Species-specific behavioral patterns correlate with differences in synaptic connections between homologous mechanosensory neurons

We characterized the behavioral responses of two leech species, Hirudo verbana and Erpobdella obscura, to mechanical skin stimulation and examined the interactions between the pressure mechanosensory neurons (P cells) that innervate the skin. To quantify behavioral responses, we stimulated both intact leeches and isolated body wall preparations from the two species. In response to mechanical stimulation, Hirudo showed local bending behavior, in which the body wall shortened only on the side of the stimulation. Erpobdella, in contrast, contracted both sides of the body in response to touch. To investigate the neuronal basis for this behavioral difference, we studied the interactions between P cells. Each midbody ganglion has four P cells; each cell innervates a different quadrant of the body wall. Consistent with local bending, activating any one P cell in Hirudo elicited polysynaptic inhibitory potentials in the other P cells. In contrast, the P cells in Erpobdella had excitatory polysynaptic connections, consistent with the segment-wide contraction observed in this species. In addition, activating individual P cells caused asymmetrical body wall contractions in Hirudo and symmetrical body wall contractions in Erpobdella. These results suggest that the different behavioral responses in Erpobdella and Hirudo are partly mediated by interactions among mechanosensory cells.     

Sensorin-A immunocytochemistry reveals putative mechanosensory neurons inLymnaea CNS

The pond snailLymnaea stagnalis is a useful model system for studying the neural basis of behaviour but the mechanosensory inputs that impact on behaviours such as respiration, locomotion, reproduction and feeding are not known. InAplysia, the peptide sensorin-A appears to be specific to a class of central mechanosensory neurons. We show that in theLymnaea central nervous system sensorin-A immunocytochemistry reveals a discrete pattern of staining involving well over 100 neurons. Identifiable sensorin positive clusters of neurons are located in the buccal and cerebral ganglia, and a single large neuron is immunopositive in each pedal ganglion. These putative mechanosensory neurons are not in the same locations as previously identified motoneurons, interneurons or neurosecretory cells. As would be expected for a mechanoafferent, sensorin positive fibres were found in nerve tracts innervating the body wall. This study lays the foundation for future electrophysiological and behavioural analysis of these putative mechanosensory neurons.     

Calcium buffering and clearance in spider mechanosensory neurons

Spider VS-3 mechanoreceptor neurons have a low-voltage-activated Ca2+ current that raises intracellular calcium concentration [Ca2+] when they are depolarized by agonists of GABAA receptors or fire action potentials. The Ca2+ rise produces negative feedback by modulating the mechanoreceptor current and regulates Ca2+- and voltage-activated K+ currents. However, nothing is known about Ca2+ buffering in VS-3 neurons. Dynamic changes in VS-3 neuron intracellular [Ca2+] were measured using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (OG488) to understand Ca2+ buffering and clearance. Intracellular OG488 concentration increased slowly over more than 2 h as it diffused through a sharp intracellular microelectrode and spread through the cell. This slow increase was used to measure endogenous Ca2+ buffering and clearance by the added buffer technique, with OG488 acting as both added exogenous buffer and Ca2+ indicator. [Ca2+] was raised for brief periods by regular action potential firing, produced by pulsed electric current injection through the microelectrode. The resulting rise and fall of [Ca2+] were well fitted by the single compartment model of Ca2+ dynamics. With earlier ratiometric [Ca2+] estimates, these data gave an endogenous Ca2+ binding ratio of 684. Strong Ca2+ buffering may assist these neurons to deal with rapid changes in mechanical inputs.     

Lateral facilitation between primary mechanosensory neurons controls nose touch perception in C. elegans

The nematode C. elegans senses head and nose touch using multiple classes of mechanoreceptor neurons that are electrically coupled through a network of gap junctions. Using in?vivo neuroimaging, we have found that multidendritic nociceptors in the head respond to harsh touch throughout their receptive field but respond to gentle touch only at the tip of the nose. Whereas the harsh touch response depends solely on cell-autonomous mechanosensory channels, gentle nose touch responses require facilitation by additional nose touch mechanoreceptors, which couple electrically to the nociceptors in a hub-and-spoke gap junction network. Conversely, nociceptor activity indirectly facilitates activation of the nose touch neurons, demonstrating that information flow across the network is bidirectional. Thus, a simple gap-junction circuit acts as a coincidence detector that allows primary sensory neurons to integrate information from neighboring mechanoreceptors and generate somatosensory perception.     

The functional organization of cutaneous low-threshold mechanosensory neurons

Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aβ-, Aδ-, and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin (guard, awl/auchene, and zigzag hairs) is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aβ-, Aδ-, and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aβ-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.     

Centrally-mediated synaptic input: Effects on an identified crayfish mechanosensory interneuron

Summary High-level mechanosensory interneurons integrate a substantial amount of polysynaptic input. We have used an identified interneuron, the crayfish (Procambarus clarki) caudal photoreceptor (CPR), to examine the extent and specificity of interneuronal input as received by a physiologically complex, smalldiameter sensory unit. Presynaptic central neurons were identified by antidromic stimulation of connective fibers and characterized physiologically relative to the bilateral responses observed in the paired CPR's. Eleven inhibitory interneurons have been identified, including cells with ipsilateral (Fig. 4), contralateral (Fig. 5) and bilateral effects (Fig. 6). Seven excitatory interneurons have been identified, including examples from each of the respective categories above (Figs. 7–9). The results of this survey are summarized in Table 1; axon locations are presented in Fig. 10.It has also been demonstrated that several of these fibers are themselves ascending mechanoreceptive interneurons (e.g., fiber 122, Fig. 1). Thus, for the encoding of environmental stimuli, these results indicate that central integration involves a lateral exchange of tactile information among a set of interrelated sensory interneurons. However, the possibility still exists that some of these fibers represent descending pathways for central influence of local (segmental) integrative processes.Abbreviations CPR caudal photoreceptor - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential This work has been supported in part by a Research Fellowship from Bryn Mawr College (to G.A.M.) and by Research Grants from NIH (NS-12971-03) and the Whitehall Foundation. This paper is a contribution of the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association (No. 123).     

Nitric oxide modulates presynaptic afferent depolarization of mechanosensory neurons

In crayfish, movement of the tailfan causes stimulation of exteroceptive sensory hairs located on its surface. Movement is monitored by a proprioceptor, the protopodite-endopodite chordotonal organ within the tailfan. Proprioceptive afferents provide indirect presynaptic inhibitory inputs to sensory hair afferents in the form of primary afferent depolarizations (PADs). Bath application of nitric oxide (NO) substrates, donors and scavengers, and nitric oxide synthase (NOS) inhibitors had no effect on the responses of proprioceptive afferents during imposed movements of the chordotonal organ. In contrast, the amplitude of PADs in exteroceptive hair afferents was dependent on NO levels. NO levels were altered by bath-application of the NO-precursor L-arginine, the NO donor SNAP, the NOS-inhibitor L-NAME, and the NO scavenger PTIO, while changes in PAD amplitude were measured. Application of L-arginine or SNAP resulted in consistent decreases in PAD amplitude, whereas L-NAME and PTIO induced increases in PAD amplitude. These results suggest that endogenous NO decreases inhibitory inputs to exteroceptive neurons, thus enhancing transmitter release at their output synapses.     

Rapid firing rates from mechanosensory neurons in copepod antennules

Small detection distances coupled with rapid movements require copepods to respond to stimuli with behavioral latencies on the order of milliseconds. Receiving adequate sensory information in such a short time necessitates extremely rapid firing rates of the efferent receptors. Here we show that copepod mechanoreceptors can fire at frequencies up to 5 kHz in response to fluid mechanical stimuli. Neural activity at these frequencies enables these animals to code for a range of fluid velocities thus providing important information regarding the nature of different fluid disturbances.     

Dynamics underlying synaptic gain between pairs of cortical pyramidal neurons

Changes in connectivity between pairs of neurons can serve as a substrate for information storage and for experience-dependent changes in neuronal circuitry. Early in development, synaptic contacts form and break, but how these dynamics influence the connectivity between pairs of neurons is not known. Here we used time-lapse imaging to examine the synaptic interactions between pairs of cultured cortical pyramidal neurons, and found that the axon-dendrite contacts between each neuronal pair were composed of both a relatively stable and a more labile population. Under basal conditions, loss and gain of contacts within this labile population was well balanced and there was little net change in connectivity. Selectively increasing the levels of activated CaMKII in the postsynaptic neuron increased connectivity between pairs of neurons by increasing the rate of gain of new contacts without affecting the probability of contact loss, or the proportion of stable and labile contacts, and this increase required Calcium/calmodulin binding to CaMKII. Our data suggest that activating CaMKII can increase synaptic connectivity through a CaM-dependent increase in contact formation, followed by stabilization of a constant fraction of new contacts.     

Signaling between glia and neurons: focus on synaptic plasticity

Glial cells are now emerging from the shadows cast by their more excitable CNS counterparts. Within the developing nervous system, astrocytes and Schwann cells actively help to promote synapse formation and function, and have even been implicated in synapse elimination. In the adult brain, astrocytes respond to synaptic activity by releasing transmitters that modulate synaptic activity. Thus, glia are active participants in brain function. Many questions remain about the identity of glial-neuronal signals and their significance.     

Effects of heterogeneity in synaptic conductance between weakly coupled identical neurons

A significant degree of heterogeneity in synaptic conductance is present in neuron to neuron connections. We study the dynamics of weakly coupled pairs of neurons with heterogeneities in synaptic conductance using Wang–Buzsaki and Hodgkin–Huxley model neurons which have Types I and II excitability, respectively. This type of heterogeneity breaks a symmetry in the bifurcation diagrams of equilibrium phase difference versus the synaptic rate constant when compared to the identical case. For weakly coupled neurons coupled with identical values of synaptic conductance a phase locked solution exists for all values of the synaptic rate constant, α. In particular, in-phase and anti-phase solutions are guaranteed to exist for all α. Heterogeneity in synaptic conductance results in regions where no phase locked solution exists and the general loss of the ubiquitous in-phase and anti-phase solutions of the identically coupled case. We explain these results through examination of interaction functions using the weak coupling approximation and an in-depth analysis of the underlying multiple cusp bifurcation structure of the systems of coupled neurons.     

Calcium channel structural determinants of synaptic transmission between identified invertebrate neurons

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.     

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.     

Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear

In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.     

Motor neurons and Schwann cells distinguish between synaptic and extrasynaptic isoforms of laminin

Laminin is a major component of all basement membranes. However, its composition varies with location because there are numerous forms of each of the three chains (α, β, and γ) that together comprise this heterotrimeric molecule. In the neuromuscular system, motor neurons and Schwann cells encounter unique trimers of laminin at different sites. The question thus arises as to whether these local differences in laminin composition act to direct the behavior of these two classes of cells. To address this question, we compared the responses of cultured rat motor neurons and Schwann cells to three forms of rodent laminin purified in our laboratory: Laminin-1 (Lmn-1; α1β1γ1); Laminin-11 (Lmn-11), a synapse-specific isoform consisting of α5β2γ1chains; and a third preparation, a mixture of three kinds of laminin (Lmn-2/4/8), that is enriched for the α2, α4, β1, β2, and γ1 subunits. Schwann cells attached best to a substrate of Lmn-2/4/8 and showed the weakest adhesion on Lmn-11. Interestingly, no such difference was seen with motor neurons; all three substrates promoted neuronal adhesion, survival, and neurite initiation equally well. With longer time in culture, however, these embryonic motor neurons extended extremely long processes on Lmn-1 and on Lmn-2/4/8, while those on Lmn-11 bore shorter neurites with unusually large, flattened growth cones. These results demonstrate that the behavior of Schwann cells and motor neurons can be regulated directly by the local laminin composition. The precise geometric relationship of these cells at the neuromuscular junction may therefore reflect the unique composition of laminin at this synapse. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 339–358, 1998     

Estimation of the number of synaptic vesicles in asymmetric synapses between hippocampal neurons

Within the framework of the quantum hypothesis of synaptic transmission, the amount of a neurotransmitter released in a unitary event of calcium-dependent exocytosis corresponds to the content of a synaptic vesicle (SV). The number of these organelles in the presynaptic terminal is an important index characterizing the functional state of the given synapse. The technique of estimation of the dimension of the total SV pool, which is based on mathematical modeling and is realized in a computer experiment, is described. This technique allows one to interpret quantitative estimations obtained in the course of the analysis of images of random ultrathin sections of presynaptic terminals in the terms of 3D space. The capabilities of this technique are illustrated using an example of estimation of the size of the total SV pool in asymmetric synapses between neurons of the radial layer of the murine hippocampal CA1 area. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 219–223, May–June, 2006.