Extrinsic Neurons Research Articles

Mushroom bodies of the cockroach: Activity and identities of neurons recorded in freely moving animals

This article describes novel attributes of the mushroom bodies of cockroaches revealed by recording from neurons in freely moving insects. The results suggest several hitherto unrecognized functions of the mushroom bodies: extrinsic neurons that discriminate between imposed and self-generated sensory stimulation, extrinsic neurons that monitor motor actions, and a third class of extrinsic neurons that predict episodes of locomotion and modulate their activity depending on the turning direction. Electrophysiological units have been correlated with neurons that were partially stained by uptake of copper ions and silver intensification. Neurons so revealed correspond to Golgi-impregnated or Lucifer yellow-filled neurons and demonstrate that their processes generally ascend to other areas of the protocerebrum. The present results support the idea of multiple roles for the mushroom bodies. These include sensory discrimination, the integration of sensory perception with motor actions, and, as described in the companion article, a role in place memory.

Mushroom bodies of the cockroach: Activity and identities of neurons recorded in freely moving animals

This article describes novel attributes of the mushroom bodies of cockroaches revealed by recording from neurons in freely moving insects. The results suggest several hitherto unrecognized functions of the mushroom bodies: extrinsic neurons that discriminate between imposed and self-generated sensory stimulation, extrinsic neurons that monitor motor actions, and a third class of extrinsic neurons that predict episodes of locomotion and modulate their activity depending on the turning direction. Electrophysiological units have been correlated with neurons that were partially stained by uptake of copper ions and silver intensification. Neurons so revealed correspond to Golgi-impregnated or Lucifer yellow-filled neurons and demonstrate that their processes generally ascend to other areas of the protocerebrum. The present results support the idea of multiple roles for the mushroom bodies. These include sensory discrimination, the integration of sensory perception with motor actions, and, as described in the companion article, a role in place memory. J. Comp. Neurol. 402:501–519, 1998. © 1998 Wiley-Liss, Inc.

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.

Effect of a serotonergic extrinsic modulatory neuron (MCC) on radula mechanoafferent function in Aplysia.

The serotonergic metacerebral cells (MCCs) and homologous neurons in related mollusks have been extensively investigated within the context of feeding. Although previous work has indicated that the MCCs exert widespread actions, MCC modulation of sensory neurons has not been identified. We characterized interactions between the MCCs and a cell that is part of a recently described group of buccal radula mechanoafferents. The cell, B21, has a peripheral process in the tissue underlying the chitinous radula [the subradula tissue (SRT)]. Previous studies have shown that B21 can fire phasically during ingestive motor programs and provide excitatory drive to the circuitry active during radula closing/retraction. We now show that activity of B21 can be modulated by serotonin (5-HT) and the MCCs. Centrally, although a slow depolarization is typically recorded in B21 as a result of MCC stimulation, this depolarization does not cause B21 to spike. It can, however, increase B21 excitability enabling a pulse that was previously subthreshold to elicit an action potential in B21. B21 is in fact rhythmically depolarized during the radula closing/retraction phase of ingestive motor programs. Thus central effects of the MCCs on radula mechanoafferent activity are only likely to be apparent while B21 is receiving input from the feeding central pattern generator. Peripherally, radula mechanoafferent neurons can be activated 1) when a mechanical stimulus is applied to the biting surface of the SRT and 2) when the SRT contracts. MCC stimulation and 5-HT modulate B21 responses to both types of stimuli. For example, MCC stimulation and low concentrations of 5-HT cause subthreshold mechanical stimuli applied to the SRT to become suprathreshold. 5-HT and MCC stimulation also enhance SRT contractility. Peripheral effects of MCC activity are also likely to be phase dependent. For example, MCC stimulation does not cause B21 to respond to peripheral stimuli with an afterdischarge. Consequently, radula mechanoafferents are likely to be activated when food is present between the radula halves during radula closing/retraction but are not likely to continue to fire as opening/protraction is initiated. In a similar vein, MCC effects on the contractility of the SRT will only be apparent when contractions are elicited by motor neuron activity. SRT motor neurons are rhythmically activated during ingestive motor programs. Thus we have shown that radula mechanoafferent activity can be modulated by the MCCs and that this modulation is likely to occur in a phase-dependent manner.

Topography of four classes of Kenyon cells in the mushroom bodies of the cockroach.

Mushroom bodies (MBs), which are higher centers in the insect brain, are implicated in associative memory and in the control of some behaviors. Intrinsic neurons of the MB, called Kenyon cells, receive synaptic inputs from axon terminals of input neurons in the calyx. Axons of Kenyon cells project into the pedunculus and to the alpha and beta lobes, where they make synaptic connections with dendrites of extrinsic (output) neurons. In this study, we examined the morphology of Kenyon cells in the cockroach by using Golgi stains and found that they can be classified into four classes (K1, K2, K3, and K4), according to the diameter, location, and morphology of the cell bodies, dendrites, and axons. The somata of Kenyon cells of different classes occupy different concentric zones; K1 cells occupy the most central zone, and K4 cells occupy the most peripheral zone. The main processes of Kenyon cells of different classes also occupy different concentric zones in the calyx. Dendrites of K2 and K3 cells are distributed throughout the calycal neuropil, whereas those of K1 and K4 cells cover the outer and inner halves of the depth of the neuropil, respectively. In the pedunculus and the alpha and beta lobes, axons of Kenyon cells of different classes occupy different zones, although the separation is not complete. A class of extrinsic neurons in the a lobe has dendrite-like arbors that cover the zones where either K1, K2, or K3 are located. These neurons probably transmit signals of each class of Kenyon cells. We conclude that, in the cockroach, four classes of Kenyon cells subdivide the cell body region, pedunculus, and lobes of the MBs, whereas subdivision is less prominent in the calycal neuropil.

Topography of modular subunits in the mushroom bodies of the cockroach.

The mushroom body (MB), a conspicuous neuropil structure in the insect brain, is implicated in associative memory and in some aspects of motor control. Intrinsic neurons of the MB (Kenyon cells) extend dendrites into the calyx, and their axons run through the pedunculus and then bifurcate to form the alpha and the beta lobes. At the pedunculus and the lobes, Kenyon cells make synaptic connections with dendrites of extrinsic (output) neurons. Previously, we reported that the alpha lobe of the cockroach MB consists of repetitive modular subunits (Mizunami et al. [1997] Neurosci. Lett. 229:153-156). Each subunit is composed of a dark layer and a light layer, and the layers are refereed to as slabs. Each slab is composed of axons of a specific subset of Kenyon cells. In the present study, we examined serial sections of reduced silver preparations and found that each dark and light slab continues throughout the length of the pedunculus and the alpha and beta lobes. We also found that Golgi-impregnated Kenyon cells often exhibit a characteristic grouping, forming a thin sheet interlaced by dozens or hundreds of axons. The sheet is much thinner than the slab, and each sheet remains within a particular slab throughout the length of the pedunculus and the lobes. Thus, the sheet is a component forming the slab. In the pedunculus and the beta lobe, a class of Golgi-impregnated extrinsic neurons exhibit segmented dendritelike arbors that interact with every other slab, i.e., either with only dark or light slabs. Because each neuron of this class interacts with each particular set of dark or light slabs, we conclude that the slabs are units for transmitting output signals from the MB.

Neurocalcin-like immunoreactivity in the rat esophageal nervous system.

Neurocalcin is a newly identified neuronal calcium-binding protein. We tried here to investigate the immunohistochemical distribution of neurocalcin in the rat esophagus. Nerve cell bodies having neurocalcin immunoreactivity were found throughout the myenteric plexus. In the myenteric ganglia, two types of nerve terminals showed neurocalcin immunoreactivity. One was varicose terminals containing numerous small clear vesicles and forming a synapse with nerve cells. The other terminals were characterized by laminar or pleomorphic structure and many mitochondria. These laminar terminals were supposed to be sensory receptors of the esophageal wall. In the motor endplates of the striated muscles, nerve terminals containing many small clear vesicles and mitochondria also had neurocalcin immunoreactivity. After left vagus nerve cutting under the nodose ganglia, the number of immunopositive thick nerve fibers, laminar endings and nerve terminals on the striated muscles decreased markedly. Retrograde tracing experiments using Fast Blue showed extrinsic innervation of esophagus from ambiguus nucleus, dorsal motor nucleus of vagus, superior cervical ganglia, celiac ganglia, nodose ganglia and dorsal root ganglia. In the celiac ganglia, nodose ganglia and dorsal root ganglia, retrogradely labeled nerve cells were neurocalcin-immunoreactive. Neurons in the celiac ganglia may project varicose terminals, while nodose and dorsal root neurons project laminar terminals. Although cell bodies of motoneurons in the ambiguus nucleus lacked neurocalcin immunoreactivity, these neurons may contain neurocalcin only in the nerve terminals in the motor endplates. Neurocalcin immunoreactivity is distributed in many extrinsic and intrinsic neurons in the esophagus and this protein may play important roles in regulating calcium signaling in the neurons.

Metamorphosis of the Mushroom Bodies; Large-Scale Rearrangements of the Neural Substrates for Associative Learning and Memory in Drosophila

Paired brain centers known as mushroom bodies are key features of the circuitry for insect associative learning, especially when evoked by olfactory cues. Mushroom bodies have an embryonic origin, and unlike most other brain structures they exhibit developmental continuity, being prominent components of both the larval and the adult CNS. Here, we use cell-type-specific markers, provided by the P[GAL4] enhancer trap system, to follow specific subsets of mushroom body intrinsic and extrinsic neurons from the larval to the adult stage. We find marked structural differences between the larval and adult mushroom bodies, arising as the consequence of large-scale reorganization during metamorphosis. Extensive, though incomplete, degradation of the larval structure is followed by establishment of adult specific alpha and beta lobes. Kenyon cells of embryonic origin, by contrast, were found to project selectively to the adult gamma lobe. We propose that the gamma lobe stores information of relevance to both developmental stages, whereas the alpha and beta lobes have uniquely adult roles.

The Organization of Extrinsic Neurons and Their Implications in the Functional Roles of the Mushroom Bodies inDrosophila melanogasterMeigen

Although the importance of the Drosophila mushroom body in olfactory learning and memory has been stressed, virtually nothing is known about the brain regions to which it is connected. Using Golgi and GAL4-UAS techniques, we performed the first systematic attempt to reveal the anatomy of its extrinsic neurons. A novel presynaptic reporter construct, UAS-neuronal synaptobrevin-green fluorescent protein (n-syb-GFP), was used to reveal the direction of information in the GAL4-labeled neurons. Our results showed that the main target of the output neurons from the mushroom body lobes is the anterior part of the inferior medial, superior medial, and superior lateral protocerebrum. The lobes also receive afferents from these neuropils. The lack of major output projections directly to the deutocerebrum's premotor pathways discourages the view that the role of the mushroom body may be that of an immediate modifier of behavior. Our data, as well as a critical evaluation of the literature, suggest that the mushroom body may not by itself be a "center" for learning and memory, but that it can equally be considered as a preprocessor of olfactory signals en route to "higher" protocerebral regions.

Integrative Properties of the Pe1 Neuron, a Unique Mushroom Body Output Neuron

A mushroom body extrinsic neuron, the Pe1 neuron, connects the peduncle of the mushroom body (MB) with two areas of the protocerebrum in the honeybee brain, the lateral protocerebral lobe (LPL) and the ring neuropil around the alpha-lobe. Each side of the bee brain contains only one Pe1 neuron. Using a combination of intracellular recording and neuroanatomical techniques we analyzed its properties of integrative processing of the different sensory modalities. The Pe1 neuron responds to visual, mechanosensory, and olfactory stimuli. The responses are broadly tuned, consisting of a sustained increase of spike frequency to the onset and offset of light flashes, to horizontal and vertical movements of extended objects, to mechanical stimuli applied to the antennae or mouth parts, and to all olfactory stimuli tested (29 chemicals). These multisensory properties are reflected in its dendritic organization. Serial reconstructions of intracellularly stained Pe1 neurons using confocal microscopy reveal that the Pe1 neuron arborizes throughout all layers of MB peduncle with finger-like, vertically oriented dendrites. The peduncle of the MB is formed by the axons of Kenyon cells, whose dendritic inputs are organized in modality-specific subcompartments of the calyx region. The peduncular arborization indicates that the Pe1 neuron receives input from Kenyon cells of all calycal subcompartments. Because the Pe1 neuron changes its odor responses transiently as a consequence of olfactory learning, we hypothesize that the multimodal response properties might have a role in memory consolidation and help to establish contextual references in the long-term trace.

Spatiotemporal Structure of Olfactory Inputs to the Mushroom Bodies

A requirement to understand mushroom body (MB) function is to characterize the operations (or transformations) that they impose on incoming signals. Understanding the nature of these integrative operations requires an understanding of the inputs from other brain areas. By inputs we mean not only the anatomical pathways leading to the MBs, but also the dynamic structure of the inflow of sensory (and other) signals. Neurons are complex, capacitative, and generally nonlinear devices that transform barrages of neurochemical packets into electrical waveforms. Their modes of operation are intrinsically time dependent and therefore, their functions or roles in a circuit cannot be inferred only from structural data. Thanks to elegant anatomical, behavioral, genetic, and molecular (for review, see Crittenden et al. 1998; Hammer and Menzel 1998; Heisenberg 1998; Wolf et al. 1998) studies, there is convincing evidence that MB circuits are involved, at least in fruit flies and honeybees, in some forms of odor integration and learning. In vivo electrophysiological studies of MB neurons, however, are rare and mainly restricted to individual (or small populations of) so-called extrinsic neurons, that is, those whose processes link MBs with other brain areas (Schildberger 1983, 1984; Homberg 1984; Hammer 1993; Mauelshagen 1993; Li and Strausfeld 1997). Kaulen et al. (1984) examined extracellular potentials in the MBs of bees, using current source density analysis, and more recently, Laurent and Naraghi (1994) provided a description of stimulus-evoked activity in Kenyon cells (KCs), the intrinsic neurons of the MBs, using intracellular recordings. In this short review we will summarize the recent results from our laboratory in an attempt to provide a description of the spatiotemporal structure of olfactory inputs to the MBs and their intrinsic neurons. We will focus only on the encoding of odor quality. We will then speculate on the possible role of MB circuits for olfactory processing.

Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat

Background: Calcitonin gene-related peptide (CGRP) is a widely distributed neuropeptide contained in intrinsic and extrinsic neurons of the gastrointestinal wall that has been shown to be released by noxious stimulation, to be involved in nociception, to inhibit gastrointestinal motility, and to partly mediate postoperative gastric ileus. We hypothesized that abdominal surgery-induced release of CGRP might inhibit postoperative colonic motility and food intake. Methods: Colonic transit, stool pellet number, stool pellet weight, and food intake were measured for 48 hours after induction of postoperative ileus in rats. CGRP was immunoneutralized by preoperative injection of CGRP monoclonal antibody, or visceral afferent nerve fibers containing CGRP were functionally ablated by topical capsaicin treatment of the vagus nerves or of the celiac/superior mesenteric ganglia before abdominal surgery. Results: Abdominal surgery increased colonic transit time and decreased 24-hour cumulative stool pellet number, stool pellet weight, and food intake. CGRP immunoneutralization reversed postoperative inhibition of colonic transit, 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 77%, 82%, 80%, and 52%, respectively. Whereas ablation of vagal afferent nerve fibers had no effect, spinal afferent nerve fiber ablation reversed postoperative inhibition of 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 41%, 38%, and 19%, respectively. Conclusions: CGRP and spinal afferent nerve fibers partly mediate postoperative colonic ileus and inhibition of food intake in the rat. By the magnitude of reversal of postoperative ileus, CGRP seems to be an important mediator of postoperative colonic ileus. Our results for the first time show involvement of a neuropeptide and spinal afferents in the mediation of postoperative colonic ileus and postoperative inhibition of food intake in rats. (Surgery 1998;123:518-27.)

Capsaicin sensitivity and epidermal growth factor

See article on page344 It is now well established that afferent neurones innervating the intestine subserve “effector” as well as sensory functions. The release of neuromodulators from the peripheral nerve...

Three classes of GABA-like immunoreactive neurons in the mushroom body of the cockroach

The mushroom body (MB) is a higher center of the insect brain and is critical to some forms of associative memory. Each MB consists of calyces connected to α and β lobes via pedunculus. In the calyces, input neurons make synaptic connections with intrinsic neurons. In the pedunculus and lobes, intrinsic neurons make synaptic connections with output neurons. Here, the distribution of γ-aminobutyric acid (GABA)-like immunoreactivity in the MB of the cockroach Periplaneta americana was investigated, using an antiserum against a GABA–protein conjugate, to elucidate inhibitory pathways of the MB. We report that three classes of extrinsic neurons of the MB exhibit GABA-like immunoreactivity. The first is four large neurons which arborize in a diffuse neuropil surrounding the α lobe and project into whole areas of the calyces. Their cell bodies are 30–50 μm in diameter, among the largest in the brain. The second group is 7–9 neurons ascending from the circumesophageal connective and projecting into the calyces, which probably represent inhibitory input neurons. The third group is ca. 40 neurons with dendritic arborizations in the junction between the pedunculus and the lobes, which probably represent inhibitory output neurons.

Dual (excitatory and inhibitory) calretinin innervation of AMPA receptor-containing neurons in the rat lateral septum

A recent study demonstrated both an extrinsic and an intrinsic calretinin (CR) innervation of the rat septal complex and that a population of the extrinsic calretinin fibers is aspartate/glutamate-containing. The aim of this study was to determine which types (GluR1, GluR2/3, or both) of AMPA receptor-containing lateral septal area neurons are innervated by extrinsic and intrinsic CR neurons and whether the intrinsic CR cells are GABAergic. Light- and electron-microscopic single immunostaining for CR, GluR1, and GluR2/3, as well as light- and electron-microscopic-double immunostaining experiments for CR plus GluR1 and CR plus GluR2/3 were performed in the lateral septal area. Furthermore, the "mirror" colocalization technique was employed on consecutive vibratome sections of the septal complex to investigate whether the intrinsic septal CR neurons are GABAergic. The results are summarized as follows: (1) both GluR1- and GluR2/3-immunoreactive neurons are innervated by CR-containing fibers; (2) the majority of these synapses, observed mainly on the soma and, to a lesser extent, on proximal dendrites of AMPA receptor-containing neurons, represent asymmetric synaptic membrane specializations; (3) a minority of CR-containing axon terminals associated with both GluR1- and GluR2/3-immunoreactive neurons form symmetric contacts, predominantly on their soma; and (4) 93% of the lateral septal area CR cells are GABAergic. These observations indicate that both GluR1- and GluR2/3-containing lateral septal area neurons receive a dual intrinsic and extrinsic CR innervation. The former (intrinsic) CR boutons are GABAergic, while the latter form asymmetric synaptic contacts, are excitatory, and probably originate in the supramammillary area, since previous work has demonstrated that a population of supramammillo-septal fibers contain aspartate and/or glutamate.

Morphology and sensory modality of mushroom body extrinsic neurons in the brain of the cockroach, Periplaneta americana

Mushroom bodies are paired centers in insect brains that are thought to be crucial in olfactory learning and memory. Early neuroanatomical descriptions suggested that the mushroom bodies comprise rather simple arrangements of nerve cells. Intrinsic neurons within each mushroom body were believed to receive olfactory afferents and to supply long, branched axons to extrinsic neurons that lead from the mushroom body into the protocerebrum. More recent suggestions that the mushroom bodies integrate several sensory modalities find support from intracellular and extracellular recordings of extrinsic neurons in the brains of crickets, honey bees, and cockroaches. Here, we describe two major classes of extrinsic neurons, simple and complex cells, in the mushroom bodies of the cockroach Periplaneta americana. Each class is defined by its pattern of branching in the brain. Simple neurons correspond to extrinsic neurons described from other species that have one set of dendrites only within the mushroom bodies. Complex extrinsic neurons possess dendrite-like branches within and outside the mushroom bodies. This arrangement may account in part for their observed multimodality, as might newly identified afferent neurons that terminate in the mushroom body lobes among the dendrites of extrinsic neurons and that respond to multimodal stimuli. Organizational complexity within the mushroom bodies is suggested by the grouping of intrinsic cell axons into discrete laminae. These are intersected by the block-like arrangements of dendritic fields of extrinsic neurons in a manner reminiscent of Purkinje cell dendrites intersecting parallel fibers in the cerebellum. The present results demonstrate that the cockroach mushroom body processes multimodal sensory information and that its neural arrangements contribute to a precise architecture consisting of discrete longitudinal and transverse subdivisions.

Morphology and sensory modality of mushroom body extrinsic neurons in the brain of the cockroach, Periplaneta americana

Mushroom bodies are paired centers in insect brains that are thought to be crucial in olfactory learning and memory. Early neuroanatomical descriptions suggested that the mushroom bodies comprise rather simple arrangements of nerve cells. Intrinsic neurons within each mushroom body were believed to receive olfactory afferents and to supply long, branched axons to extrinsic neurons that lead from the mushroom body into the protocerebrum. More recent suggestions that the mushroom bodies integrate several sensory modalities find support from intracellular and extracellular recordings of extrinsic neurons in the brains of crickets, honey bees, and cockroaches. Here, we describe two major classes of extrinsic neurons, simple and complex cells, in the mushroom bodies of the cockroach Periplaneta americana. Each class is defined by its pattern of branching in the brain. Simple neurons correspond to extrinsic neurons described from other species that have one set of dendrites only within the mushroom bodies. Complex extrinsic neurons possess dendrite-like branches within and outside the mushroom bodies. This arrangement may account in part for their observed multimodality, as might newly identified afferent neurons that terminate in the mushroom body lobes among the dendrites of extrinsic neurons and that respond to multimodal stimuli. Organizational complexity within the mushroom bodies is suggested by the grouping of intrinsic cell axons into discrete laminae. These are intersected by the block-like arrangements of dendritic fields of extrinsic neurons in a manner reminiscent of Purkinje cell dendrites intersecting parallel fibers in the cerebellum. The present results demonstrate that the cockroach mushroom body processes multimodal sensory information and that its neural arrangements contribute to a precise architecture consisting of discrete longitudinal and transverse subdivisions. J. Comp. Neurol. 387:631–650, 1997. © 1997 Wiley-Liss, Inc.

Neuroprotective fibroblast growth factor type-2 down-regulates the c-Jun transcription factor in axotomized sympathetic preganglionic neurons of adult rat

The immediate-early gene encoded transcription factor c-Jun is highly inducible following axotomy and therefore serves as a valuable marker in neuronal de- and regeneration. As the signals that may trigger c-Jun expression are still obscure, molecules derived from lesioned neurons and/or their targets such as growth factors or cytokines have been proposed as candidates for interneuronal transcriptional regulation in vivo. We therefore tested whether local administration of the neuroprotective cytokine fibroblast growth factor type-2 in vivo has an effect on the axotomy-induced nuclear expression patterns of the activator protein-1 transcription factors c-Fos and JunB, or c-Jun in the spinal cord–intermedolateral nucleus–adrenal axis lesion paradigm in the rat. Partial axotomy of preganglionic nerve fibres by selective unilateral removal of the adrenal medulla resulted in strong staining patterns of c-Jun in the nuclei of preganglionic cell bodies located in the spinal intermediolateral cell column identified by in vivo retrograde prelabelling with the fluorescent tracer Fast Blue prior to lesion. Axotomy-induced nuclear c-Jun expression was highly increased when compared with the moderate baseline expression in normal or sham-operated animals. In animals treated with fibroblast growth factor-2 gelfoams implanted to the lesioned adrenal gland the nuclear c-Jun staining pattern is reduced or even absent from these neurons. By contrast, c-Fos and JunB induction did not occur in the intermediolateral nucleus in the lesion paradigm investigated. These results support the idea of functional links between neurotrophic cytokines such as fibroblast growth factor-2 and transcriptional effectors such as c-Jun. The target derived fibroblast growth factor-2 thus may signal the intactness of the neuron–target axis resulting in suppression of central extrinsic neurons and promotion of neuroprotective gene activation. Neuronal survival in absence of c-Jun indicates that c-Jun exerts negative actions in vulnerated neurons.

ANG II modifies cardiomyocyte function via extracardiac and intracardiac neurons: in situ and in vitro studies.

To determine whether angiotensin II (ANG II) affects cardiac performance via neurons in intrathoracic cardiac ganglia, studies were performed on anesthetized dogs. To exclude possible vascular regulatory effects of ANG II, experiments were also performed using long-term cultures of adult guinea pig ventricular cardiomyocytes with or without intrathoracic neurons. 1) In in situ experiments in 10 anesthetized dogs, cardiac augmentation occurred when ANG II (10 microl or 0.1 ml; 10-100 microM) was administered into limited loci within acutely decentralized stellate or middle cervical ganglia that were neurally connected to, but not those disconnected from, the heart. In another 18 dogs, ANG II increased intrinsic cardiac neuronal activity when administered adjacent to such neurons or into their local arterial blood supply. Ventricular ionotropic effects elicited by ANG II were eliminated by timolol, whereas increases in intrinsic cardiac neuronal activity were not affected. Effects elicited by ANG II were eliminated by administration of a selective AT1 receptor antagonist (losartan) but not by a selective AT2 receptor antagonist (PD-123319). 2) In in vitro experiments, ANG II (100 nM) induced positive chronotropic effects on cultured adult guinea pig cardiomyocytes innervated with adult extrinsic or intrinsic cardiac neurons, but not those cultured without neurons. The frequency of calcium inward current (Ca(i)) transients (recorded by fura 2 fluorescence) increased in innervated cocultures but not in the noninnervated cardiomyocyte cultures; however, the amplitude of Ca(i) transients was not affected by ANG II in cultures or in freshly isolated adult guinea pig cardiomyocytes. ANG II-induced effects in cocultures were blocked by losartan but not PD-123319 or timolol. Thus 1) ANG II-sensitive neurons exist in intrathoracic extracardiac and intrinsic cardiac ganglia; 2) these neurons possess AT1 receptors; and 3) these neurons appear to act directly and indirectly via adrenergic neurons to enhance cardiomyocyte function.

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro.

The mechanism(s) by which intestinal smooth muscle tension is signaled to extrinsic primary afferent neurons is poorly understood. In order to characterize myocyte-neuron communication, we developed a coculture system using rat dorsal root ganglion (DRG) neurons and myocytes obtained from the circular muscle layer of the rat distal colon. Both cell types maintained their phenotype in culture, as demonstrated by positive immunocytochemical staining for neuron-specific enolase and smooth muscle actin. Myocytes showed mechanosensitivity in the form of increases in [Ca2+]i in response to light mechanical touch of the plasma membrane. This increase in [Ca2+]i was independent of extracellular Ca2+ and passed as a propagated wave from muscle cells into adjacent DRG neurites. The inhibitory effect of octanol on this intercellular propagation suggests propagation of [Ca2+]i gradients via heterologous gap junctions. This preparation may serve a useful model system for the study of the interaction of visceral afferents and their target cells.