1Department of Physiology, University of Melbourne, Parkville, Victoria, Australia; 2Department of Internal Medicine, Sahlgrenska University Hospital, Göteborg, Sweden; 3Howard Florey Institute, Parkville, Victoria, Australia
Submitted 29 October 2004 ; accepted in final form 23 December 2004
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ABSTRACT |
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neural networks; recurrent excitation; computational modeling; enteric nervous system; submucous plexus; hypersecretion
Vasoactive intestinal peptide (VIP) neurons, which make up almost one-half the neurons in the submucous plexus (19), are the most likely candidates for the final secretomotor neuron population involved in neurogenic secretory diarrhea (9, 28, 35). In addition to projecting to the mucosa, the axons of VIP neurons can ramify through the submucous plexus for up to 3.5 mm (41) and project preferentially in a circumferential direction. When passing through adjacent ganglia, the VIP neurons have axonal varicosities and occasional varicose collaterals (14, 38). Furthermore, dual impalement experiments have shown that stimulating one VIP neuron will cause a slow excitatory postsynaptic potential (EPSP) in a nearby VIP neuron (38). This structural and electrophysiological evidence indicates that the population of VIP neurons form a recurrent network and that neurons in this network are likely to be very excitable because of this positive feedback (44).
Neurons with Dogiel type II morphology and large afterhyperpolarizing potentials (AHPs) in the submucous plexus are almost certainly intrinsic primary afferent (sensory) neurons (28), and they are also organized in recurrent networks. They have projections that can travel through the submucous plexus for up to 3 mm, have a preference for circumferential projection (41), and provide a large number of varicosities to their own and neighboring ganglia (14, 38). Myenteric sensory neurons transmit to each other by slow EPSPs (29), so it is likely that submucous sensory neurons do also. Unlike VIP neurons, however, sensory neurons have a prominent source of inhibition in the AHP (1, 4, 14, 20, 42), which causes strong accommodation in action potential firing. In the myenteric plexus, the slow EPSP suppresses the AHP (22), and it is likely the same thing occurs in the submucosa. In the myenteric plexus, the degree of suppression of the AHP very strongly influences the excitability of the sensory neuron network (44, 46).
The ENS also plays a major part in the hypersecretion induced by luminal secretagogues in the small intestine (12, 15, 16, 31). According to the prevailing hypothesis, the signal transduction between large luminal toxins like cholera toxin and the ENS is conveyed by the release of serotonin from enterochromaffin cells, which in turn leads to activation of the ENS via 5-hydroxytryptamine type 3 receptors (36, 48, 49), a nicotinic transmission step that may involve sensory neurons, interneurons, and/or secretomotor neurons (10, 11), and a final VIP-mediated secretomotor mechanism (9, 28, 35). This simple model disregards the network behavior of VIP neurons and/or sensory neurons and requires the ongoing release of serotonin to continuously activate the ENS. Currently there is a disagreement in the literature as to whether continued activation of the ENS is required for hypersecretion in the presence of luminal secretagogues (16, 31).
The present study investigated quantitatively how the recurrent networks in the submucous plexus respond to stimuli and maintain control of firing. It also examined whether sustained sensory input is required for overactivity or whether other conditions must be met for this to happen. This was done using a realistic computer model based on physiological and neuroanatomical data. The simulated neural circuit we studied incorporates the neurons in preparations of mucosa/submucosa used for the study of noncholinergic neurogenic secretion in Ussing chambers. In the Ussing chamber preparation, a segment of intestine was cut open and stripped of the circular and longitudinal muscle layers, which also removes the myenteric plexus and other exogenous input, leaving only the mucosa and submucous plexus. The resulting flat sheet is then mounted in Ussing flux chambers, which allow simultaneous recording of transmucosal current and ion fluxes resulting from electrogenic chloride transport across the mucosal epithelium. Firing in the network of VIP neurons in our model is a surrogate for the component of electrogenic chloride secretion that is not sensitive to muscarinic blockade in Ussing chamber studies (8, 13, 26).
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MATERIALS AND METHODS |
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The details of the model used to simulate individual neurons have been previously published (2, 6, 4446) and so are only summarized here. The model neurons are similar to "leaky integrate and fire" neurons (21), but the membrane potential, which is described by the usual equivalent circuit equation, is calculated from the underlying conductance changes and driving voltages rather than directly from currents. The conductances used in this model include those responsible for slow EPSPs, AHPs, action potentials, proximal process potentials (PPPs), and fast EPSPs.
Synaptic Transmission Model
Fast EPSPs were modeled by a stereotyped conductance change with a time course given by an -function (44, 46). Similarly, PPPs were modeled with a stereotyped conductance change with a more rapid time course also given by an
-function (44, 46). The PPPs were large enough to evoke an action potential in a neuron at rest, but not if the membrane was hyperpolarized (e.g., by an AHP).
Slow EPSPs were modeled by a set of equations chosen to reproduce the experimentally observed nonlinear stimulus response relationship (2), and parameters of the equations were systematically varied to reproduce EPSPs with time courses within the physiological range (8120 s, Table 1) (44, 46). Synaptic strength is a variable that represents receptor occupation, receptor-second messenger coupling, and formation of second messengers (2). Increasing the synaptic strength, while holding other parameters constant, increases the rate of rise of the slow EPSP, increases the membrane potential change for submaximal events and increases the duration of a slow EPSP but does not alter the maximum depolarization that can be achieved. Synaptic strength was varied (Table 1) to give slow EPSPs, in response to five stimuli at 1 Hz, with amplitudes between 1 mV and the maximum slow EPSP amplitude and durations between 5 and 120 s.
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Sensory neurons display a prominent AHP after the action potential, which is in turn suppressed by the slow EPSP (22). The AHP and its suppression by the slow EPSP was included in the model as previously described (44, 46).
Network Construction
A novel algorithm was developed to produce anatomically realistic networks of submucosal neurons. Ganglia were randomly assigned a position on a two-dimensional plane so that no ganglia were further than 700 µm apart (50) and no closer than 200 µm, because a single ganglion can be up to 200 µm in length (14). Internodal strands between ganglia were created so that each ganglion had between three and six internodal strands connecting it to ganglia within 700 µm. Each ganglion was filled with 8.15 ± 4.62 (all numbers are expressed as means ± SD) neurons (50). The combination of ganglia distribution and cell distribution within each ganglia gave an overall cell density that matched the observed 129.9 ± 16 neurons/mm2 (41). Each neuron was assigned as a sensory, VIP, cholinergic-secretomotor, or vasodilator neuron, while keeping the overall proportion of each class of neuron in close accordance with reported values (18, 41).
Pathways and synapses were created for the VIP neurons and sensory neurons by first assigning a projection length following the normally distributed patterns reported in the literature (41) and selecting a ganglion close to this distance from the cell body. The procedure for selecting the ganglion was set so that the projection length was within the maximum projection distances for the circumferential, oral, and anal directions (41). The algorithm introduced a bias so that neurons projected more circumferentially than longitudinally and slightly more anally than orally, in accordance with observations (41).
An axon, traveling along the internodal strands from the source neuron to the target ganglion, was created. The algorithm tried to match the reported values for the number of ganglia traversed (14, 38). However, it was not possible to closely match these reported values, while also keeping in close accordance with reported values for projection lengths and ganglia positions. Slightly increasing the number of ganglia traversed above the reported values enabled neurons to form either direct or indirect projections to their terminations that matched observations (3). Because it is hard to follow the dye-filled axons in the submucous plexus (3) and the number of ganglia traversed has only been reported twice (14, 38), it is quite possible that the number of ganglia traversed has been underestimated to date.
Making the assumption that varicose structures observed on axons are synaptic connections, each VIP neuron was set to make 0.50.75 synapses per ganglion traversed to match the reported value of 0.5 ± 0.15 varicosities per ganglion traversed for these neurons. Sensory neurons were set to make 11.012.0 per ganglion traversed to match the published value of 11.64 ± 2.04 varicosities per ganglion traversed (38).
Simulations
Networks representing a 5 x 5-mm (length by circumference) section of the submucous plexus were used in simulations of network activity for VIP neurons or VIP and sensory neurons. Networks were connected on the circumferential side, mimicking an intact tube. The 5 x 5-mm networks of VIP neurons contained 1,0001,200 neurons and networks of VIP neurons and sensory neurons contained 1,2001,500 neurons. A previous study simulating the myenteric plexus using similar models found that networks of 5 x 5 mm are large enough to avoid size-dependent artifacts and small enough to avoid excessive run times (46). Network activity is reported as action potential rate average over a class of neurons.
Several different stimulus protocols were used. For networks containing only VIP neurons, fast, slow, or both types of EPSPs were imposed on these neurons. Networks containing sensory neurons and VIP neurons were stimulated via PPPs applied to the sensory neurons (47). These inputs were randomly applied to the appropriate neurons but with a constant average frequency.
The following parameters were systematically varied as part of the study: maximum slow EPSP amplitude, synaptic strength, slow EPSP duration, and the relative size of the AHP after suppression by the slow EPSP () (Table 1). The first three parameters were varied for both VIP and sensory neurons, as appropriate. To a lesser extent the network structure was also varied.
Simulations were undertaken in software developed for this purpose and written in the C and Python programming languages. The differential equations describing neuron dynamics were integrated by using a third order, explicit, adaptive, Runge-Kutta method (39). Software is freely available by contacting the authors.
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RESULTS |
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Behavior of isolated VIP neuron networks. We numerically simulated large networks of VIP neurons. Random trains of exogenous EPSPs with constant mean frequency were played into neurons initially at rest. This input is the equivalent for the myenteric plexus to submucous plexus pathways that play a major role in the regulation of the submucous neural circuits (5, 33). The type of EPSP input was varied systematically, as was the maximum amplitude of the slow EPSP (range, 1025 mV).
Activity in the network reached a stable firing rate after 320 s of simulated time (18 h on an Intel Pentium-4 2.0 GHz processor). Increasing the frequency of the input caused an increase in stable firing rate (Fig. 1A). The average neuron firing rate in the network (10 Hz) was well below the maximum firing rate of these neurons in response to a current injection of
20 Hz.
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Once the network activity reached a stable firing rate, the input into the networks was removed and the resulting changes in network activity were observed. Three qualitative types of network activity were observed after removal of the input: uncontrolled, long decay, and quiescent (Fig. 2A). With uncontrolled behavior, activity settled to a high stable firing rate independent of the input frequency (Fig. 2B). The second type was called long decay, because the network activity decreased to zero or a low stable firing rate over a period of time longer than durations of individual slow EPSPs. Quiescent behavior was a rapid return to rest within a time period shorter than the duration of the slow EPSP.
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Interaction between VIP neuron and sensory neuron networks. The sensory neurons display a large number of varicose structures around all neuron types in the submucous plexus (14, 38) and, in this analysis, we assumed that sensory neurons can transmit to each other via slow EPSPs in analogy with equivalent neurons in the myenteric plexus (29). We also assumed there are connections from VIP neurons to sensory neurons that induce slow EPSPs in their targets (38). We performed numerical simulations of the combined networks stimulated by playing PPPs into the sensory neurons. This simulated the effect of a sensory stimulus lasting several seconds, equivalent to a sustained distension or chemical activation of the sensory neurons either by a nutrient or a toxin.
In this case, we varied the nature of transmission between the two populations of neurons and the maximum amplitudes of slow EPSPs in VIP neurons only. The maximum amplitudes of slow EPSPs in sensory neurons were held constant at threshold. We also varied the amount by which a slow EPSP suppressed the AHP in sensory neurons. This has been shown to be an important regulator of excitability in myenteric networks (44, 46).
When the AHP was not suppressed by the slow EPSP in sensory neurons, activity in these neurons quickly returned to rest after removal of the input stimulus (Fig. 3A). In this case, the VIP neurons behaved just as they did when the sensory neurons were not present and no stimulus was present (see above). However, when the slow EPSP was able to suppress the AHP in sensory neurons, the interactions between the two recurrent networks changed the behavior of the VIP neurons after the stimulus was removed.
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After cessation of the input into the sensory neurons, firing of VIP neurons returned to a stable nonzero rate over a substantially longer time than the duration of slow EPSPs in these neurons (Fig. 3D). This was reminiscent of the long decay seen in isolated VIP neuron networks. Ongoing sensory neuron activity or the interaction between the two populations of neurons drove ongoing activity in the VIP neurons for a network that would otherwise return to rest in the absence of input.
We tested how the combined networks responded to a second stimulus once both populations of neurons had reached a low stable firing rate (Fig. 4). This is the equivalent of initiating a second stimulus period in an Ussing chamber experiment. The response of the network during the second stimulus was dependent on the stimulus frequency. This graded firing occurred in both populations of neurons, and there was an amplification of activity from sensory neurons to VIP neurons. Once the second stimulus was withdrawn, the networks returned to the same low stable firing rate. This dependence on the input stimulus frequency and the return to the same low stable firing rate was observed whether or not there was transmission between sensory neurons; whether the transmission from sensory neurons to VIP neurons was via fast or slow EPSPs, or both; and for threshold or suprathreshold slow EPSP maximum depolarizations in VIP neurons.
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Behavior of isolated VIP networks. We systematically varied a number of neuron and network properties and determined their effects on network firing during combined fast and slow EPSP input. Varying the maximum slow EPSP amplitude from 10 to 25 mV could induce dramatic changes in the stable firing rates reached during the input (Fig. 5A). Increasing the synaptic strength at synapses between VIP neurons from 10 to 50 (Fig. 5B), increasing the slow EPSP duration from 10 to 120 s (Fig. 5C), or varying the network structure by increasing the number of synapses per ganglion traversed from 0.5 to 0.7 (Fig. 6) only produced small changes in the stable firing rate during fast and slow EPSP input.
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Figure 3B indicates that increased activity in VIP neurons leads to increased activity for sensory neurons, and Fig. 7A shows that increased activity in VIP neurons (by changing the type of transmission from sensory neurons to VIP neurons) results in increased activity in sensory neurons for the same degree of suppression of the AHP. When transmission between sensory neurons is blocked, the VIP neurons still drive activity in the sensory neurons (Fig. 3C). When the AHP was largely unsuppressed, there was little activity in the VIP neurons. Reducing the AHP (by increasing the degree of suppression) enabled the feedback of fast EPSPs to drive the VIP neurons to a moderate stable firing rate when the slow EPSP amplitude in VIP neurons was threshold. Increasing the maximum slow EPSP amplitude in VIP neurons to suprathreshold caused the VIP neurons to reach a high stable firing rate. In this analysis, when the slow EPSP amplitude in VIP neurons was set at threshold or suprathreshold, the synaptic strength and slow EPSP duration were set at values that would have resulted in a return to rest, if the VIP neurons were not receiving input from sensory neurons. Thus the high stable firing rates in VIP neurons depended on the ability of slow EPSPs in sensory neurons to suppress their characteristic AHPs and on transmission from sensory neurons to VIP neurons including a component mediated by fast EPSPs.
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DISCUSSION |
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Modeling in the Physiological Range
We studied the properties of isolated VIP neuron networks and their response to excitatory stimuli, which would represent input from either submucous sensory neurons or the myenteric plexus (5, 33). We varied the maximum depolarization of the slow EPSP and the type of input into VIP neurons (fast and/or slow EPSPs). When a stimulus was applied to the VIP neurons, the stimulus strength and transmission type largely determined the response for all mean maximum amplitudes of slow EPSPs. This graded response indicates that the VIP neurons are able to encode useful information without inhibition to control firing.
There were three qualitative types of network behavior after removal of the input stimulus to the VIP neurons, which indicates the significance of the positive feedback between VIP neurons. The three states are 1) quiescent state in which activity returned very rapidly to zero after removal of the stimulus, 2) long decay in which activity returned to a low or zero firing rate over a period of time longer than the slow EPSP duration, and 3) uncontrolled in which activity settled on a high firing rate. In quiescent networks, the positive feedback was insignificant in determining the activity for these neurons. The long decay state occurred because the VIP neurons almost, but not quite, had enough positive feedback to sustain network activity in the absence of any input. Finally, in uncontrolled networks, the positive feedback was sufficient to maintain activity for the population of VIP neurons.
Of these three different states, the long decay occurred when the slow EPSP parameters were in the previously described physiological range for a slow EPSP evoked in a VIP neuron by intracellular stimulation of another VIP neuron (38). This decay provides a possible explanation for the long decay in chloride secretion across the mucosa after electrical field stimulation (13, 26). This decay in chloride secretion is TTX-sensitive and has cholinergic and noncholinergic components (13, 26). We predict that blocking transmission between VIP neurons will abolish or decrease the noncholinergic component of the long decay in chloride secretion.
A major aim of the study was to perform simulations on the complex VIP and sensory neuron network. The sensory neuron network probably has recurrent positive feedback, and both the VIP and sensory neuron network are cross connected by excitatory connections (37, 38). Sensory neurons have a prominent AHP (1, 4, 14, 20, 42), which probably interacts with the slow EPSP to regulate firing in this network (44, 46). Physiologically, the AHP (in myenteric neurons) is suppressed by 90% in the presence of a slow EPSP (Johnson PJ and Bornstein JC, unpublished observations). When this was simulated in network of submucosal neurons, sensory neurons alone in the network reached a low stable firing rate. This is different from a previous study with myenteric sensory neurons (46) because of the variance in electrophysiological parameters of the sensory neurons and the structure of the sensory neuron network (see MATERIALS AND METHODS). This low stable firing rate in sensory neurons results in the VIP neurons being driven at low stable firing rates under conditions that would allow the VIP neurons to return to rest on their own (either quiescent or long decay, see above). Even when transmission between sensory neurons was blocked, the combined networks of sensory neurons and VIP neurons were able to reach a low stable firing rate because of the positive feedback between the two different populations of neurons.
Regardless of whether the low stable firing rate was due to the positive feedback within the sensory neurons or between the VIP neurons and sensory neurons, it was very robust and the networks could still encode useful information. Similarly, the low stable firing rate showed similar characteristics when the transmission from sensory neurons to VIP neurons was via fast or slow EPSPs or both (although the magnitude of the response changed as it did when stimulating the VIP neurons with similar exogenous synaptic inputs). This is important because there may be two subsets of submucosal sensory neurons distinguished by the presence of substance P or calcitonin gene-related peptide (CGRP) (20, 28, 30, 37). The sensory neurons with substance P, and not CGRP, are unlikely to transmit slow EPSPs to VIP neurons because VIP neurons do not have neurokinin receptors (34). It also means that addition of fast or slow EPSP input from other submucosal neurons should not affect the ability of these neurons to encode useful information, but only affect the magnitude and duration of the response.
The low stable firing rates in both populations of neurons are likely to be the basis of the observed tonic activity of submucosal neurons seen in Ussing chambers (7, 26). The tonic activity observed in Ussing chambers is TTX-sensitive and appears to be atropine-insensitive (7, 26). It is also depressed by norepinephrine and somatostatin, both of which hyperpolarize VIP neurons (40, 43) via mechanisms requiring neuronal activity (25). We predict that basal secretion will continue when sensory input is blocked. Furthermore, according to our model, tonic or spontaneous activity may arise from the sensory neurons or because of an interaction between the sensory neurons and VIP neurons. These two possibilities can be distinguished by examining the effect on basal electrogenic secretion of blocking transmission between sensory neurons and comparing this to blocking transmission from VIP to sensory neurons. Of course, testing these predictions will require identification of suitable antagonists specific to these types of synapses.
Whereas we predict basal secretion will continue in the absence of sensory input, inhibitory input onto the VIP neurons from sympathetic or myenteric ganglia may abolish this basal secretion. The effect of inhibitory input will depend on the size and duration of the inhibition, whether the tonic or spontaneous activity arises from the sensory neurons or from an interaction between the sensory neurons and VIP neurons and the presence of any sensory input or input from other neurons. Such variations mean that this basal secretion may or may not be observed in vivo.
Modeling Overactivity in the VIP Neurons
It is almost certain that the hypersecretion is due to overactivity in VIP neurons rather than cholinergic secretomotor neurons (9, 28, 35). Therefore, we investigated the conditions that produced overactivity in VIP neurons.
A high frequency input stimulus readily produced overactivity in the VIP neurons for isolated VIP neuron networks and for VIP and sensory neuron networks. When applying an exogenous stimulus to the VIP neurons in our model, fast EPSP transmission onto these neurons was required to achieve high rates over 60% of the maximum for these neurons. Similarly, when PPPs were played into the sensory neurons, fast EPSP transmission from sensory neurons to VIP neurons was required to achieve high firing rates. These results are in accordance with the observation that administration of hexamethonium prevents manifestation of cholera-induced hypersecretion (11) and indicates that hexamethonium is acting, at least in part, to block fast EPSP transmission onto the VIP neurons.
However, under these conditions, once the high frequency input stimulus is removed, the activity in VIP neurons quickly returned to low or zero firing rates. If Farthing (16) is correct in suggesting that the ENS does not require constant sensory drive during hypersecretion, then overactivity in the VIP neurons would continue in the absence of any input stimulus. Whereas it has been reported that preparations devoid of the myenteric plexus are unable to produce a hypersecretion response to cholera toxin (24), the role of the myenteric plexus may well be to amplify and spread the activity arising in the submucous plexus.
VIP neuron networks switched from the long decay type to the uncontrolled type by increasing the slow EPSP duration or increasing the synaptic strength between VIP neurons. However, the VIP neurons had two requirements for uncontrolled firing. The slow EPSPs in the VIP neurons needed to either have greater than normal amplitudes or greater than normal durations, and the VIP neurons need to make 0.75 synapses per ganglion traversed (or 34 synapses per VIP neuron, which is at the highest end of the reported range). The former requirement therefore leads to the prediction that, if continuous restimulation of the enteric neural circuits is not required, then toxins evoking such can directly affect the properties of the VIP neurons as reported by Jiang et al. (23). The latter suggests that hypersecretion would only occur under certain neuroanatomical conditions, which may explain segmental and species differences in the neurogenic component of cholera secretion.
The AHP may play a role in controlling activity in the submucous plexus. Increasing the amount by which the slow EPSP suppresses the AHP increases the low stable firing rate in both populations of neurons. Therefore, an alternative hypothesis for ongoing high firing within the VIP network is that the toxin can modulate the suppression of the AHP by slow EPSPs in the sensory neurons either via a direct effect on the sensory neurons themselves, or perhaps via an action of serotonin on a receptor other than the 5-HT3 receptor. For the change in AHP suppression to increase the activity in VIP neurons, one of two conditions had to be satisfied: transmission from sensory neurons to VIP neurons must be fast or the slow EPSPs in VIP neurons must be able to evoke action potentials. Both of these conditions are likely to be met under normal physiological circumstances making the suppression of the AHP a critical issue for further physiological experimentation.
In summary, our modeling study shows that positive feedback within submucous neuron networks provides an explanation for the observed decaying secretion response elicited by electrical stimuli and tonic activity leading to a basal secretion rate in the small intestine. The firing rate of the combined network in response to a given stimulus depends strongly on the amplitude of the slow EPSPs in VIP neurons and the interaction between the AHP and slow EPSPs in sensory neurons, but fast transmission onto VIP neurons also has an important role. Under certain conditions, the combined network can go into uncontrolled firing that persists even if the initial stimulus is removed. We propose that this is, in part, the mechanism behind the neurogenic component of cholera secretion in the small intestine in vivo.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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