Structure and development of the subesophageal zone of the Drosophila brain. II. Sensory compartments.

The subesophageal zone (SEZ) of the Drosophila brain processes mechanosensory and gustatory sensory input from sensilla located on the head, mouth cavity and trunk. Motor output from the SEZ directly controls the movements involved in feeding behavior. In an accompanying paper (Hartenstein et al., ), we analyzed the systems of fiber tracts and secondary lineages to establish reliable criteria for defining boundaries between the four neuromeres of the SEZ, as well as discrete longitudinal neuropil domains within each SEZ neuromere. Here we use this anatomical framework to systematically map the sensory projections entering the SEZ throughout development. Our findings show continuity between larval and adult sensory neuropils. Gustatory axons from internal and external taste sensilla of the larva and adult form two closely related sensory projections, (a) the anterior central sensory center located deep in the ventromedial neuropil of the tritocerebrum and mandibular neuromere, and (b) the anterior ventral sensory center (AVSC), occupying a superficial layer within the ventromedial tritocerebrum. Additional, presumed mechanosensory terminal axons entering via the labial nerve define the ventromedial sensory center (VMSC) in the maxilla and labium. Mechanosensory afferents of the massive array of chordotonal organs (Johnston's organ) of the adult antenna project into the centrolateral neuropil column of the anterior SEZ, creating the antenno-mechanosensory and motor center (AMMC). Dendritic projections of dye back-filled motor neurons extend throughout a ventral layer of the SEZ, overlapping widely with the AVSC and VMSC. Our findings elucidate fundamental structural aspects of the developing sensory systems in Drosophila.

Development, plasticity and modulation of visceral afferents.

Visceral pain is the most common reason for doctor visits in the US. Like somatic pain, virtually all visceral pain sensations begin with the activation of primary sensory neurons innervating the viscera and/or the blood vessels associated with these structures. Visceral afferents also play a central role in tissue homeostasis. Recent studies show that in addition to monitoring the state of the viscera, they perform efferent functions through the release of small molecules (e.g. peptides like CGRP) that can drive inflammation, thereby contributing to the development of visceral pathologies (e.g. diabetes Razavi, R., Chan, Y., Afifiyan, F.N., Liu, X.J., Wan, X., Yantha, J., Tsui, H., Tang, L., Tsai, S., Santamaria, P., Driver, J.P., Serreze, D., Salter, M.W., Dosch, H.M., 2006. TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes, Cell 127 1123-1135). Visceral afferents are heterogeneous with respect to their anatomy, neurochemistry and function. They are also highly plastic in that their cellular environment continuously influences their response properties. This plasticity makes them susceptible to long-term changes that may contribute significantly to the development of persistent pain states such as those associated with irritable bowel syndrome, pancreatitis, and visceral cancers. This review examines recent insights into visceral afferent anatomy and neurochemistry and how neonatal insults can affect the function of these neurons in the adult. New approaches to the treatment of visceral pain, which focus on primary afferents, will also be discussed.

Activity-dependent reorganization of local circuitry in the developing visceral sensory system.

Neural activity during critical periods could fine-tune functional synaptic connections. N-methyl-d-aspartate (NMDA) receptor activation is critically implicated in this process and blockade leads to disruption of normal circuit formation. This phenomenon has been well investigated in several neural systems including the somatosensory system, but not yet evidenced in the visceral sensory system. Ultrastructural analysis of GABAergic synapses and electrophysiological analysis of inhibitory and excitatory postsynaptic currents of the rat caudal nucleus tractus solitarii (NTS) cells revealed that developmental changes in the synaptic organizations were blocked by MK-801, an NMDA receptor antagonist, when administered at postnatal days 5-8, a presumed critical period for the visceral sensory system. Normal synapse reorganization during postnatal development dictates undifferentiated neonatal caudal NTS neurons in terms of synaptic input patterns measured by electron microscopy and electrophysiology into two cell groups: small and large cells under far stronger excitatory and inhibitory influence, respectively. Blockade by MK-801 during the critical period might leave adult neurons wired in the undifferentiated synaptic networks, possibly preventing synapse elimination and subsequent stabilization of the proper wiring.

Gastric preloads of corn oil and mineral oil produce different patterns of increases of c-Fos-like immunoreacitve cells in the brain of 9-12 day-old rats.

Equivolumetric gastric preloads of corn oil and mineral oil administered to rats on postnatal day 12 (P12) inhibited intake equally during a 30-min test of independent ingestion (II), but preloads of corn oil inhibited intake significantly more than preloads of mineral oil on P15 and P18 [Weller, A., Gispan, I.H., Armony-Sivan, R., Ritter, R.C., Smith, G.P., 1997. Preloads of corn oil inhibit independent ingestion on postnatal day 15 in rats. Physiol. Behav. 62, 871-874]. It is possible that the equivalent inhibition of intake by the oil preloads on P12 resulted from the failure of the preabsorptive sensory properties of the preloads to be discriminated by peripheral or central sensory mechanisms. To investigate this possibility, we administered equivolumetric gastric preloads of 25% corn oil and 25% mineral oil to pups on P9-12 and counted the number of c-Fos-like immunoreactive (CFLI) cells in central sites that are activated by food intake and postingestive preabsortive mechanisms in adult rats and in pups on P10-11. The major result was that preloads of 25% corn oil and 25% mineral oil that produced equivalent inhibition of II intake produced differential increases of CFLI cells in the forebrain and hindbrain. Specifically, preloads of corn oil increased the number of CFLI cells in the caudal Nucleus Tractus Solitarius significantly more than preloads of mineral oil. Furthermore, preloads of corn oil increased the number of CFLI cells in the Paraventricular and Supraoptic nuclei, but preloads of mineral oil did not. This differential pattern of increases of CFLI cells is evidence that the brain discriminates the preabsorptive sensory properties of preloads of corn oil and mineral oil on P9-12.

Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling.

Early dietary sodium restriction has profound influences on the organization of the gustatory brainstem. However, the anatomical relationships among multiple gustatory nerve inputs have not been examined. Through the use of triple-fluorescence labeling and confocal laser microscopy, terminal fields of the greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves were visualized concurrently in the nucleus of the solitary tract (NTS) of developmentally sodium-restricted and control rats. Dietary sodium restriction during pre- and postnatal development resulted in a twofold increase in the volume of both the CT and the IX nerve terminal fields but did not affect the volume of the GSP terminal field. In controls, these nerve terminal fields overlapped considerably. The dietary manipulation significantly increased the overlapping zones among terminal fields, resulting in an extension of CT and IX fields past their normal boundaries. The differences in terminal field volumes were exaggerated when expressed relative to the respective NTS volumes. Furthermore, increased terminal field volumes could not be attributed to an increase in the number of afferents because ganglion cell counts did not differ between groups. Taken together, selective increases in terminal field volume and ensuing overlap among terminal fields suggest an increased convergence of these gustatory nerve terminals onto neurons in the NTS. The genesis of such convergence is likely related to disruption of cellular and molecular mechanisms during the development of individual terminal fields, the consequences of which have implications for corresponding functional and behavioral alterations.

Patent ductus arteriosus remodels cardiac sensory neuronal chemotransduction.

We sought to determine the capacity of neonatal ventricular sensory nerve endings (neurites) to transduce the cardiac milieu in the presence of cardiovascular pathology. The spontaneous activity generated by nodose ganglion cardiac afferent neurons was identified in situ using extracellular recording techniques in two groups of piglets approximately 2 weeks old: (i). controls that underwent sham operations (n=19 piglets) 2 weeks earlier and (ii). a pathological model of patent ductus arteriosus stented open for about 2 weeks (n=16 piglets). The capacity of ventricular sensory neurites associated with nodose ganglion afferent neurons to transduce local mechanical (including alterations in right or left ventricular volumes) or chemical stimuli was studied in both groups. The average conduction velocity of afferent axons associated with identified neuronal somata was estimated to be 1.5+/-0.6 or 2.9+/-1.3 m s(-1). Ventricular afferent neurons transduced mechanical stimuli similarly in both groups. In control animals, ventricular afferent neurons transduced the following chemicals: the sodium channel modifier veratridine (delta 23+/-7 impulses min(-1)), the P(1)-purinoceptor agonist adenosine (Delta 24+/-8 impulses min(-1)), and the beta-adrenoceptor agonist isoproterenol (delta 18+/-7 impulses min(-1)). On the other hand, patent ductus arteriosus cardiac afferent neurons did not transduce these chemicals. It is concluded that neonatal cardiac afferent neuronal chemosensory-as opposed to mechanosensory-transduction remodels in the presence of a patent ductus arteriosus. The reduced capacity of neonatal cardiac afferent neurons to transduce chemicals in the presence of a patent ductus arteriosus should be taken into account when considering neonatal cardiovascular control in such a state.

Adult-like complexity of the larval antennal lobe of D. melanogaster despite markedly low numbers of odorant receptor neurons.

We provide a detailed analysis of the larval head chemosensory system of Drosophila melanogaster, based on confocal microscopy of cell-specific reporter gene expression in P[GAL4] enhancer trap lines. In particular, we describe the neuronal composition of three external and three pharyngeal chemosensory organs, the nerve tracts chosen by their afferents, and their central target regions. With a total of 21 olfactory and 80 gustatory neurons, the sensory level is numerically much simpler than that of the adult. Moreover, its design is different than in the adult, showing an association between smell and taste sensilla. In contrast, the first-order relay of the olfactory afferents, the larval antennal lobe (LAL), exhibits adult-like features both in terms of structure and cell number. It shows a division into approximately 30 subunits, reminiscent of glomeruli in the adult antennal lobe. Taken together, the design of the larval chemosensory system is a "hybrid," with larval-specific features in the periphery and central characteristics in common with the adult. The largely reduced numbers of afferents and the similar architecture of the LAL and the adult antennal lobe, render the larval chemosensory system of Drosophila a valuable model system, both for studying smell and taste and for examining the development of its adult organization.

Proliferation patterns of dorsal root ganglion neurons of cutaneous, muscle and visceral nerves in the rat.

In a previous study we provided evidence that dorsal root ganglion (DRG) neurons of different phenotypes have different birthdates. The present study aimed at determining if birthdates of DRG neurons are related to different types of peripheral nerves, namely cutaneous versus muscle, and somatic versus visceral. Pregnant rats were injected intraperitoneally with bromodeoxyuridine (BrdU) to label the neurons on one of the embryonic days E12-E16. When the progeny rats reached adulthood, a mixture of 1% B-fragment of cholera toxin and 1% isolectin B4 from Griffonia simplicifolia I was injected into the peripheral nerves, or a 5% Fluoro-Gold solution was applied to the transected end of the nerves. The saphenous and sural nerves were used as cutaneous nerves, the gastrocnemius nerve as a muscle nerve, the intercostal nerves T9-11 as somatic nerves and the greater splanchnic nerve as a visceral nerve. Cell size measurements were made of DRG neurons labeled from the two cutaneous nerves and the muscle nerve, as well as of neurons of the saphenous and gastrocnemius nerves labeled by BrdU at different embryonic stages. Most of the DRG neurons of the muscle and intercostal nerves were generated early, with peaks at E13, and those of the cutaneous and visceral afferent nerves later, with peaks at E14. The temporal differences were reflected in the cell size spectrum, the muscle nerve having a greater proportion of large neurons compared to the cutaneous nerves. The findings add to previous knowledge regarding the sequence of development of different DRG phenotypes.

Dual effects of NMDA receptor activation on polysialylated neural cell adhesion molecule expression during brainstem postnatal development.

Here we show a dual role of N-methyl-d-aspartate receptor (NMDAR) activation in controlling polysialylated neural cell adhesion molecule (PSA-NCAM) dynamic expression in the dorsal vagal complex (DVC), a gateway for many primary afferent fibres. In this structure the overall expression of PSA-NCAM decreases during the first 2 weeks after birth to persist only at synapses in the adult. Electrical stimulation of the vagal afferents causes a rapid increase of PSA-NCAM expression both in vivo and in acute slices before postnatal day (P) 14 whereas a similar stimulation induces a decrease after P15. Inhibition of NMDAR activity in vitro completely prevented these changes. These regulations depend on calmodulin activation and cGMP production at all stages. By contrast, blockade of neuronal nitric oxide synthase (nNOS) prevented these changes only after P10 in agreement with its late expression in the DVC. The pivotal role of NMDAR is also supported by the observation that chronic blockade induces a dramatic decrease in PSA-NCAM expression.

Functional analysis of GABA(A) receptors in nucleus tractus solitarius neurons from neonatal rats.

To gain insight into specific GABA(A) receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1-14 day old rats using whole-cell patch clamp recordings. GABA(A-) receptor-mediated currents were detected in all neurons tested, with an average EC(50) of 22.2 microM. GABA currents were consistently stimulated by diazepam (EC(50)=63 nM), zolpidem (EC(50)=85 nM), loreclezole (EC(50)=10.1 microM) and the neurosteroid 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn(2+) (IC(50)=33.6 microM) picrotoxin (IC(50)=2.4 microM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10-1000 microM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant alpha subunit isoform present in GABA(A) receptors of the NTS appears to be the alpha1 and/or alpha2 subunit. beta2 and/or beta3 subunits are the major beta isoform, while the predominant gamma subunit is likely gamma2. Our data suggest the contribution to NTS GABA currents by alpha3-alpha6, beta1, gamma1 and delta subunits, if present, is minor by comparison.