Vladimir Brezina

Analyzing the functional consequences of transmitter complexity

Neurons and other cells are regulated by a great multiplicity of neurotransmitters, modulators, hormones and other chemical messengers, which, through complex networks of extensively diverging and converging pathways, exert a multiplicity of effects. How do we analyze the functioning of such a complex network? If the effects of a transmitter depend on the presence of many other transmitters, how can we predict what they will be? If multiple transmitters act through the same network, how can their actions be specific? If they converge on the same effects, are some of the transmitters redundant? Why are there so many transmitters? Such questions can be addressed using an analytical approach that examines, qualitatively or quantitatively, how the operation of the network globally maps a multidimensional input space of transmitters to a multidimensional output space of effects.

Functional uncoupling of linked neurotransmitter effects by combinatorial convergence.

Physiological signaling pathways both diverge and converge—a single neurotransmitter can have multiple effects and multiple transmitters can have the same effects—in the same target cell. Divergence couples the effects of a transmitter together in a relatively fixed ratio. Different physiological circumstances may require a different ratio, however; the coupling must be made modifiable. This can be achieved through convergence. If two transmitters couple the effects in different ratios, then combinations of the transmitters can yield all intermediate ratios of the effects, thus functionally uncoupling them. This mechanism is analyzed in a well-understood, simple invertebrate neuromuscular circuit.

Beyond the wiring diagram: signalling through complex neuromodulator networks

During the computations performed by the nervous system, its ‘wiring diagram’—the map of its neurons and synaptic connections—is dynamically modified and supplemented by multiple actions of neuromodulators that can be so complex that they can be thought of as constituting a biochemical network that combines with the neuronal network to perform the computation. Thus, the neuronal wiring diagram alone is not sufficient to specify, and permit us to understand, the computation that underlies behaviour. Here I review how such modulatory networks operate, the problems that their existence poses for the experimental study and conceptual understanding of the computations performed by the nervous system, and how these problems may perhaps be solved and the computations understood by considering the structural and functional ‘logic’ of the modulatory networks.

Physiology and biochemistry of peptidergic cotransmission in Aplysia

The marine mollusc Aplysia, whose simple nervous system facilitates study of the neural basis of behavior, was used to investigate the role of peptidergic cotransmission in feeding behavior. Several novel modulatory neuropeptides were purified and localized to identified cholinergic motoneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motoneurons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change.

A specific synaptic pathway activates a conditional plateau potential underlying protraction phase in the Aplysia feeding central pattern generator

A common feature in the architecture of neuronal networks is a high degree of seemingly redundant synaptic connectivity. In many cases, the synaptic inputs converging on any particular neuron all use the same neurotransmitter and appear to be fundamentally equivalent. Here, we analyze a striking counterexample in which such inputs are not equivalent and, as a result, play very different roles in the generation of the pattern of activity produced by the network. In the feeding central pattern generator of Aplysia, the pattern-initiating neuron B50 elicits motor programs by exciting the plateauing neuron B31/B32 in two ways: directly and indirectly through neuron B63. All of the synaptic connections use ACh. Despite the direct input of B50 to B31/B32, the indirect pathway of exciting B31/B32 through B63 is required for B50 to elicit the B31/B32 plateau potential and the motor program. We dissect this requirement using the muscarinic cholinergic antagonist pirenzepine. Pirenzepine blocks the B50-elicited motor program, the plateau potential in B31/B32, and, notably, a slow component of the EPSP elicited in B31/B32 by B63 but not that elicited by B50. The muscarinic agonist oxotremorine restores the plateau potential in B31/B32 and eliminates the necessity for B63 in B50-elicited motor programs. Together, our analysis shows that the plateau potential in B31/B32 is not endogenous but conditional, furthermore conditional on one particular synaptic input, that from B63. Thus, among several inputs to B31/B32 that use the same transmitter, the input from B63 is functionally distinct in its preferential access to the plateau potential that represents the committed step toward the initiation of a motor program.

Distinct mechanisms produce functionally complementary actions of neuropeptides that are structurally related but derived from different precursors

Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.

State Dependence of Network Output: Modeling and Experiments

Emerging experimental evidence suggests that both networks and their component neurons respond to similar inputs differently, depending on the state of network activity. The network state is determined by the intrinsic dynamical structure of the network and may change as a function of neuromodulation, the balance or stochasticity of synaptic inputs to the network, and the history of network activity. Much of the knowledge on state-dependent effects comes from comparisons of awake and sleep states of the mammalian brain. Yet, the mechanisms underlying these states are difficult to unravel. Several vertebrate and invertebrate studies have elucidated cellular and synaptic mechanisms of state dependence resulting from neuromodulation, sensory input, and experience. Recent studies have combined modeling and experiments to examine the computational principles that emerge when network state is taken into account; these studies are highlighted in this article. We discuss these principles in a variety of systems (mammalian, crustacean, and mollusk) to demonstrate the unifying theme of state dependence of network output.

Changes of Internal State Are Expressed in Coherent Shifts of Neuromuscular Activity in Aplysia Feeding Behavior

The multitasking central pattern generator (CPG) that drives consummatory feeding behaviors of Aplysia can produce ingestive, egestive, and intermediate motor programs. External stimuli trigger the programs but, remarkably, do not directly specify which type of program is produced. Rather, recent work has proposed, the type of program is determined by the internal network state of the CPG that has developed in response to the previous history of the stimulation. Here we have tested a key prediction of this network-state hypothesis. If the network state has a real existence and governs real functional behavior, changes in the state should be seen as coherent, coordinated changes along many dimensions of interneuron and motor neuron activity, muscle contraction, and ultimately movement, that underlie functional behavior. In reduced neuromuscular preparations, we elicited repetitive motor programs by continued stimulation of the esophageal nerve while recording the firing of motor neurons B8, B15, B16, B4/5, and B48, and contractions of the accessory radula closer and I7-I10 muscles that respectively close and open the animals food-grasping organ, the radula. Using sonomicrometric techniques, we similarly recorded the movement of the radula in the complete buccal mass. Successive esophageal nerve programs indeed exhibited clear progressive changes in motor neuron firing, muscle contractions, and the phasing of radula movements within each cycle, from an initially intermediate or even ingestive character to a strongly egestive character. We conclude that the Aplysia feeding CPG really has a coherent internal network state whose dynamics are likely to be reflected in the real behavior of the animal.

Variability of Motor Neuron Spike Timing Maintains and Shapes Contractions of the Accessory Radula Closer Muscle of Aplysia

The accessory radula closer (ARC) muscle of Aplysia has long been studied as a typical “slow” muscle, one that would be assumed to respond only to the overall, integrated spike rate of its motor neurons, B15 and B16. The precise timing of the individual spikes should not much matter. However, but real B15 and B16 spike patterns recorded in vivo show great variability that extends down to the timing of individual spikes. By replaying these real as well as artificially constructed spike patterns into ARC muscles in vitro, we examined the consequences of this spike-level variability for contraction. Replaying the same pattern several times reproduces precisely the same contraction shape: the B15/B16–ARC neuromuscular transform is deterministic. However, varying the timing of the spikes produces very different contraction shapes and amplitudes. The transform in fact operates at an interface between “fast” and “slow” regimens. It is fast enough that the timing of individual spikes greatly influences the detailed contraction shape. At the same time, slow integration of the spike pattern through the nonlinear transform allows the variable spike timing to determine also the overall contraction amplitude. Indeed, the variability appears to be necessary to maintain the contraction amplitude at a robust level. This phenomenon is tuned by neuromodulators that tune the speed and nonlinearity of the transform. Thus, the variable timing of individual spikes does matter, in at least two, functionally significant ways, in this “slow” neuromuscular system.

Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of aplysia

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.