GABA- and glycine-immunoreactive synapses in spinal cord of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Using the method of the double immune label combined with two antibodies, i.e., monoclonal antibodies to gamma-aminobutyric acid (GABA) and polyclonal antibodies to glycine, the distribution of gamma-aminobutyric acid- and glycine-immunoreactive synapses on motoneurons and primary afferent axons was studied in the frog Rana temporaria spinal cord. An analysis of all labeled boutons on the dendrites and soma of motoneurons showed the existence of three categories of immunoreactive synapses as follows: 7% were labeled for GABA, 23% were labeled for glycine, and approximately 70% were immunoreactive to both GABA and glycine. These results confirm the predominant role of glycine in the postsynaptic inhibition of motoneuronal activity. Three similar populations of synaptic boutons were also founded on primary afferent axons, including one GABA-immunoreactive (25%) and one glycine-immunoreactive (5%); the majority of the immunoreactive synapses had the colocalization of two inhibitory transmitters. As a rule, the higher proportion of axo-axonal synapses was organized in synaptic triads. The possible simultaneous roles of glycine as a transmitter of postsynaptic inhibition and as a transmitter that mediates the process of the autoreception of glutamate in the axo-axonal synapses on the primary afferent fibers are discussed.     

Synaptic and electotonic contacts on primary afferent axons in the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> spinal cord

Distribution of GABA and glycine immunoreactivity was studied in synapses on primary afferent axons of the lamprey Lampetra fluviatilis spinal cord using a double labelling technique. Approximately 25% of synapses exhibit GABA immunoreactivity, while more than 70% are immunoreactive to both neurotransmitters. As in other vertebrates, axo-axonal contacts represent three-component synaptic complexes, the so-called triads, where the immunoreactive terminal make synaptic contact simultaneously with the afferent axon and the dendrite contacting this afferent. Contact zones with gap junction-like cell membrane specializations were found between adjacent afferents suggesting the presence of electrotonic interaction between them. This interaction appears to serve for the synchronization of the afferent flow and represents a structural correlate of the mechanism of rapid interneuronal communication between functionally uniform neurons, which is an important element in the organization of coordinated locomotor acts. Besides, our studies provide evidence that afferent–afferent interaction may be mediated not only electrotonically but also with the aid of chemical synapses. This finding suggests that glutamate-induced depolarization of primary afferents results not only from autoreception but also from the direct effect of glutamate on the afferent’s cell membrane.     

Light-optic, electron microscopic and electrophysiological study of centrifugal fibers of the retina in the lamprey Lampetra fluviatilis

Light and electronmicroscopic studies have been made on retinal structures in the lamprey labeled by horseradish peroxidase injected into the peripheral end of the cut optic nerve or to the midbrain tectum. On total retinal preparations, labeled axons were revealed together with dendrites and ganglionic cell bodies, as well as branching (presumably retinopetal) fibers, fine endings of which come closely to the labeled dendrites of the ganglionic cells. Electron microscopic data indicate that the labeled terminations of afferent fibers from synapses with both labeled and unlabeled dendrites, as well as with unlabeled neuronal bodies. It is concluded that centrifugal fibers in lamprey retina form contacts with the bodies and dendrites of the amacrine cells and dendrites of the ganglionic cells. Results of intracellular registration of responses of various retinal elements to the electrical stimulation of the optic nerve support this conclusion.     

Central Connectivity of Transient Receptor Potential Melastatin 8-Expressing Axons in the Brain Stem and Spinal Dorsal Horn

Transient receptor potential melastatin 8 (TRPM8) ion channels mediate the detection of noxious and innocuous cold and are expressed by primary sensory neurons, but little is known about the processing of the TRPM8-mediated cold information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized TRPM8-positive (+) neurons in the trigeminal ganglion and investigated the distribution of TRPM8+ axons and terminals, and their synaptic organization in the TSN and in the DH using light and electron microscopic immunohistochemistry in transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. TRPM8 was expressed in a fraction of small myelinated primary afferent fibers (23.7%) and unmyelinated fibers (76.3%), suggesting that TRPM8-mediated cold is conveyed via C and Aδ afferents. TRPM8+ axons were observed in all TSN, but at different densities in the dorsal and ventral areas of the rostral TSN, which dominantly receive sensory afferents from intra- and peri-oral structures and from the face, respectively. While synaptic boutons arising from Aδ and non-peptidergic C afferents usually receive many axoaxonic contacts and form complex synaptic arrangements, TRPM8+ boutons arising from afferents of the same classes of fibers showed a unique synaptic connectivity; simple synapses with one or two dendrites and sparse axoaxonic contacts. These findings suggest that TRPM8-mediated cold is conveyed via a specific subset of C and Aδ afferent neurons and is processed in a unique manner and differently in the TSN and DH.     

Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.     

Integration of Sensory Quanta in Cuneate Nucleus Neurons In Vivo

Discriminative touch relies on afferent information carried to the central nervous system by action potentials (spikes) in ensembles of primary afferents bundled in peripheral nerves. These sensory quanta are first processed by the cuneate nucleus before the afferent information is transmitted to brain networks serving specific perceptual and sensorimotor functions. Here we report data on the integration of primary afferent synaptic inputs obtained with in vivo whole cell patch clamp recordings from the neurons of this nucleus. We find that the synaptic integration in individual cuneate neurons is dominated by 4–8 primary afferent inputs with large synaptic weights. In a simulation we show that the arrangement with a low number of primary afferent inputs can maximize transfer over the cuneate nucleus of information encoded in the spatiotemporal patterns of spikes generated when a human fingertip contact objects. Hence, the observed distributions of synaptic weights support high fidelity transfer of signals from ensembles of tactile afferents. Various anatomical estimates suggest that a cuneate neuron may receive hundreds of primary afferents rather than 4–8. Therefore, we discuss the possibility that adaptation of synaptic weight distribution, possibly involving silent synapses, may function to maximize information transfer in somatosensory pathways.     

Presynaptic mitochondria and the temporal pattern of neurotransmitter release

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.     

Speaking out of turn: a role for silent synapses in pain

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.     

Extrastriate feedback to primary visual cortex in primates: a quantitative analysis of connectivity.

Knowledge-based or top-down influences on primary visual cortex (area V1) are believed to originate from information conveyed by extrastriate feedback axon connections. Understanding how this information is communicated to area V1 neurons relies in part on elucidating the quantitative as well as the qualitative nature of extrastriate pathway connectivity. A quantitative analysis of the connectivity based on anatomical data regarding the feedback pathway from extrastriate area V2 to area V1 in macaque monkey suggests (i) a total of around ten million or more area V2 axons project to area V1; (ii) the mean number of synaptic inputs from area V2 per upper-layer pyramidal cell in area V1 is less than 6% of all excitatory inputs; and (iii) the mean degree of convergence of area V2 afferents may be high, perhaps more than 100 afferent axons per cell. These results are consistent with empirical observations of the density of radial myelinated axons present in the upper layers in macaque area V1 and the proportion of excitatory extrastriate feedback synaptic inputs onto upper-layer neurons in rat visual cortex. Thus, in primate area V1, extrastriate feedback synapses onto upper-layer cells may, like geniculocortical afferent synapses onto layer IVC neurons, form only a small percentage of the total excitatory synaptic input.     

Synaptic morphology of the carotid body of the domestic fowl

Efferent and reciprocal synapses have been demonstrated in the carotid body of the domestic fowl (Gallus gallus domesticus). Synapses were also found with purely afferent morphology, but were probably components of reciprocal synapses. The general morphology of the endings suggested the presence of two types of axon, afferent axons making reciprocal and perhaps afferent synapses with Type I cells, and efferent axons making efferent synapses with Type I cells. A few axo-dendritic synapses were also found. The dense-cored vesicles associated with the afferent components of reciprocal synapses and with the possible true afferent synapses varied in diameter and core but could belong to one population of pre-synaptic vesicles. These observations are consistent wtih a new theory for the carotid body receptor mechanism. This proposes a spontaneously discharging afferent axon inhibited by an inhibitory transmitter substance released by the Type I cell via the "afferent" component of its reciprocal synapse, the "efferent" component inhibiting this release. Besides this chemoreceptor modulation of its afferent axon, the Type I cell may also have a general secretory function.     

Electrical excitability of taste cells. Mechanisms and possible physiological significance

Neuronal, muscle and some endocrine cells are electrically excitable. While in muscle and endocrine cells AP stimulates and synchronizes intracellular processes, neurons employ action potentials (APs) to govern discontinuous synapses located distantly. Meanwhile, such axonless sensory cells as photoreceptors and hair cells exemplify afferent output, which is not driven by APs; instead, gradual receptor potentials elicited by sensory stimuli control the release of afferent neurotransmitter glutamate. Mammalian taste cells of the type II and type III are electrically excitable and respond to stimulation by firing APs. Since taste cells also have no axons, physiological significance of the electrical excitability for taste transduction and encoding sensory information is unclear. Perhaps, AP facilitates transmitter release, ATP in type II cells and 5-HT in type III cells, although via different mechanisms. The ATP release is mediated by connexin hemichannels, does not require a Ca²⁺ trigger, and largely gated by membrane voltage. 5-HT secretion is driven by intracellular Ca²⁺ and involves VG Ca²⁺ channels. Here, we discuss ionic mechanisms of excitability of taste cells and speculate on a likely role of APs in mediating their afferent output.     

Neural pathways and innervation of cnidocytes in tentacles of sea anemones

Our previously published studies are here reviewed detailing neuro-cnidocyte synapses, demonstrating putative neurotransmitter substances, and identifying complex neural pathways in sea anemones. Synapses were traced to their contacts on nematocytes and spirocytes by transmission electron microscopy of serial thin sections of tentacles. In five animals, cells containing microbasic p-mastigophores had synapses with clear vesicles, whereas cells containing basitrichous isorhizas had synapses with dense-cored vesicles, providing preliminary evidence for a selectivity of neurotransmitter types for different nematocysts. Either clear or dense-cored synaptic vesicles were also present at neuro-spirocyte contacts. Antho-RFamide immunoreactivity occurred in some anthozoan synaptic vesicles and immunogold labeling of serotonin was found at a neuro-spirocyte synapse. Neural pathways included direct innervation of spirocytes by sensory cells, sequential neuro-neuro-spirocyte and neuro-neuro-nematocyte synapses and reciprocal synapses involving axons of both sensory cells and ganglion cells. Such synaptic patterns resemble neuro-effector pathways found in higher animals and lay to rest the independent effector hypothesis for cnidocyte discharge in tentacles of sea anemones.     

Neuromodulation of Transduction and Signal Processing in the End Organs of Taste

Chemical synapses transmit gustatory signals from taste receptorcells to sensory afferent axons. Chemical (and electrical) synapsesalso provide a lateral pathway for cells within the taste budto communicate. Lateral synaptic pathways may represent someform of signal processing in the peripheral end organs of taste.Efferent synaptic input may also regulate sensory transductionin taste buds. To date, the synaptic neurotransmitter(s) orneuromodulator(s) released at chemical synapses in taste budshave not been identified unambiguously. This paper summarizesthe attempts that have been made over the past 40 years to identifythe neuroactive substances acting at taste bud synapses. Wereview the four traditional criteria for identifying chemicaltransmitters elsewhere in the nervous system—localization,uptake/degradation, release and physiological actions—andapply these criteria to neuroactive substances in taste buds.The most complete evidence to date implicates serotonin as aneuromodulator of taste transduction in the end organs. However,studies also suggest that adrenergic, cholinergic and peptidergicneurotransmission may be involved in taste buds. Chem. Senses21: 353–365, 1996.     

Rat gustatory neurons in the geniculate ganglion express glutamate receptor subunits

Taste receptor cells are innervated by primary gustatory neurons that relay sensory information to the central nervous system. The transmitter(s) at synapses between taste receptor cells and primary afferent fibers is (are) not yet known. By analogy with other sensory organs, glutamate might a transmitter in taste buds. We examined the presence of AMPA and NMDA receptor subunits in rat gustatory primary neurons in the ganglion that innervates the anterior tongue (geniculate ganglion). AMPA and NMDA type subunits were immunohistochemically detected with antibodies against GluR1, GluR2, GluR2/3, GluR4 and NR1 subunits. Gustatory neurons were specifically identified by retrograde tracing with fluorogold from injections made into the anterior portion of the tongue. Most gustatory neurons in the geniculate ganglion were strongly immunoreactive for GluR2/3 (68%), GluR4 (78%) or NR1 (71%). GluR1 was seen in few cells (16%). We further examined if glutamate receptors were present in the peripheral terminals of primary gustatory neurons in taste buds. Many axonal varicosities in fungiform and vallate taste buds were immunoreactive for GluR2/3 but not for NR1. We conclude that gustatory neurons express glutamate receptors and that glutamate receptors of the AMPA type are likely targeted to synapses within taste buds.     

Efferent projections of dorsal root afferents in the spinal cord of the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Arborization of dorsal root afferents was studied in the lamprey spinal cord by the method of horseradish peroxidase transport. Direct evidence was obtained for the presence of efferent fibers in dorsal roots, representing collaterals that depart from ascending and descending intraspinal branches of sensory axons and travel towards the periphery through the adjacent roots.     

The distribution of and interactions between GABA-immunoreactive and non-immunoreactive processes presynaptic to afferents from campaniform sensilla on the trochanter of the locust leg

Summary Campaniform sensilla on the trochanter of the mesothoracic legs of the locust were backfilled with cobalt salts or horseradish peroxidase for light and electron microscopy. The distribution of the terminal branches of afferent neurones in the thoracic ganglia were described from wholemount preparations and from thick slices through the ganglia. Ultrathin sections of identified branches were processed with GABA antibodies using a post-embedding immunogold technique and examined in the electron microscope. Input synapses were observed on fine varicose branches in all regions of the terminal arborisations close to the sites of afferent output. The major branches neither make nor receive synapses. Seventy-two percent of the input synapses are made by processes immunoreactive for GABA. Immunoreactive and non-immunoreactive processes synapse onto afferent terminals in close proximity. In some instances GABA-immunoreactive processes presynaptic to an afferent are also presynaptic to a non-immunoreactive presynaptic processes strongly suggesting that different presynaptic influences can interact directly with each other.     

Bar synapses in the end buds of lamprey skin

Summary The end buds of lamprey epidermis have been considered to be similar to taste buds, but the synapses on the receptor cells are of the type with a dense mass surrounded by lucent vesicles, which is not found in gustatory cells. It is suggested that the end buds may belong to the lateralis sensory system and be involved in the light sensitivity of lamprey skin.     

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.     

Neurotransmission in the vestibular endorgans--glutamatergic transmission in the afferent synapses of hair cells.

In the sensory pathways the first synapse is that between hair cells and primary afferent neurons and its most likely neurotransmitter candidate has long been thought to be glutamate. A number of pharmacological and electrophysiological studies have lent credence to this theory (reviewed by Bledsoe et al. 1988, Bobbin 1979, Ehrenberger and Felix 1991, Puel et al. 1991; Puel 1995) as has recent neurochemical and immunocytochemical work (reviewed by Ottersen et al. 1998; Usami et al. 2000). These recent studies reveal that the afferent hair cell synapse resembles the central glutamate synapses in many ways. Of the proteins confirmed to be involved in signal transduction and transmitter metabolism at most central synapses, many are also seen in the afferent hair cell synapse, and have an analogous compartmentation. On the other hand, there are also important differences, especially those related to the molecular mechanisms that underlie transmitter release.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.