eminence (LGE), the neuroepithelial precursor of the neostriatum. We show that dopamine arrives in the LGE via the nigrostriatal pathway early in the embryonic period and that neostriatal neurogenesis progresses in a dopamine-rich milieu. Dopamine D1-like receptor activation reduces entry of progenitor cells from the G(1)- to S-phase of the cell cycle, whereas D2-like receptor activation produces the opposite effects by promoting G(1)- to S-phase entry. D1-like effects are prominent in the ventricular zone, and D2-like effects are prominent in the subventricular zone. The overall effects of dopamine on the cell cycle are D1-like effects, most likely because of the preponderance of D1-like binding sites in the embryonic neostriatum. These data reveal a novel developmental role for dopamine and underscore the relevance of dopaminergic signaling in brain development.

The lateral ganglionic eminence (LGE) is known to give rise to striatal projection neurons as well as interneurons, which migrate in the rostral migratory stream (RMS) to populate the granule cell and glomerular layers of the olfactory bulb. Because all of these neuronal subtypes express Distalless-related (DLX) homeobox proteins during their differentiation, we set out to further characterize progenitors in the Dlx-positive domain of the LGE. Previous studies have shown that the LIM homeobox protein Islet1 (ISL1) marks the LGE subventricular zone (SVZ) and differentiating striatal projection neurons. However, ISL1 is not expressed in neurons of the developing olfactory bulb or the RMS. We show here that the dorsal-most portion of the Dlx-expressing region of the LGE SVZ lacks ISL1 cells. This dorsal domain, however, contains cells that express the ETS transcription factor Er81, which is also expressed in granule and periglomerular cells of the developing and adult olfactory bulb. Moreover, the adult SVZ and RMS contain numerous Er81-positive cells. Fate-mapping studies using Dlx5/6-cre transgenic mice demonstrate that Er81-positive cells in the granule cell and glomerular layers of the olfactory bulb derive from the Dlx-expressing SVZ region. These findings suggest that the LGE SVZ contains two distinct progenitor populations: a DLX(+);ISL1(+) population representing striatal progenitors and a DLX(+);Er81(+) population comprising olfactory bulb interneuron progenitors. In support of this, mice mutant for the homeobox genes Gsh2 and Gsh1/2, which show olfactory bulb defects, exhibit dramatically reduced numbers of Er81-positive cells in the LGE SVZ as well as in the olfactory bulb mantle.

Decision making can be a complex task involving a sequence of subdecisions. For example, we decide to pursue a goal (e.g., get something to eat), then decide how to accomplish that goal (e.g., go to a restaurant), and then make a sequence of more specific plans (e.g., which restaurant to go to, how to get there, what to order, etc.). In characterizing the effects of stimulating individual brain neurons in the isolated nervous system of the leech Hirudo medicinalis, we have found evidence that leeches also make decisions sequentially. In this study, we describe a pair of interneurons that elicited locomotory motor programs, either swimming or crawling, in isolated nerve cords. In semi-intact animals, stimulating the same neurons also produced either swimming or crawling, and which behavior was produced could be controlled experimentally by manipulating the depth of saline around the intact part of the leech. These same neurons were excited and fired strongly when swimming or crawling occurred spontaneously or in response to mechanosensory stimulation. We conclude that these brain interneurons help to decide on locomotion (i.e., they are "locomotory command-like neurons") and that the ultimate behavior is determined downstream, in a part of the decision-making hierarchy that monitors stimuli related to the depth of fluid surrounding the leech.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.

Glutamate, the major excitatory neurotransmitter in the CNS, is also an excitatory neurotransmitter in the enteric nervous system (ENS). We tested the hypothesis that excessive exposure to glutamate, or related agonists, produces neurotoxicity in enteric neurons. Prolonged stimulation of enteric ganglia by glutamate caused necrosis and apoptosis in enteric neurons. Acute and delayed cell deaths were observed. Glutamate neurotoxicity was mimicked by NMDA and blocked by the NMDA antagonist D-2-amino-5-phosphonopentanoate. Excitotoxicity was more pronounced in cultured enteric ganglia than in intact preparations of bowel, presumably because of a reduction in glutamate uptake. Glutamate-immunoreactive neurons were found in cultured myenteric ganglia, and a subset of enteric neurons expressed NMDA (NR1, NR2A/B), AMPA (GluR1, GluR2/3), and kainate (GluR5/6/7) receptor subunits. Glutamate receptors were clustered on enteric neurites. Stimulation of cultured enteric neurons by kainic acid led to the swelling of somas and the growth of varicosities ("blebs") on neurites. Blebs formed close to neurite intersections and were enriched in mitochondria, as revealed by rhodamine 123 staining. Kainic acid also produced a loss of mitochondrial membrane potential in cultured enteric neurons at sites where blebs tended to form. These observations demonstrate, for the first time, excitotoxicity in the ENS and suggest that overactivation of enteric glutamate receptors may contribute to the intestinal damage produced by anoxia, ischemia, and excitotoxins present in food.

Previous results have indicated that the bilateral cerebral interneuron CC5 mediates the pedal artery shortening that is a component of defensive withdrawal responses involving the head. Current studies suggest that CC5 contributes to aspects of at least six different behaviors: locomotion, head turning, defensive head withdrawal, local tentacular withdrawal, rhythmic feeding, and head lifting. In addition to receiving input from mechanoreceptors in the head, CC5 receives synaptic input during fictive locomotor and feeding programs. Firing of CC5 produces widespread monosynaptic or polysynaptic actions in all ganglia in the animal. CC5 excites presumptive motor neurons for the neck, and its activity can contract neck muscles. The pedal artery shortener motor neuron (PAS), a key excitatory follower cell of CC5, fires during ipsilateral head turning, head withdrawal, tentacle withdrawal, feeding, and locomotion. For all behaviors, except locomotion and biting, responses of PAS were eliminated by cutting the ipsilateral-pleural connective, which interrupts the only direct connection of CC5 to the ipsilateral PAS. The data suggest that CC5 is a multifunctional interneuron that plays different roles during different behaviors. The neuron appears to be involved in producing coordinated movements of the head, involving both somatic and visceral muscles. For some behaviors, or for certain aspects of behaviors, CC5 appears to act as an individual command-like neuron; for other behaviors, CC5 appears to act more as an element of a distributed circuit and is neither necessary nor sufficient for any aspects of the behavior.

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.

We examined the effects of denervation on clusters of nicotinic acetylcholine receptors (AChRs) on autonomic neurons in the frog heart using immunofluorescence techniques and laser scanning confocal microscopy. We showed previously that normally innervated neurons have both large, brightly stained AChR clusters and small, dim AChR clusters. A majority (80%) of the large/bright AChR clusters are located at synaptic sites, whereas the small/dim clusters are distributed widely over the cell surface. Here, we use image analysis to identify these two classes of clusters on images generated from stacks of optical sections through neuronal cell bodies and to examine the effects of denervation on their number, size, and brightness (pixel intensity). Denervation reduces the number of large/ bright AChR clusters per cell to < 10% of sham-operated values and increases the number of small/dim clusters per cell by two- to threefold. These changes occur at 4 d of denervation, the earliest time examined, and are sustained for 6 weeks. The size of large/bright AChR clusters is decreased compared with sham-operated controls, and their brightness is unchanged. The size of small/dim AChR clusters is unchanged by denervation, but their brightness is increased. Denervation results in a shift in the contribution of each AChR cluster class to the total measurable AChR pool-from one dominated by large/bright clusters to one dominated by small/dim clusters. These results show that the nerve terminals on cardiac ganglion neurons appear to exert a continual and reversible influence on the organization of the postsynaptic membrane.

The promoter region of the human dopamine beta-hydroxylase (DBH) gene was analyzed in transgenic mice to identify DNA sequences responsible for the tissue- and cell-specific expression of the gene. Transgenic mice were generated that carried the Escherichia coli lacZ gene under control of DBH promoter fragments between 0.6 and 5.8 kilobases (kb) in length. Sequences required for expression in adult and fetal noradrenergic neurons were located between 0.6 and 1.1 kb 5' to the DBH transcriptional start site. Sequences in this region and farther upstream also directed expression to dopaminergic and noncatecholaminergic brain neurons that was repressed by negative elements elsewhere in the gene. The results indicate that the neuron-specific expression of the DBH gene is mediated by positive regulatory elements but that negative elements are required to restrict expression to the proper subset of neurons.

We have studied the effect of human recombinant neurotrophin-4/5 (NT-4/5) on the survival of developing PNS neurons from embryonic mice and chickens. NT-4/5 transiently supported mouse NGF-dependent trigeminal and jugular neurons at early stages of target field innervation and mouse brain-derived neurotrophic factor (BDNF)-dependent no-dose neurons during the phase of naturally occurring cell death. NT-4/5 was as potent as BDNF in supporting the survival of these neuronal populations. Surprisingly, NT-4/5 was 3 orders of magnitude less potent than BDNF as a survival factor for early chick dorsomedial trigeminal sensory neurons and did not support the survival of chick BDNF-dependent trigeminal mesencephalic or ventrolateral trigeminal sensory neurons at any of the developmental stages tested. Thus, NT-4/5 is a survival factor for certain embryonic mouse cranial sensory neurons. It is the first species-specific neurotrophin to be identified and it can discriminate at high concentrations between different BDNF-responsive chick neurons.

Global observations of neuronal response in the Aplysia abdominal ganglion were made during habituation of the gill withdrawal reflex using voltage-sensitive dye recording. This technique makes it possible to measure the spike activity of 30-50% of the 1000 neurons present in the ganglion. Our experiments address the issue of how habituation is expressed in the activity of the population of neurons responding to siphon stimulation. Several classes of neurons exhibited characteristically distinct responses to the stimuli and to habituation training. One class of neurons (group I) responded to the onset and offset of the sensory stimulus although they are probably not primary sensory neurons. They habituate only partially when the behavioral reflex has already habituated completely. Two other classes (groups II and III) both have sustained responses to the touch, but habituate differently. Members of group III habituate completely while those in group II habituate only partially. Another class of neurons are inhibited by the stimulus (group IV). They become less inhibited after habituation. The response of both group I and group IV are new classes of response that have not been previously reported.

Simultaneous endoscopic and electrophysiological recordings were used to observe the behavior of the gastric mill complex while recording the motor output of the stomatogastric ganglion (STG) in intact crabs. In the crab STG, many pattern-generating neurons are able to fire in several distinct rhythmic motor patterns. Specifically, many neurons can switch between firing in time with the rapid pyloric rhythm to firing in time with the slower gastric mill rhythm (Weimann et al., 1991). We now correlate behaviorally relevant movements of the gastric mill with some of the modifications of neuronal firing patterns previously characterized using in vitro STG preparations. The intracellular and extracellular recordings from the intact crab are largely indistinguishable from those obtained from in vitro preparations. For the first time, we describe the movements that result as neurons switch their activity patterns associated with activation of the gastric mill rhythm. Extracellular stimulation and intracellular depolarization of individual motor neurons is used to determine the relationship between frequency of firing and movement in behaving animals.

Retzius (Rz) neurons in the midbody ganglia of medicinal leeches responded to ACh, applied to their somata, in a manner that depended upon the neuron's segmental location: Rz neurons in ganglia from midbody segments 5 and 6 [Rz(5,6)] hyperpolarized, whereas Rz neurons from all other segments [Rz(X)] depolarized. Midbody segments 5 and 6 are notable because they contain the male and female reproductive organs. Both types of Rz neurons responded to ACh in a complex way, but the initial phase of each response appeared to be nicotinic because nicotinic agonists evoked the responses and nicotinic antagonists blocked them. The reversal potentials of the responses and the effects of changing the internal and external Cl- concentration indicated that the hyperpolarizing response of Rz(5,6) neurons depended upon Cl- whereas the depolarizing response of Rz(X) neurons did not. The segmentally characteristic responses of Rz neurons arose during embryonic development. Removing the reproductive ducts [the peripheral targets of Rz(5,6)] early in embryogenesis caused the Rz(5,6) neurons to depolarize in response to ACh rather than to hyperpolarize. This result indicates that development of the characteristic response of Rz neurons to ACh is strongly influenced by interactions between the neurons and their appropriate target tissues.

The aim of these experiments was to analyze how depolarization influences neurite outgrowth in leech neurons and what role the substrate and Ca2+ play in this response. Neurons in culture were exposed to 60 mM extracellular K+ for 30 min, which induced retraction of a subset of neurites growing on extracellular matrix substrate (ECM), a response comparable to that observed after electrical stimulation (Grumbacher-Reinert and Nicholls, 1992). After normal medium had been restored, the neurites continued to retract for about 1 hr to approximately 80% of the total starting neurite length. Retraction was reversible and regrowth began after the cells had been in normal medium for about 3 hr. Similar depolarization-induced neurite retraction was observed in both Retzius and anterior pagoda cells. Retraction was inhibited by raised extracellular Mg2+, suggesting a mechanism dependent on calcium. The effect of high K+ on neurite outgrowth was also influenced by the substrate on which the cells were plated. Cells plated on concanavalin A (ConA) did not retract but continued to extend processes during exposure to high K+. To understand the different behavior of cells grown on ECM and ConA, the morphology of growth cones was analyzed by scanning electron microscopy. The growth cones of cells grown on ECM and exposed to high K+ revealed retraction of lamellipodial and filopodial structures. On ConA, however, no differences were observed between growth cones of cells exposed to high K+ and those of control cells. These results demonstrate the importance of substrate molecules in the responses of growth cones to depolarization and therefore in the differentiation of neurons.

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.

Tyrosine hydroxylase (TH) is transiently detectable in cells distributed throughout cranial sensory ganglia during early stages of gangliogenesis [embryonic day (E) 10.5-15.5]. Although TH cells appear in embryonic ganglia of both neural crest and placode origin, mature cranial sensory neurons that express catecholaminergic properties are restricted to placode derivatives. The mechanism(s) underlying the loss of TH expression in crest-derived sensory ganglia is unknown, and the present study was undertaken to define the temporal regulation of this phenotype. Our data indicate that transient TH cells belong to a large subset of primary sensory neurons that exhibit the capability to express TH throughout development. The lack of TH expression after E15.5 appears to be due to modulation of this catecholaminergic potential. The phenotype reappears, however, when E16.5 and older ganglia are dissociated in culture into single cells, suggesting that factors associated with cell aggregation modulate TH expression. In support of this hypothesis, sensory neurons grown at high cell density exhibit lower levels of TH expression than low-density cultures. The decrease in TH levels seen at high density was associated with changes in sensory neuron morphology that are characteristic of ganglion cell maturation in vivo; therefore, modulation of TH expression may be only one facet of a more general program of sensory neuron differentiation associated with cell aggregation in developing ganglia. In contrast to the effects of cell aggregation, treatment with NGF increased the proportion of TH cells in dissociate cultures of E14.5 and E16.5 cranial sensory ganglia. Our findings indicate that sensory transmitter phenotype may be modulated by multiple factors during gangliogenesis, including cellular interactions intrinsic to the developing ganglionic microenvironment.

We identified candidate neurons in the cerebral ganglion that regulate feeding responses mediated by the buccal ganglion. Backfilling the cerebral-buccal connectives revealed that each cerebral hemi-ganglion contains approximately 20 neurons that project axons to the buccal ganglion. Three M-cluster neurons (CBI-1, CBI-2, CBI-3) and one E-cluster neuron (CBI-4) were identified as cerebral-to-buccal interneurons (CBIs) based on position, morphology, synaptic connections, and ability to drive buccal motor programs (BMPs). CBI-1 responds to touch of the tentacles, lips, and buccal mass. It receives monosynaptic EPSPs from interganglionic, cerebral-to-buccal mechanoafferent (ICBM) neurons and monosynaptically excites buccal cells, some of which are also excited by the ICBMs. Tonic firing of CBI-1 usually evokes a single cycle of BMP activity. CBI-1 phase-shifts the rhythmic BMP driven by firing a dopaminergic neuron in the buccal ganglion. CBI-1 itself exhibits dopamine-like histofluorescence following formaldehyde-glutaraldehyde fixation. CBI-2 is excited by food stimuli applied to the lips. Constant-current intracellular stimulation of CBI-2 produces phasic firing of the cell that reliably evokes a rhythmic BMP that incorporates buccal and cerebral motor neurons, putative pattern-generating and pattern-initiating neurons, and neuromodulatory cells (metacerebral cells). CBI-4 also evokes a rhythmic BMP, but the details of its actions and synaptic effects differ from that of CBI-2. CBI-3 does not evoke a BMP, even though it is excited by food stimuli applied to the lips, and it makes monosynaptic connections (both excitatory and inhibitory) to many follower cells of the other CBIs. Firing of CBI-3 phase-delays the BMP driven by CBI-2. Since its activity is incorporated into BMPs and it provides direct inputs to elements of the feeding circuitry, it may play a role in pattern generation. The distinctive features of the CBIs suggest that the consummatory phase of feeding may be controlled by a population of interneurons that subserve different roles.

The majority of the neurons in the adult nervous system of Manduca sexta are born postembryonically, during larval life. Stereotypic arrays of identifiable neuroblasts generate their clonal families or lineages commencing at the end of the second larval instar through pupal day 2, when the neuroblasts die (Booker and Truman, 1987a). We have used immunohistochemical techniques to follow the neurochemical differentiation of GABA and a peptide similar to molluscan small cardioactive peptide B (SCPB) in identified lineages. We report here the distribution and developmental acquisition of the expression of these putative transmitters. There are 24 postembryonic lineages in the second thoracic ganglion of the larvae (Booker and Truman, 1987a). Immunoreactivity against GABA and SCPB is seen only in a subset of these 24 clonal families. GABA immunoreactivity is confined to the progeny of the E, K, M, N, T, and X neuroblasts and is expressed by most or all of the neurons in these lineages. The SCPB-like immunoreactivity is found in a subset of the neurons in only two clonal groups, the K and M groups, and is colocalized with GABA. These results show that, though heterogeneity in transmitter type exists (GABA, GABA/SCPB), members of a given lineage share at least some features (GABA) in common. The onset of transmitter expression was followed in detail for the K- and M-lineage neurons. During the larval stages, the postembryonic lineage cells are developmentally arrested in a partially differentiated state (Booker and Truman, 1987a) and do not express transmitter immunoreactivity at this time. Their maturation resumes with the onset of metamorphosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of GABA is restricted to the progeny of only six of the 24 identified postembryonic lineages in the thoracic ganglia of the tobacco hornworm, Manduca sexta (Witten and Truman, 1991). It is colocalized with a peptide similar to molluscan small cardioactive peptide B (SCPB) in some of the neurons in two of the six lineages. By combining chemical ablation of the neuroblasts at specific larval stages with birth dating of the progeny, we tested whether the expression of GABA and the SCPB-like peptide was determined strictly by cell lineage or involved cellular interactions among the members of individual clonal groups. Chemical ablation of the six specific neuroblasts that produced the GABA-positive neurons (E, K, M, N, T, and X) or of the two that produced the GABA + SCPB-like-immunoreactive neurons (K, M) prior to the generation of their lineages resulted in the loss of these immunoreactivities. These results suggest that regulation between lineages did not occur. Ablation of the K and M neuroblasts after they had produced a small portion of their lineages had no effect on the expression of GABA, but did affect the pattern of the SCPB-like immunoreactivity. Combining birth-dating techniques with transmitter immunocytochemistry revealed that it was the position in the birth order and not interactions among the clonally related neurons that influenced the peptidergic phenotype. These results suggest that cell lineage is involved in establishing the GABAergic phenotype and that both cell lineage and birth order influence the determination of the peptidergic phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial cells are a large component of the developing nervous system, appearing before the onset of axon outgrowth in a variety of developing systems. Their time of appearance and their location in conjunction with developing axon pathways may allow them to define the position of axon pathways. Specific glial cells may be utilized as guideposts by growing axons, allowing them to recognize the appropriate pathway, or conversely, glial cells may inhibit axons from growing along an inappropriate pathway. The 7F7 monoclonal antibody labels a subset of glial cells in grasshopper embryos that may play a role in defining the location of selected axonal pathways. This antibody recognizes the REGA-1 molecule, a cell-surface antigen with a molecular weight of 60 kDa, which is regionally expressed on developing glial cells. REGA-1 is expressed around the edges of clusters of glial cells and on lamellae extending from glial cells to line the edges of some axonal pathways. REGA-1 expression is first seen in the neuroblast sheet, surrounding neuroblast 4-1. Slightly later in development, 2 glial cells extend processes that express REGA-1 and demarcate the caudal edge of the anterior commissure. As the animal matures, cell processes expressing REGA-1 line the edges of the longitudinal connective, then expand to surround the central neuropil of the segmental ganglia. REGA-1 expression is also seen in conjunction with axons leaving the segmental ganglia via the segmental nerves and the intersegmental connectives. REGA-1 expression is limited to a subset of glial cells; some known glial cells such as the segment boundary cell do not express REGA-1. Glial cell processes expressing REGA-1 are seen only in association with axons, which suggests that these processes may act as borders or guard rails confining axons to the appropriate regions of the developing CNS. Axons navigating a path through the CNS may be prohibited from growing into inappropriate regions based on their inability to cross the boundaries established by glial cells expressing REGA-1.

Antibodies to small cardioactive peptide (SCP) label a segmentally iterated subset of cells in the leech nerve cord, including the previously identified alternating SCP (AS) neurons. Unlike the majority of leech neurons, these cells are asymmetrically distributed in the adult nerve cord. Moreover, each AS neuron shows a strong tendency to lie on alternate right and left sides in successive ganglia. Previous work has shown that these unpaired neurons arise from bilaterally paired embryonic homologues, only 1 of which takes on the mature immunoreactive phenotype. The 2 AS homologues within a ganglion compete for this fate, in that either the right or the left homologue will become a mature AS neuron with a high degree of reliability if its contralateral homologue is ablated during embryogenesis. In this paper, we demonstrate the existence of interactions between neurons in adjacent ganglia that could account for the alternation of sides observed during normal development. The unilateral ablation of a single AS homologue neuron forced its contralateral homologue to take on the mature AS fate, and this consistently biased the side of AS development in adjacent, unlesioned ganglia both anterior and posterior to the lesion. One of the AS neurons, the caudal alternating SCP (CAS) cell, was injected with Lucifer yellow in adult nerve cords and was shown to have a large primary axon that extends into more anterior ganglia, as well as other, finer axons that are variable in number and arrangement. If the interganglionic interaction of AS neuron homologues is mediated by their primary axons, signals of developmental import must be transmitted both anterogradely and retrogradely along the axon's length. The present results indicate that the development of individual AS neurons is influenced by homologous cells located in the same and neighboring ganglia and suggest that the final, multisegmental patterning of the AS neuron distribution is not predetermined, but rather, arises as an emergent property of the cell interactions that occur during nervous system differentiation.

Cyclic nucleotides have been implicated in many long-lasting transmitter-induced effects on membrane conductance. One previously observed effect of cAMP on molluscan neurons is to induce a slow inward current, which has been further evaluated here in depolarized anterior and medial cells of the pleural ganglion of Aplysia californica in order to understand better its underlying ionic mechanisms and its sensitivity to a variety of pharmacological agents. This current, which appears to be the only cAMP-induced current seen in the anterior cells, was shown to invert at about +25 mV, that is, approximately 25-30 mV inferior to ENa. This reversal potential was lowered by about 15-16 mV when half of the extracellular Na was replaced by either mannitol or N-methyl-D-glucamine, whereas it was unaffected by changes in extracellular Cl, Ca, or Mg. The response persisted in seawater in which the Na had been totally replaced by K, and its reversal potential shifted towards more negative values. These data are consistent with the hypothesis that both Na and K ions permeate the channel, with a Na/K permeability ratio of approximately 2. Ca ions do not appear to permeate the channel, but they do have a marked inhibitory effect on the response amplitude, as do Mg ions when Ca is not present. Caffeine, intracellular acidification, and phosphodiesterase inhibitors enhance and prolong the response without changing its reversal potential. Previous studies have shown that both caffeine and intracellular acidification inhibit phosphodiesterase, and it is assumed that the common effect of these manipulations on the cAMP-induced inward current is mediated, at least partially, by the inhibition of that enzyme. In the medial cells of the pleural ganglion, this slow inward current is present, but is dominated in the depolarized cell by a cAMP-induced diminution in a Ca-activated K conductance (Kehoe, 1985b). This K conductance and, consequently, the noninverting, cAMP-induced inward current that reflects its diminution, were shown to disappear in Ca-free solutions, in the presence of isobutyl-1-methylxanthine (IBMX) or caffeine, and upon acidification of the cytoplasm. When this cAMP-sensitive K conductance is blocked, the presence of the inverting cAMP-induced cationic current is unmasked. The cAMP-induced cationic current is shown to have many properties in common with cyclic nucleotide-induced currents described in photoreceptors, olfactory receptor cilia, and cardiac myocytes, all of which have been shown to be outwardly rectifying cationic currents that are inhibited by divalent cations and do not involve the activation of a cAMP-dependent kinase.

An analysis has been made of intrinsic mechanisms influencing growth patterns of 2 identified leech interneurons in tissue culture. These small cells (known as DL and VL) display unusual arborizations in the ganglion. The distinctive branching patterns resemble the letter "T" for VL and the letter "Y" for DL. DL and VL cells contain serotonin and can be identified in situ with Neutral red. 1. DL and VL cells were isolated and cultured on 2 homogeneous substrates, concanavalin A (Con A) and leech extracellular matrix extract (ECM). Individual DL and VL cells in culture retained their ability to fire in spontaneous, rhythmical bursts. The synaptic connections formed in culture were specific and differed from those made by other serotonergic neurons. 2. On both Con A and ECM the neurons sprouted to reproduce their characteristic patterns. Every DL or VL cell that grew processes on ECM developed the appropriate Y- or T-shaped pattern. On Con A, most but not all of the cells showed specific Y or T patterns. The probability of sprouting was higher on Con A than on ECM. 3. Disruption of microtubular assemblies in freshly isolated DL and VL cells by treatment with nocodazole did not affect the later outgrowth of specific patterns in culture. 4. These results provide evidence that adult DL and VL leech neurons retain intrinsic information for determining a variety of their properties in culture: In particular, they reproduce type-specific neuritic patterns in the absence of extrinsic guidance cues. Moreover, the intrinsic pattern determining mechanism is not lost after the destruction of microtubular arrays within the cytoskeleton.

Bath application of muscarinic agonists induced rhythmic motor activity in an in vitro preparation of the thoracic nervous system of the crayfish, Procambarus clarkii. In 70% of the cases, the rhythm was organized into 1 of the 2 normal patterns: "backward" walking or "forward" walking. In the rest (30%), the ganglion produced either a series of bursts of impulses or no rhythm at all, just an increase in the tonic activity. When it was isolated from all ascending and descending afferents, the fourth thoracic ganglion was still able to generate rhythmic motor output during bath application of muscarinic agonists. In certain motor neurons, muscarinic agonists induced plateau potentials. Under these conditions, some of these motor neurons were able to change the period of the motor pattern, which might suggest that these motor neurons were part of the central pattern generator (CPG) for locomotion. In the presence of 5 x 10(-6)M TTX, the membrane potential of these motor neurons continued to oscillate with organized rhythmic membrane potential oscillations into 1 of the 2 patterns. Under these conditions, current injection into certain motor neurons demonstrated that they continued to affect the CPG. Two classes of walking leg interneurons have been found. First, there are those with a sustained membrane potential: injection of a steady depolarizing current into some of these interneurons induced rhythmic activity in all thoracic motor nerves, even in the absence of any pharmacological activation. Second, there are those with an oscillating membrane potential: these seemed to enable silent motor neurons to be involved in an ongoing rhythm.

Many neurons are now known to undergo dramatic morphological or biochemical changes long after they have completed differentiation and maturation. The ability of fully mature neurons to alter their transmitter phenotype has been amply demonstrated in culture, but direct in vivo data on single neurons have been difficult to obtain. Here we show that a set of 4 individually identified neurosecretory neurons in the moth Manduca sexta, previously demonstrated to contain the peptide hormone bursicon, also stain with a monoclonal antibody directed against 2 insect cardioacceleratory peptides (CAPs). These lateral cells exhibit CAP-like immunoreactivity in larvae but not in pupae or adults, in contrast to other CAP-containing neurons which are strongly immunoreactive in all postembryonic stages. Biochemical analyses using high-pressure liquid chromatography confirm that the lateral neurons in larvae contain CAP2, one of the CAPs. CAP measurements of cell clusters containing these cells indicate high levels only in caterpillars. When the same neurosecretory cells are individually dissected and assayed for CAP bioactivity, high CAP levels are again found in larvae, whereas the same neurons in pupae show no such CAP bioactivity. Simultaneous determinations of both bursicon and CAP levels in single lateral cells indicate that these cells express high levels of CAP activity and low amounts of bursicon in larvae yet are solely bursicon-containing in pupae and adults. Thus, by demonstrating that these cells alter their secretory profile in vivo during metamorphosis, our results confirm the notion that functionally mature neurons are capable of altering their transmitter expression after the completion of embryonic development.

The RB cells, an identified cluster of serotonergic neurons in the abdominal ganglion of Aplysia californica, are excited by ACh. We have found that serotonin (5-HT) enhances the ACh responses recorded from the RB cells. The locus and specificity of the serotonergic interaction with the ACh response were analyzed using the conventional two-electrode voltage-clamp technique. We compared the current responses evoked in RB cells by constant-current iontophoretic pulses of ACh in control solutions and in solutions containing various concentrations of 5-HT. Serotonin consistently enhances the ACh evoked response in a dose-dependent manner while simultaneously directly activating the RB cells by turning on a small steady-state inward current. The dose-response characteristics for the 5-HT-mediated steady-state current differ, however, from those of the 5-HT-induced ACh response facilitation. The ACh response enhancement appears to be an effect of 5-HT upon RB cell ACh receptors since the enhancement is present under conditions that eliminate presynaptic input, and 5-HT is still capable of enhancing the iontophoretically evoked response when the AChE-resistant agonist carbachol is substituted for ACh. We tested other neurons in the Aplysia CNS exhibiting ACh evoked responses qualitatively similar to the RB cell ACh responses; 5-HT does not enhance the iontophoretically evoked ACh responses of these cells. Also, although the RB cells are responsive to dopamine and histamine, these putative neurotransmitters do not enhance the ACh response.

Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplysia. Using SDS-PAGE, we have compared the proteins which comprise the cytoskeletons of cell bodies and axons. Separate populations of cell bodies and axons were collected and the proteins stained by the Coomassie brilliant blue method. Individual identified cell bodies, with long segments of their axons attached, were isolated, and the proteins were labeled with the [125I]Bolton-Hunter reagent. The proteins which are stably associated with the cytoskeleton were obtained by extracting the neuronal material in a physiological buffer containing Triton X-100. As a correlative measure to the biochemical analyses, electron microscopy was performed on the cell body and axonal fractions. Our results demonstrate that the composition and biochemical properties of the cytoskeletal proteins in the neuron cell bodies differ from those associated with axons. Specifically, the amount of neurofilament proteins, designated NF60 and NF65 , is 5 times more abundant in the axon than in the cell body. The relative amounts of actin and tubulin are comparable in these two regions of the neuron. In addition, the ratio of NF60 and NF65 is different in the cell body and axon. The cell bodies contain proportionally more NF60 than the axons. However, the physical properties of the tubulin in the cell body, as measured by relative solubility, differ from that of the axon. The substantial differences between the composition of the cytoskeleton of the cell bodies and axons of Aplysia suggests that at least two distinct cytoskeletal networks exist in these neurons, one specific for the cell body and the other specific for the axon.

The giant neurons R3-14 in the parietovisceral ganglion of Aplysia, originally proposed to be a homogeneous group of neuroendocrine cells, are likely candidates for a multifunctional and multiple messenger status. The studies reported here suggest that individual R3-14 giant neurons not only innervate specific target tissues but appear to operate more autonomously than previously thought. Identified members of the group were traced into peripheral tissues by electrophysiological, autoradiographic, and intracellular cobalt staining techniques. Five neurons (numbered R6, R7, R8, R11, and R14) were identified on the basis of their unique patterns of axonal projections. R6 innervates the ganglionic artery and pericardial area; R7 and R8, the heart; R11, the kidney; and R14, a large number of vascular tissues. The wide distribution of R3-14 terminals innervating a variety of vascular tissues indicates that several general and local aspects of circulatory physiology are likely to be regulated by these neurons. R3-14 contain the free amino acid glycine, a putative neuromodulator that potentiates cardiac and vascular smooth muscle contraction and several small peptides of unknown, but probably neurohormonal, function. A model is proposed in which R3-14 release glycine to modulate local (e.g., hemolymph pressure and distribution) cardiovascular performance and, indirectly, metabolic homeostasis as well.

Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells. Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well. Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture. The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated. Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5-9 S range. Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour. The transit time for newly synthesized receptor to reach the cell surface appears to be 2-3 hr. These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture. The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr. Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

To survey the distribution of neuronal receptors for NGF, sections of the rat brain, spinal cord, and peripheral ganglia were incubated in vitro with radioiodinated NGF and examined by autoradiography. NGF binds selectively with high affinity to most sympathetic neurons and many primary sensory neurons together with their intraspinal or intramedullary axons. In autoradiographs of the brain, labeled neuronal perikarya are seen in the basal forebrain, the caudate-putamen, the medulla oblongata, the ventral cochlear nucleus, and the dorsal nucleus of the lateral lemniscus. The distribution of neurons binding NGF resembles the distribution of cholinergic neurons in the forebrain, but these 2 systems overlap very little in the brain stem. In extracts of the brain or spinal cord enriched for plasma membranes, avid binding sites are regionally manifest with properties similar to those of fetal peripheral neurons. The localization of neurons expressing the high-affinity receptor for NGF defies simple correlation with neurotransmitter function or embryogenesis.

Heart interneurons (HN cells) in isolated ganglia of the medicinal leech were voltage-clamped with single microelectrodes. Hyperpolarizing voltage steps elicited a slow inward current (Ih), which underlies the characteristic depolarizing response of HN cells to injection of prolonged hyperpolarizing current pulses (Arbas and Calabrese, 1987a). The conductance underlying Ih begins to activate near -mV and is fully activated between -70 and -80 mV. The activation kinetics of Ih are slow and voltage dependent. The activation time constant (tau h) ranges from approximately 2 sec at -60 mV to near 700 msec at -100 mV. Ih persists in low Ca2+ (0.1 mM), 5 mM Mn2+ saline and exhibits a reversal potential of -21 +/- 5 mV. The reversal potential is shifted by altering [Na+]o or [K+]o but is unaffected by changes in [Cl-]o. Ih is blocked by extracellular Cs+ (1-5 mM) but not Ba2+ (5 mM) or TEA (25 mM). Low concentrations of Cs+ (100-200 microM) cause a partial block that exhibits strong voltage dependence. Temperature changes were also shown to affect Ih. Both the rate of activation and the steady-state amplitude of Ih are enhanced by temperature increases. HN cells are interconnected by inhibitory chemical synapses, and their normal electrical activity consists of bursts of action potentials separated by periods of inhibition. During the inhibitory phase of rhythmic bursting activity, HN cells hyperpolarize to a voltage range where Ih is activated. Block of Ih with extracellular Cs+ (4 mM) disrupted the normal bursting activity of HN cells. These results are consistent with the hypothesis that Ih contributes to escape from inhibitory inputs during normal bursting activity.

Serotonin (5-HT) has previously been shown to evoke an increase in the duration of the Ca2+-dependent spike of molluscan neurons by decreasing the S current (Klein et al., 1982), a K+ current controlled by cAMP. However, in a group of identified ventral neurons of the snail Helix aspersa in which 5-HT (1-10 microM) also prolonged the duration of the Ca2+-dependent action potential, no 5-HT-induced depression of S current or of any other outward current was observed. Instead, 5-HT was found to evoke the prolongation of the somatic spike by inducing an increase in Ca2+ membrane conductance. This 5-HT-induced increase of Ca2+-current was mimicked neither by the intracellular injection of cAMP nor by the extracellular application of forskolin (20 microM). In contrast, it was mimicked by the intracellular injection of cGMP and by the extracellular application of 100 nM zaprinast, a cGMP-phosphodiesterase inhibitor. The extracellular application of phorbol ester TPA (100 nM), an activator of protein kinase C, was also found to increase the Ca2+ current in the identified snail ventral neurons, but this enhancing effect had a different time course from that induced by 5-HT. These results indicate that there is a second mechanism for prolonging the Ca2+ spike of molluscan neurons, consisting of an increase in Ca2+ current, in which cGMP may play a role as second messenger.

In identified Helisoma neurons, intracellular calcium can regulate neurite elongation and growth cone motility. Neurotransmitters such as 5-HT suppress both neurite elongation and the filopodial and lamellipodial movements of growth cones by causing increases in intracellular calcium (Haydon et al., 1984; Cohan et al., 1987; Mattson and Kater, 1987). Since an additional second messenger, cyclic AMP (cAMP), is known to mediate many physiological effects of neurotransmitters, we tested (1) the possible involvement of cAMP in the regulation of neurite outgrowth from Helisoma buccal neurons and (2) calcium-cAMP interrelationships in the regulation of outgrowth. The cAMP-elevating agents forskolin (5 x 10(-6)-10(-4) M) and dibutyryl cAMP (dbcAMP; 5 x 10(-3)-10(-2) M) suppressed neurite elongation and growth cone movements in identified neurons B19 (5-HT sensitive) and B5 (5-HT insensitive); the suppression was reversible. Exposure of these particular identified neurons to the calcium channel blocker La3+ (10(-5) M) or a culture medium with reduced calcium prevented and reversed the suppressive effects of forskolin and dbcAMP. In order to determine if the results on neurons B5 and B19 were representative of all neurons or only a subset, we examined a larger population of neurons. Calcium ionophore A23187 suppressed outgrowth from all neurons in mass dissociate cultures of buccal neurons, while forskolin or dbcAMP plus IBMX suppressed outgrowth from only one-half of buccal neurons. Finally, we found that 2 subpopulations exist among the neurons whose outgrowth is suppressed by cAMP: One subpopulation requires calcium influx for cAMP to act, while the other does not. Thus, even within the relatively small population of neuronal types comprising the buccal ganglion of Helisoma, second messengers within different neurons can act and interact in different ways to regulate outgrowth.

We previously isolated and characterized a cDNA clone specifically expressed in neurons R3 to R8 and R14 of the Aplysia abdominal ganglion (Nambu, J.R., R. Taussig, A.C. Mahon, and R.H. Scheller (1983) Cell 35: 47-56). The cDNA nucleotide sequence and the inferred protein amino acid sequence suggest that this gene encodes the precursor for neuroactive peptides used by these cells. Peptides corresponding to three regions of the precursor were synthesized, coupled to a protein carrier, and used to generate antibodies. These antibodies stain a set of cell bodies, R3 to R14, and their processes in the abdominal ganglion; no other cells in the nervous system or the periphery are immunoreactive. R3 to R14 send numerous fine immunoreactive processes into the vascularized sheath that surrounds the ganglion. Each of these cells also has a large axon which exits the ganglion via the branchial nerve and terminates on the heart. In addition, R14 is anatomically distinct from R3 to R13 in that it sends additional immunoreactive processes to the vasculature near the ganglion. Immunoreactive processes and varicosities were observed on the efferent vein of the gill, the abdominal ganglion artery, and the anterior aorta. These data are consistent with previous studies suggesting that one or more neuropeptides released from R3 to R14 function as modulators of cardiovascular physiology.

Following brief stimulation of an afferent pathway, the bag cell neurons of Aplysia undergo a dramatic change in excitability, resulting in a prolonged discharge of spontaneous action potentials. During the discharge, the action potentials of the bag cell neurons become enhanced in height and width. The afterdischarge triggers release of neuroactive peptides that initiate egg-laying behavior in this animal. Evidence suggests that changes in excitability of the bag cell neurons may be mediated by activation of protein kinase C (PKC) and cAMP-dependent protein kinase (cAMP-PK). PKC activators, such as the phorbol ester TPA (12-O-tetradecanoyl-13-phorbol acetate), enhance the amplitude of action potentials in isolated bag cell neurons in cell culture. These agents act by unmasking a previously covert species of voltage-dependent calcium channel resulting in an increase in calcium current. In the accompanying paper (Conn et al., 1989), we showed that H-7, a protein kinase inhibitor, inhibits the effect of TPA, and is a selective inhibitor of PKC relative to cAMP-PK in these cells. We now report that another PKC inhibitor, sphinganine, also inhibits the effect of TPA on action potential height and calcium current in cultured bag cell neurons, and that N-acetylsphinganine, an inactive sphinganine analog, fails to inhibit the effects of PKC activators. Although both H-7 and sphinganine prevent the effects of TPA when added prior to TPA addition, neither compound reverses the effects of TPA when added after the action potentials have already become enhanced by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Brief electrical or hormonal stimulation of the bag cell neurons of Aplysia triggers a long-lasting discharge during which alpha bag cell peptide (alpha-BCP) and other neuropeptides are released from the cells. We have carried out experiments, using both intact abdominal ganglia and isolated neurons, demonstrating that alpha-BCP acts directly on the bag cell neurons to influence cAMP levels and voltage-dependent potassium currents. Exposure of the bag cell neurons within intact ganglia to alpha-BCP, at concentrations greater than 1 nM, inhibited an ongoing discharge. alpha-BCP also significantly reduced both basal and forskolin-stimulated levels of cAMP in bag cell clusters. The inhibition of the discharge by alpha-BCP could be prevented and reversed by pharmacological elevation of intracellular cAMP levels. Immunohistochemical staining of neurons maintained in cell culture showed that all isolated bag cell neurons exhibit immunoreactivity with antisera against alpha-BCP. Application of the adenylate cyclase activator forskolin to such isolated cells, in the presence of a phosphodiesterase inhibitor, attenuates the amplitude of the delayed voltage-dependent outward currents measured in voltage-clamp experiments. Pretreatment of the cells with alpha-BCP significantly reduced the ability of forskolin to attenuate these currents, demonstrating that alpha-BCP acts directly at autoreceptors on bag cell neurons. Experiments with the isolated cells showed that a second autoreceptor-mediated effect of alpha-BCP was the enhancement of an inwardly rectifying potassium current that was activated at potentials more negative than -40 mV. The reversal potential and conductance of the current induced by alpha-BCP were dependent on the external K+ concentration. This response to alpha-BCP could be blocked by rubidium, cesium, and barium ions. Our data demonstrate that alpha-BCP can exert inhibitory biochemical and electrophysiological actions on the bag cell neurons that release it and suggest that autoreceptors for alpha-BCP play an important role in the termination of a discharge in the bag cell neurons.

An examination of the cellular properties and synaptic outputs of mechanoafferent neurons found on the ventrocaudal surface of the cerebral ganglion of Aplysia indicated that the cerebral mechanoafferent (CM) neurons are a heterogeneous population of cells. Based on changes in action potential duration in response to bath applications of 5-HT in the presence of TEA, CM neurons could be divided into 2 broad classes: mechanoafferents whose spikes broaden in response to 5-HT (CM-SB neurons) and mechanoafferents whose spikes narrow in response to 5-HT (CM-SN neurons). Morphological and electrophysiological studies of the CM-SN neurons indicated that they were comprised of previously identified interganglionic cerebral-buccal mechanoafferent (ICBM) neurons and a novel set of sensory neurons that send an axon into the LLAB cerebral nerve and have perioral zone receptive fields that are similar to those of ICBM neurons. Changes in spike width due to 5-HT were correlated with changes in synaptic output as indicated by the magnitudes of EPSPs evoked in postsynaptic neurons. Electrical stimulation of cerebral nerves and connectives also produced spike narrowing or broadening, and the sign of the effect was a function of the parameters of stimulation. Both heterosynaptic facilitation and heterosynaptic depression of EPSPs evoked in follower cells could be demonstrated. A variety of putative neuromodulators other than 5-HT were also found to affect the duration of action potentials in both classes of CM neurons. FMRFamide had effects opposite to that of 5-HT. SCPB and a recently characterized Aplysia neuropeptide, buccalin, broadened the spikes of both CM classes. Another neuropeptide, myomodulin, decreased the duration of CM-SB neuron spikes but had no effect on CM-SN spikes. Since the CM neurons appear to mediate a variety of competing behaviors, including feeding, locomotion, and defensive withdrawal, the various neuromodulator actions may contribute to the mechanisms whereby behaviors are selected and modified.

A correlated biochemical and histochemical study was undertaken to identify and quantify the presence of different biogenic amines and a substance P-like peptide within the parasympathetic cardiac ganglion of the mudpuppy (Necturus maculosus). Tissue extracts of the cardiac septum containing the parasympathetic cardiac ganglia from control animals were found, by high-pressure liquid chromatography, to contain significant amounts of norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-HT. To allow neural elements of extraganglionic origin to degenerate, ganglia were explanted and maintained in organ culture for 8 d. Extracts from these explanted preparations had no detectable level of E, and NE was reduced, whereas DA and 5-HT levels were similar to those of control preparations. The results indicated that some of the neurons intrinsic to the cardiac septum contain DA and 5-HT and that most (greater than 70%) of the E and NE found in this tissue is of extrinsic origin. Histochemistry of control and explanted preparations showed 5-HT-immunoreactive and catecholamine-containing intrinsic neurons. A substance P-like peptide was identified by radioimmune assay in septal extracts. The peptide content diminished by one-third to one-fifth in preparations maintained in organ culture for 8-14 d, suggesting that a significant amount of the substance P-like peptide is derived from extraganglionic sources. Immunocytochemical studies demonstrated the presence of numerous long substance P-immunoreactive fibers coursing across the septum, branching over cardiac muscle fibers, and forming pericellular networks around individual parasympathetic ganglion cells and clusters of ganglion cells. In addition, numerous small intrinsic neurons exhibited immunoreactivity for substance P. Comparison of the substance P-staining patterns in control and explanted ganglia suggests that the majority of the long substance P-immunoreactive fibers innervating the mudpuppy cardiac ganglion cells are not parasympathetic preganglionic fibers. Rather, it is hypothesized that these fibers are processes of primary sensory fibers. The present observations indicate that the mudpuppy cardiac ganglion exhibits a complex organization similar to that of mammalian sympathetic and enteric ganglia.

The posteromedial, serotonin-containing (PMS) neurons are found in the ventral aspect of certain anterior segmental ganglia of adult leeches. With one exception, these cells are unpaired in all the ganglia where they are found. During early embryogenesis in Hirudo medicinalis, however, a bilateral pair of PMS neurons appears and differentiates in each of the 21 segmental ganglia (SG1-SG21). Over the next several days, one of the pair dies in SG3-SG21. Examination of the PMS neuron in any one of these segments reveals that either the right or the left cell remains, with equal probability, suggesting that the elimination of one of the pair is a random process. When unpaired PMS neurons are examined in pairs of adjacent ganglia, however, the cells are from opposite sides in the majority of cases (approximately 88%). This observation implies that the death of a PMS neuron in 1 ganglion strongly biases which member of the pair of PMS neurons degenerates in adjacent ganglia. Detailed examination of the sequence of degeneration shows that it begins at several separate loci in the nerve cord. We propose that the mechanism responsible for the death of one of the pair of PMS neurons in a segmental ganglion is competition between these 2 cells for some trophic factor, but that the outcome of this competition is predetermined if one of the PMS neurons in an adjacent ganglion has already begun to degenerate.