Granular Cells Of The Mormyrid Electrosensory Lobe And Postsynaptic Control Over Presynaptic Spikes Through Electric Synapse: With References To Computerized Modeling On This Action And Molecular Analysis Of Gap Junction In The Mormyrid Brain

Mormyrid electric fish, which come from South America and Africa, not only posses electric organs for generating moderate current in the form of electric organ discharges (EODs) but also have electroreceptors for sensing current in the water. Such fish detect nearby objects by distortions in the pattern of self-generated current that flows through their skin. People have been interested in mormyrid electric fish for more than 150 years because of its active electrosensory systems and its extraordinary size of cerebellum and cerebellum-like structure which the inputs from electroreceptors project to.Primary afferent fibers from electroreceptors in the skin terminate in the deep and superficial granular layers of the electrosensory lobe (ELL). The granular cells of these two layers relay electrosensory information to Purkinje-like cells and other higher order cells of ELL. Although much is known about the characteristics of both afferent fibers and higher order ELL cells, almost nothing is known about the granular cells, a key point that transfer information from primary afferents to higher order cells in ELL. Electromicroscopic study shows that primary afferent fibers terminate on granular cells with morphologically mixed electrical and chemical synapses. Electrical recording in vivo reveals that the afferent fibers from electroreceptors encode the intensity of local EOD current flow by the number of spikes and especially by the latency of the first spike in the EOD evoked afferent burst because there exhibits a smooth shortening of this latency with increasing stimulus intensity. Thus, it is very interesting for us to clarify how the granular cells decode the information from the afferents and relay information to higher order cells of ELL and what happens in detail at the synapse between afferent terminals and postsynaptic membrane of the granular cells.As we have known, granular cells receive both centrally originating input (corollary discharges from command nucleus which also project to electric organ, EOCD) and peripherally coming input. The EOCD input is considered to be an input with minimal variation and is hypothesized to be the centrally originating timing signal, while the peripheral input depends on the intensity of local EOD current that flows through electroreceptors. After comparing the latency of the first afferent spike with that of centrally originating EOCD signals, granular cells decode afferent latency and transfer the integrative information to higher order of ELL. Under this hypothesis, the granular cells will be an ideal and suitable point to investigate the coincidence detection. One goal of the present study was to determine how these functional properties control the synaptic transmission between afferent terminal and granular cell in ELL by using in vitro whole cell patch clamp recording in slices. The modulation of membrane potential of granular cells on the size of presynaptic spikes through electrical synapse (i.e. ephapse) was observed. Moreover, in order to test the hypothesis further, we used the data obtained from the first set of experiments to build a compartmental model that computerizes the mechanism upon the postsynaptic control over the occurrence and amplitude of presynaptic spike through electrical synapse.Although electrical synapses have been identified at a number of sites within electrosensory systems, and electromotor pathways of mormyrid electric fish, almost nothing is known about the structure of connexon which may be composed of different connexins attributing different permeability to the connexon or electrical synapse. Therefore, the present study also analysed the subtype of Connexin presenting at these sites and whole brain by using immunocytochemical techniques, with antibody against Connexin 36 (Cx36), and molecular biological assays.Part I: Granular Cells of the Mormyrid Electrosensory Lobe and Postsynaptic Control over Presynaptic Spike Occurrence and Amplitude through an Electrical SynapseWe examined the morphology and electrophysiology of superficial and deep granular cells in slice preparation with whole-cell patch clamp recordings combined with electrical stimulation of afferent fibers. Then, biocytin was injected into cells by iontophoresis after electrophysiological examinations on them being completed. Cell clamping and biocytin injection were carried out in slice under direct visual control and so that we could definitely determine the superficial and deep granular cells layer entirely. In some experiments, neurobiotin was placed on the cut end of the posterior lateral line nerve on one side and biotinylated dextran amine (BDA) was placed on the other side. Confocal microscope was used to examine dye-filled cells and dye coupling between labeled terminals and cells with fluorescent markers. Our results showed that:1. The superficial granular layer is thinner than the deep granular layer and contains cells that are smaller (3-5μm) and more densely packed. Superficial granular layer cells have basal dendrites that descend into the deep granular layer and may branch quite extensively. The axons ascend and may branch as they do so. The axons of superficial granular cells often pass through the ganglion layer to end in the lower molecular layer. The deep granular cells have round cell bodies (5-7μm) and 2 to 4 short, unbranched dendrites of approximately equal length extending out in all directions from the cell body. The dendrites end in claws that are similar to the claws at the ends of cerebellar granule cell dendrites. The axons of deep granular cells ascend, passing through the superficial granular layer to terminate with tight clumps of synaptic swellings in the plexiform and ganglion layers of ELL. The axons do not branch in traversing the granular layers and do not cross the ganglion layer into the molecular layer. Superficial granular cells showed clear and strong labeling on the side of ELL in which the neurobiotin-filled nerve terminated. Dye coupling was much less marked in the deep granular layer where only a few cells were lightly labeled.2. An afferent stimulation evoked a large "all-or-none" EPSP in granular cells and we proposed that the transmission is ephaptic in nature. Comparing these EPSPs with the responses evoked by a graded series of brief current injected into the same cells provides evidence that the "all-or-none" EPSPs are evoked by presynaptic spike rather than the graded rseponses as evoked by injected currents in the recorded cell. Pharmacological tests were also consistent with an ephaptic rather than a chemical transmission. The gap junction antagonist, carbenoxolone, reduced the responses, whereas the blockers against chemical transmission, just as cadmium or a combined application of NMDA and AMPA glutamate receptor antagonists, had no discernible effect on the EPSPs or EPSCs evoked by afferent stimulation. In addition, the short latency, fast rising time, and high following frequency also indicate that the EPSPs are electrically rather than chemically mediated.3. The amplitude of the EPSPs depended on postsynaptic membrane potential, with maximum amplitudes at membrane potentials between -65 and -110 mV. Hyperpolarization beyond this level resulted in either an abrupt disappearance of EPSPs, a step-like reduction to a smaller EPSP, or a graded reduction in EPSP amplitude. Depolarization to membrane potentials lower than that yielding a maximum caused a linear decrease in EPSP amplitude, with EPSP amplitude reaching zero mV at potentials between -55 and -40 mV.4. Afferent stimulation evoked large all-or-none EPSPs and large all-or-none GABAergic IPSPs in both superficial and deep granular cells. In some cells, both all-or-none IPSPs and all-or-none EPSPs were recorded on the same cell at the same or different stimulation position. Some classical IPSPs have expecting reversal potentials and can be blocked by Bicuculline, but some EPSPs only happened at more depolarized membrane potential. These results indicate that lateral inhibition at this first stage of the system is mediated by GABAa receptors. Also, some inhibitory transmitter may be released, at least in part, by a nonsynaptic excitation of the terminals.The most striking finding of the present study was that the amplitude and even the occurrence of the EPSP depended on the membrane potential of the postsynaptic cell. We hypothesize that the dependence of EPSP size on postsynaptic membrane potential is due to close coupling between the membrane potentials of pre- and postsynaptic elements. Our hypothesis is that when the postsynaptic membrane is strongly depolarized the presynaptic membrane is also depolarized and sodium channels in the presynaptic terminal are inactivated. Such presynaptic inactivation is removed with hyperpolarization, and the presynaptic spike becomes larger with increasing hyperpolarization, resulting in a larger EPSP. When the presynaptic membrane is too hyperpolarized, however, the incoming spike cannot reach threshold in the terminal and is blocked. Some of the EPSPs we recorded decreased abruptly in amplitude when the cell was hyperpolarized beyond the potential yielding a maximum EPSP. We suggest that two different afferent fibers were activated and that postsynaptic hyperpolarization blocked one before the other. Finally, other EPSPs decreased linearly in amplitude with hyperpolarization beyond the membrane potential yielding a maximum EPSP. We suggest that in these latter cases the presynaptic terminal may be unmyelinated for some distance distal to the terminal. Increasing hyperpolarization in an unmyelinated preterminal axon could push the occurrence of the spike further and further away from the terminal. The passively propagated spike at the terminal (and the EPSP in the postsynaptic cell) will be attenuated in proportion to the distance between the spike location and the terminal.ELL granular cells receive hyperpolarizing inhibitory input from ELL interneurons and depolarizing excitatory input from central sources linked to the EOD motor command, in addition to their primary afferent input. The effect of small changes in membrane potential on the size and occurrence of the EPSP suggests the possibility of various forms of non-linear interactions between the EPSP and the cell's other synaptic inputs.The cellular properties of ELL granular cells are consistent with measurement of relative timing of the afferent spike with that of a centrally originating EOCD signal. Such cellular properties include the following: fast rise times of the EPSPs involved; non -linear interactions among synaptic inputs; a capacity to generate spike bursts; and minimal convergence. Minimal convergence would be important to maintain good spatial resolution and because the capacity to measure afferent latency precisely would be reduced if a large number of afferents with different latencies converged onto the same cell. These features were indeed observed in our recordings from ELL granular cells. Part II: Postsynaptic Modulation of EPSP Size: a Modeling Study by Computerized SimulationPrevious experiments strongly support the hypothesis that the latency of afferent spike is used as a code for stimulus intensity in the active electrosensory system of mormyrid fish. From in vitro whole cell patch clamp recording experiment, we found that membrane potential of the granular cells control over presynaptic spike occurrence and amplitude. In order to test the mechanism underlies these phenomena we observed, we used the data obtained from the first set of experiments to build a computerized compartmental model that simulates the mechanism by which postsynaptic electrical changes modulates the size of presynaptic spike through electrical synapse, and thus the relative timing of two afferents inputs to granular cells also determines the effect of the afferent spike through the electrical synapse or ephapse. Our model has been implemented in the simulation environment provided by the software package NEURON.All our original choices for parameter values were estimated from cerebellar granule cells, which formed the original starting point for our model parameters due to their morphological similarity to the granular cells in the ELL. The design of the currents in the soma relied on the physiological properties of outward rectification and long time constant features. We assigned a non-inactivating potassium current, I_kv, to the soma and tuned the activation rates and maximum conductance to fit the data. We inserted an initial segment between the soma and axon with a high resistance and with exclusively passive properties. The axon was assigned as active membrane conductance to generate spikes, a fast sodium current I_Na and a delayed-rectifier current, I_K, with maximum conductance set to yield fast repetitive spiking at a rate proportional to the membrane depolarization, and we defined in terms of the Hodgkin-Huxley formalism for the afferent. We have tuned the model parameters to reflect the electrical properties of the neuronal circuit and have investigated weather the measured parameters of the cells could give rise to the relationship between the soma membrane potential and the size of the EPSP at the time of the arrival of afferent spikes.The model simulates many of the properties of the EPSP and its modulation by the membrane potential of the granular cell with only minimal assumptions. The results showed that: (1) we found a sharp rise time, large current, and long time constant of the EPSP when we injected current into afferent and generated a spike; (2) the current-voltage relationship at the model granular soma exactly reflects the I-V curve we got from the in vitro patch recording; (3) our model reproduced the dependence of the size of EPSP in granular cell on the holding membrane potential. We observed EPSP abrupt disappear at membrane potential about -90mV due to inhibition of the presynaptic spike, and the gradual reduction of EPSP size when depolarize membrane gradually, mainly due to Na⁺ channels inactivation.Our model replicates the essential features of the experimental data. In particular, we have shown that a reasonable choice of parameters can replicate the dependence of the EPSP size on the membrane potential, including the abrupt disappearance of the EPSP when the granular cell is extremely hyperpolarized. The model thus supports our hypothesis regarding the ephaptic transmission and its modulation.Part III: Identification of Gap Junctions and Some Circuitry Tracing in the Brain of Mormyrid Electric Fish: Immunocytochemical, Molecular Biological, and Tract Tracing AnalysesAlthough electric synapses have been identified morphologically and physiologically at a number of sites within the electrosensory systems, and electromotor pathways of mormyrid electric fish, almost nothing is known about the structure of connexon which may be composed of different connexins attributing different permeability to the connexon or electric synapse. Therefore, the present study also analysed the type of Connexin presenting at all these sites by using immunocytochemical techniques, with antibody against Connexin 36 (Cx36), and using molecular biological assays. The goals of the present study were to use immunocytochemistry and molecular biology techniques to determine the Connexin present at known gap junctions and to indicate hitherto unrecognized gap junction sites within the electrosensory and electromotor systems of mormyrid fish.Our results showed that: (1) The Western blots with the anti-Cx36 antibody showed specific labeling on Connexin proteins with molecular weight of about 35 kD in the mormyrid tissue, but show no labeling with the Cx45 antibody in the mormyrid tissue, though the specific labeling with the Cx45 antibody exhibits in the rat cerebellum. (2) The Cx36 antibody labeled several gap junctions in the mormyrid brain, that had been previously recognized in the electron microscope, including: synapses of mormyromast afferents in the electrosensory lobe (ELL); synapses of knollenorgan afferents in the nucleus of ELL (nELL); synapses of nELL axons in the anterior exterolateral nucleus of the mesencephalon (ELa); synapses in the electric organ discharge (EOD) command nucleus; synapses in the medullary relay nucleus (MRN); and eighth nerve synapses on Mauthner cells. (3) Staining with the antibody also indicated hitherto unrecognized gap junctions in the electrosensory preeminential nucleus and in the precommand (PCN) and ventral posterior nuclei (VPN). PCN and VPN were labeled retrogradely by injections in the command nucleus label terminals of these cells. (4) No staining was present in superficial granular cells of the electrosensory lobe on which primary afferent fibers terminate with gap junctions as indicated by electron microscopy, dye coupling, and electrophysiology. (5) We identified structures of the EOCD pathway as well as mesencephalic cells projecting to the command nucleus by injecting biotinylated dextran amine into the region of the medullary relay nucleus (MRN) and the nearby command nucleus. BCA, MCA and PCA all were anterogradely labeled by injection of BDA into the command nucleus. Our results were completely negative with regard to the possible presence of Connexin 35 in nuclei of the EOCD pathway.The present study using immunocytochemistry determined the Connexin 35 present at previously known gap junctions and found several unrecognized gap junction sites within the whole brain of mormyrid fish. The negative results in EOCD pathway and in superficial granular cells of the ELL quite surprise us. It suggests the possibility of a different gap junction protein at these sites. These results contribute to our understanding of the electrosensory and electromotor circuitry of the mormyrid fish.In conclusion, we first found membrane potential of postsynaptic granular cell can control over occurrence and amplitude of the presynaptic spike, and this dependence may result in functionally important non-linear interactions among synaptic inputs. Then, the compartmental model simulates many of the properties of the ephaptic transmission and its modulation by the membrane potential of the granular cell with only minimal assumptions. Our model supports the hypothesis we proposed from results of in vitro whole-cell patch clamp recording. At last, our immunocytochemical and molecular biological results contribute to our understanding of the distribution and function of gap junctions in electrosensory and electromotor circuitry of the mormyrid fish.