Non-telecentric two-photon microscopy for 3D random access mesoscale imaging.

Diffraction-limited two-photon microscopy permits minimally invasive optical monitoring of neuronal activity. However, most conventional two-photon microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric optical design, we present a low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.

Persistent sodium current is a target for cAMP-induced neuronal plasticity in a state-setting modulatory interneuron.

We have identified a TTX-resistant low-threshold persistent inward sodium current in the cerebral giant cells (CGCs) of Lymnaea, an important state-setting modulatory cell type of molluscan feeding networks. This current has slow voltage-dependent activation and de-activation kinetics, ultra-slow inactivation kinetics and fast de-inactivation kinetics. It activates at approximately -90 mV, peaks at approximately -30 mV, reverses at approximately +35 mV and does not show full voltage-dependent inactivation even at positive voltage steps. Lithium-sodium replacement experiments indicate that the persistent sodium current makes a significant contribution to the CGC membrane potential. Injection of cyclic adenosine monophosphate (cAMP) into the CGC cell body produces a large increase in the persistent sodium current that lasts for several hours. cAMP injection also leads to increased bursting, a significant decrease in the resistance and a significant depolarization of the soma membrane, indicating that cAMP-dependent mechanisms induce prolonged neuronal plasticity in the CGCs. Our observations provide the first link between cAMP-mediated modulation of a TTX-resistant persistent sodium current and prolonged neuronal plasticity in an identified modulatory cell type that plays an important role in behavioral state setting.

Automatic classification of interference patterns in driven event series: application to single sympathetic neuron discharge forced by mechanical ventilation.

This study proposes a method for the automatic classification of nonlinear interactions between a strictly periodical event series modelling the activity of an exogenous oscillator working at a fixed and well-known rate and an event series modelling the activity of a self-sustained oscillator forced by the exogenous one. The method is based on a combination of several well-known tools (probability density function of the cyclic relative phase, probability density function of the count of forced events per forcing cycle, conditional entropy of the cyclic relative phase sequence and a surrogate data approach). Classification is reached via a sequence of easily applicable decision rules, thus rendering classification virtually user-independent and fully reproducible. The method classifies four types of dynamics: full uncoupling, quasiperiodicity, phase locking and aperiodicity. In the case of phase locking, the coupling ratio (i.e. n: m) and the strength of the coupling are calculated. The method, validated on simulations of simple and complex phase-locking dynamics corrupted by different levels of noise, is applied to data derived from one anesthetized and artificially ventilated rat to classify the nonlinear interactions between mechanical ventilation and: (1) the discharges of two (contemporaneously recorded) single postganglionic sympathetic neurons innervating the caudal ventral artery in the tail and (2) arterial blood pressure. Under central apnea, the activity of the underlying sympathetic oscillators is perturbed by means of five different lung inflation rates (0.58, 0.64, 0.76, 0.95, 1.99 Hz). While ventilation and arterial pressure are fully uncoupled, ventilation is capable of phase locking sympathetic discharges, thus producing 40% of phase-locked patterns (one case of 2:5, 1:1, 3:2 and 2:2) and 40% of aperiodic dynamics. In the case of phase-locked patterns, the coupling strength is low, thus demonstrating that this pattern is sliding. Non-stationary interactions are observed in 20% of cases. The two discharges behave differently, suggesting the presence of a population of sympathetic oscillators working at different frequencies.

Resetting of sympathetic rhythm by somatic afferents causes post-reflex coordination of sympathetic activity in rat.

1. We have proposed previously that graded synchronous activity is produced by periodic inputs acting on weakly coupled or uncoupled oscillators influencing the discharges of a population of cutaneous vasoconstrictor sympathetic postganglionic neurones (PGNs) in anaesthetized rats. 2. Here we investigated the effects of somatic afferent (superficial radial nerve, RaN) stimulation, on the rhythmic discharges of this population. We recorded (1) at the population level from the ventral collector nerve and (2) from single PGNs focally from the caudal ventral artery of the tail. 3. Following RaN stimulation we observed an excitatory response followed by a period of reduced discharge and subsequent rhythmical discharges seemingly phase-locked to the stimulus. 4. We suggest that the rhythmical discharges following the initial excitatory response (conventional reflex) result from a resetting of sympathetic rhythm generators such that rhythmic PGN activity is synchronized transiently. We also demonstrate that a natural mechanical stimulus can produce a similar pattern of response. 5. Our results support the idea that in sympathetic control, resetting of multiple oscillators driving the rhythmic discharges of a population of PGNs may provide a mechanism for producing a sustained and coordinated response to somatic input.

Multiple types of control by identified interneurons in a sensory-activated rhythmic motor pattern.

Modulatory interneurons that can drive central pattern generators (CPGs) are considered as good candidates for decision-making roles in rhythmic behaviors. Although the mechanisms by which such neurons activate their target CPGs are known in detail in many systems, their role in the sensory activation of CPG-driven behaviors is poorly understood. In the feeding system of the mollusc Lymnaea, one of the best-studied rhythmical networks, intracellular stimulation of either of two types of neuron, the cerebral ventral 1a (CV1a) and the slow oscillator (SO) cells, leads to robust CPG-driven fictive feeding patterns, suggesting that they might make an important contribution to natural food-activated behavior. In this paper we investigated this contribution using a lip-CNS preparation in which feeding was elicited with a natural chemostimulant rather than intracellular stimulation. We found that despite their CPG-driving capabilities, neither CV1a nor SO were involved in the initial activation of sucrose-evoked fictive feeding, whereas a CPG interneuron, N1M, was active first in almost all preparations. Instead, the two interneurons play important and distinct roles in determining the characteristics of the rhythmic motor output; CV1a by modulating motoneuron burst duration and SO by setting the frequency of the ongoing rhythm. This is an example of a distributed system in which (1) interneurons that drive similar motor patterns when activated artificially contribute differently to the shaping of the motor output when it is evoked by the relevant sensory input, and (2) a CPG rather than a modulatory interneuron type plays the most critical role in initiation of sensory-evoked rhythmic activity.

Multiple oscillators provide metastability in rhythm generation.

Biological rhythms such as cardiac and circadian rhythms arise from activity of multiple oscillators with dispersed intrinsic frequencies. It has been proposed that a stable population rhythm, fundamental to normal physiological processes, can be achieved in these systems by synchronization, through mutual entrainment, of individual oscillators. Mutual entrainment, however, is unlikely to be the mechanism underlying the generation of a stable rhythm in a population of multiple weakly coupled or uncoupled oscillators. We have recently identified such a population that is involved in the sympathetic regulation of vascular tone in a thermoregulatory circulation. In this paper, we investigate the stability of the output rhythm of these sympathetic oscillators by subjecting the system to a periodic driving force (the lung inflation cycle-related activity). We show that a population rhythm coupled to the drive can remain stable over a much wider driving frequency range compared with that of any one of its constituent oscillators. This population rhythmicity still exists despite the fact that the dominant frequencies of individual oscillators are not necessarily 1:1 frequency-locked to the drive. We provide evidence to show that this population metastability is achieved through linear and nonlinear dynamic interactions between the driving force and single sympathetic oscillators. Our study suggests that the generation of a stable population rhythm can exist even in the absence of mutual entrainment of its constituents, and this allows the population to generate a stable and flexible patterned response.

A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea.

We show that appetitive and aversive conditioning can be analyzed at the cellular level in the well-described neural circuitries underlying rhythmic feeding and respiration in the pond snail, Lymnaea stagnalis. To relate electrical changes directly to behavior, the snails were first trained and the neural changes recorded at multiple sites in reduced preparations made from the same animals. Changes in neural activity following conditioning could be recorded at the level of motoneurons, central pattern generator interneurons and modulatory neurons. Of significant interest was recent work showing that neural correlates of long-term memory could be recorded in the feeding network following single-trial appetitive chemical conditioning. Available information on the synaptic connectivity and transmitter content of identified neurons within the Lymnaea circuits will allow further work on the synaptic and molecular mechanisms of learning and memory.

Sympathetic neuronal oscillators are capable of dynamic synchronization.

In this paper we show that the discharges of sympathetic neurons innervating an identified peripheral target are driven by multiple oscillators that undergo dynamic synchronization when an entraining force, central respiratory drive (CRD), is increased. Activity was recorded from postganglionic sympathetic neurons (PGNs) innervating the caudal ventral artery of the rat tail: (1) at the population level from the ventral collector nerve (VCN); and (2) from pairs of single PGNs recorded simultaneously using a focal recording technique. Autospectral analysis of VCN activity revealed a more prominent rhythmical component in the presence of CRD than in its absence, suggesting that (1) multiple oscillators drive the discharges of PGNs and (2) these oscillators can be entrained and therefore synchronized by CRD. This interpretation was supported by analysis of the firing behavior of PGN pairs. Autocorrelation and cross-correlation analysis showed that pairs were not synchronized in the absence of CRD but showed significant synchronization when CRD was enhanced. Time-evolving spectral analysis and raster plots demonstrated that the temporal stability of PGN-to-PGN and CRD-to-PGN interactions at a given level of CRD were also dynamic in nature, with stable constant phase relationships predominating as CRD was increased. This is the first reported example of dynamic synchronization in populations of single postganglionic sympathetic neurons, and we suggest that, as in sensory processing and motor control, temporal pattern coding may also be an important feature of neuronal discharges in sympathetic pathways.

Electrophysiological and behavioral analysis of lip touch as a component of the food stimulus in the snail Lymnaea.

Electrophysiological and video recording methods were used to investigate the function of lip touch in feeding ingestion behavior of the pond snail Lymnaea stagnalis. Although this stimulus was used successfully as a conditioning stimulus (CS) in appetitive learning experiments, the detailed role of lip touch as a component of the sensory stimulus provided by food in unconditioned feeding behavior was never ascertained. Synaptic responses to lip touch in identified feeding motoneurons, central pattern generator interneurons, and modulatory interneurons were recorded by intracellular electrodes in a semi-intact preparation. We showed that touch evoked a complex but characteristic sequence of synaptic inputs on each neuron type. Touch never simply activated feeding cycles but provided different types of synaptic input, determined by the feeding phase in which the neuron was normally active in the rhythmic feeding cycle. The tactile stimulus evoked mainly inhibitory synaptic inputs in protraction-phase neurons and excitation in rasp-phase neurons. Swallow-phase neurons were also excited after some delay, suggesting that touch first reinforces the rasp then swallow phase. Video analysis of freely feeding animals demonstrated that during normal ingestion of a solid food flake the food is drawn across the lips throughout the rasp phase and swallow phase and therefore provides a tactile stimulus during both these retraction phases of the feeding cycle. The tactile component of the food stimulus is strongest during the rasp phase when the lips are actively pressed onto the substrate that is being moved across them by the radula. By using a semi-intact preparation we demonstrated that application of touch to the lips during the rasp phase of a sucrose-driven fictive feeding rhythm increases both the regularity and frequency of rasp-phase motoneuron firing compared with sucrose applied alone.

Cellular traces of behavioral classical conditioning can be recorded at several specific sites in a simple nervous system.

We used a behavioral learning paradigm followed by electrophysiological analysis to find sites in the Lymnaea feeding network in which electrical changes could be recorded after appetitive conditioning. Specifically, we analyzed conditioning-induced changes in cellular responses in the mechanosensory conditioned stimulus (CS) pathway, in the central pattern generator (CPG) network, and in feeding motoneurons. During training, experimental animals received 15 pairings of lip touch (the CS) with sucrose (the unconditioned stimulus, US). Control animals received 15 random CS and US presentations. Electrophysiological tests on semi-intact preparations made from conditioned animals demonstrated a network correlate of the overall feeding conditioned response, a touch-evoked CPG-driven fictive feeding rhythm. At the motoneuronal level, we found significant conditioning-induced increases in the amplitude of an early touch-evoked EPSP and spike activity, recorded from the B3 feeding motoneuron. Increases in EPSP amplitude and motoneuronal spike activity could occur independently of conditioned fictive feeding. These changes in response recorded at the level of CPG interneurons, and motoneurons were preceded by changes recorded in the CS pathway. This was demonstrated by recording a conditioning-induced increase in the number of touch-evoked spikes in the cerebrobuccal connective, which forms part of the CS pathway. The finding that electrophysiological changes after conditioning can be recorded at multiple sites in this simple system provided an important intermediate level of analysis between whole animal behavior and cellular studies on the synaptic sites of plasticity.

Pattern-generating role for motoneurons in a rhythmically active neuronal network.

The role of motoneurons in central motor pattern generation was investigated in the feeding system of the pond snail Lymnaea stagnalis, an important invertebrate model of behavioral rhythm generation. The neuronal network responsible for the three-phase feeding motor program (fictive feeding) has been characterized extensively and divided into populations of central pattern generator (CPG) interneurons, modulatory interneurons, and motoneurons. A previous model of the feeding system considered that the motoneurons were passive followers of CPG interneuronal activity. Here we present new, detailed physiological evidence that motoneurons that innervate the musculature of the feeding apparatus have significant electrotonic motoneuron-->interneuron connections, mainly confined to cells active in the same phase of the feeding cycle (protraction, rasp, or swallow). This suggested that the motoneurons participate in rhythm generation. This was assessed by manipulating firing activity in the motoneurons during maintained fictive feeding rhythms. Experiments showed that motoneurons contribute to the maintenance and phase setting of the feeding rhythm and provide an efficient system for phase-locking muscle activity with central neural activity. These data indicate that the distinction between motoneurons and interneurons in a complex CNS network like that involved in snail feeding is no longer justified and that both cell types are important in motor pattern generation. This is a distributed type of organization likely to be a general characteristic of CNS circuitries that produce rhythmic motor behavior.

Neurophysiological correlates of unconditioned and conditioned feeding behavior in the pond snail Lymnaea stagnalis.

We used a behavioral appetitive learning paradigm followed by electrophysiological analysis to investigate the neuronal expression of appetitive conditioning in Lymnaea. We first established the levels of unconditioned and conditioned feeding responses in intact animals. We then demonstrated that neuronal correlates of both unconditioned responses to touch and food and a conditioned response to touch could be found in semi-intact preparations of the same animals that had been subjected to behavioral tests and conditioning trials. In the conditioning experiments, the experimental animals received 15 trials in which touch to the lips, the conditioned stimulus (CS), was paired with sucrose, the unconditioned food stimulus (US). Control animals received 15 presentations of either CS or US, or both, applied in a random manner. After training, a strong conditioned response to touch was established in the experimental but not in the control groups. For subsequent electrophysiological analysis of posttraining neuronal responses to the touch CS, semi-intact preparations were set up from the same animals that had been behaviorally conditioned or subjected to control procedures. Intracellular recordings, made from previously identified motoneurons of the feeding system, allowed the fictive feeding response to the CS to be monitored. In experimental preparations, touch applied to the lips evoked significantly more fictive feeding cycles than in controls, and this demonstrated the existence of a neurophysiological correlate of the appetitively conditioned response observed in the whole animals.

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis.

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis. J. Neurophysiol. 78: 2351-2362, 1997. An in vitro preparation was developed that allowed electrophysiological analysis of appetitive conditioning of feeding in the model molluscan system, Lymnaea. The network generating the feeding motor program (fictive feeding) is well characterized at the cellular level and consists of identified central pattern generator (CPG) interneurons, motor neurons, and modulatory interneurons. Activation of a modulatory interneuron, the slow oscillator (SO), evokes the three-phase fictive feeding rhythm in the same semi-intact preparations where tactile stimuli can be applied to the lips. By pairing touch as a conditioned stimulus (CS) with stimulation of the SO as an unconditioned stimulus (US), we established an effective in vitro paradigm for appetitive conditioning. Before training, touch to the lips evoked only brief and weak activity in the feeding interneurons and motor neurons. After 6-10 conditioning trials, there was a significant enhancement in the fictive feeding response to CS alone. This was not seen in controls (CS only, US only, random CS and US) and in preparations where there was no initial brief response to touch before conditioning. Direct recordings from the protraction phase N1M interneurons during in vitro conditioning indicated that the enhancement of the fictive feeding is due to an increased activation of these CPG cells by mechanosensory inputs from the lips. We also found that the conditioned response was not due to a facilitated activation of modulatory neurons in the feeding network, such as the SO or the cerebral giant cells (CGCs), because the activity of these cells remained unchanged after conditioning.

Behavioral function of glutamatergic interneurons in the feeding system of Lymnaea: plateauing properties and synaptic connections with motor neurons.

Intracellular recording techniques were used to examine the electrical properties and behavioral function of a novel type of retraction phase interneuron, the N2 ventral (N2v) cells in the feeding network of the snail Lymnaea. The N2vs were compared with the previously identified N2 cells that now are renamed the N2 dorsal (N2d) cells. The N2vs are a bilaterally symmetrical pair of electrotonically coupled plateauing interneurons that are located on the ventral surfaces of the buccal ganglia. Their main axons project to the opposite buccal ganglion, but they have an additional fine process in the postbuccal nerve. N2v plateaus that outlast the duration of the stimulus can be triggered by depolarizing current pulses and prematurely terminated by applied hyperpolarizing pulses. Gradually increasing the amplitude of depolarizing pulses reveals a clear threshold for plateau initiation. N2v plateauing persists in a high Mg2+/nominally zero Ca2+ saline that blocks chemical synaptic connections, suggesting an endogenous mechanism for plateau generation. The N2vs fire sustained bursts of action potentials throughout the N2/rasp phase of the fictive feeding cycle and control the retraction phase feeding motor neurons. The N2vs excite the B3 and B9 feeding motor neurons to fire during the rasp phase of the feeding cycle. They also inhibit the B7 and B8 feeding motor neurons. The B8 cells recover from inhibition and fire during the following swallowing phase. These synaptic connections appear to be monosynaptic as they persist in high Mg2+/high Ca2+ (HiDi) saline that blocks polysynaptic pathways. Strong current-induced plateaus in the N2vs generate brief inhibitory postsynaptic responses in the B4CL rasp phase motor neurons, but this was due to the indirect N2v --> N2d --> B4CL pathway. The N2vs are coupled electrotonically to the N2d cells, and triggering plateau in a N2v usually induced one or two spikes in a N2d. Previous experiments showed that the N2ds generate plateau potentials during a fictive feeding cycle. Here we show that the main component of the "plateauing" waveform is due to the electrotonic coupling with the N2v cells. The differential synaptic connections of the N2v and N2d cells with retraction phase motor neurons results in a sequence of motor neuron burst activity B9 --> B4CL --> B8 that produces the full retraction (rasp --> swallow) movements of the feeding apparatus (buccal mass). We conclude that the N2v cells are an essential component of the interneuronal network required to produce feeding motor neuron activity.

Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc.

A role for the NO-cGMP pathway in mediating chemosensory activation of feeding is suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase. In preparations reduced to isolated lips and CNS, intracellular recordings were made from motoneurons driven by the interneurons of the central pattern generator (CPG) for feeding. Fictive feeding in such preparations can be recorded from these motoneurons following the application of sucrose to the lips. Sucrose activation of fictive feeding is inhibited by the NO scavenger hemoglobin, the NO synthase inhibitor N omega-Nitro-L-Arginine Methyl Ester (L-NAME) and by methylene blue, an inhibitor of guanylyl cyclase. Fictive feeding in isolated lip-CNS preparations can be activated without sucrose by superfusion of NO donor molecules such as SNAP and hydroxylamine and by the nonhydrolyzable analog of cGMP, 8-bromo-cGMP. The feeding CPG can also be activated centrally by depolarizing a modulatory interneuron, the slow oscillator (SO). When the CPG is activated in this way, fictive feeding is not susceptible to inhibition by hemoglobin, the most potent of the inhibitors of sucrose-activated fictive feeding. Behavioral experiments on intact snails confirm the findings from in vitro experiments and show that hemoglobin prevents feeding and methylene blue significantly delays the onset of feeding. These results indicate (1) that NO is a putative chemosensory transmitter in the snail L. stagnalis, (2) that the NO-cGMP pathway can mediate chemosensory activation of specific patterns of centrally generated behavior, (3) that NO is not involved in transmission within the central network of neurons responsible for the behavior, and more generally (4) that a freely diffusing and highly reactive gaseous signalling molecule can have restricted and specific behavioral functions.