Neural correlates of habituation of the proleg withdrawal reflex in larvae of the hawk moth, Manduca sexta

The larval proleg withdrawal reflex of the hawk moth, Manduca sexta, exhibits robust habituation. This reflex is evoked by deflecting one or more mechanosensory planta hairs on a proleg tip. We examined neural correlates of habituation in an isolated proleg preparation consisting of one proleg and its segmental ganglion. Repeated deflection of a single planta hair caused a significant decrease in the number of action potentials evoked in the proleg motor nerve (which carries the axons of proleg retractor motor neurons). Significant response decrement was seen for interstimulus intervals of 10 s, 60 s and 5 min. Response decrement failed to occur in the absence of repetitive stimulation, the decremented response recovered spontaneously following a rest, and electrical stimulation of a body wall nerve facilitated the decremented response (a neural correlate of dishabituation). Adaptation of sensory neuron responses occurred during repeated hair deflections. However, when adaptation was eliminated by direct electrical stimulation of sensory neurons, the response in the proleg motor nerve still decreased significantly. Muscle recordings indicated that the response of an identified proleg retractor motor neuron decreased significantly during habituation training. Thus, habituation of the proleg withdrawal reflex includes a central component that is apparent at the level of a single motor neuron. Accepted: 20 December 1996     

Transfer of habituation in Aplysia: contribution of heterosynaptic pathways in habituation of the gill-withdrawal reflex

Habituation of the Aplysia gill-withdrawal reflex (and siphon-withdrawal reflex) has been attributed to low-frequency homosynaptic depression at central sensory-motor synapses. The recent demonstration that transfer of habituation between stimulation sites occurs in this model system has prompted the hypothesis that heterosynaptic inhibitory pathways also play a role in the mediation of habituation behavior. To test this hypothesis, the sites and mechanisms of neural plasticity which underlie transfer of habituation in Aplysia were examined. Transfer of habituation is a reduction in the reflex evoked at one stimulation site (siphon) due to repeated presentation of a stimulus to a second site (gill). Centrally mediated transfer of habituation, measured in a preparation lacking the siphon-gill peripheral nervous system (PNS), was associated with a reduced excitatory response in central motor neurons. Repeated tactile stimulation of the gill did not attenuate the gill response evoked by electrical stimulation of the branchial nerve nor the mechanoreceptor response recorded in LE sensory neurons. In contrast, repeated stimulation of siphon or gill at a site which was "off" the sensory field of a specific mechanoreceptor led to a diminution in synaptic transmission between that sensory neuron and its followers (motor neurons and inter-neurons). These data demonstrate that centrally mediated transfer of habituation results from heterosynaptic modulation of synaptic transmission at the sensory-motor (and sensory-interneuron) synapses. Therefore, habituation behavior in Aplysia is mediated through the conjoint action of homosynaptic and heterosynaptic inhibitory processes.     

Associative neural network model for the generation of temporal patterns. Theory and application to central pattern generators.

Cyclic patterns of motor neuron activity are involved in the production of many rhythmic movements, such as walking, swimming, and scratching. These movements are controlled by neural circuits referred to as central pattern generators (CPGs). Some of these circuits function in the absence of both internal pacemakers and external feedback. We describe an associative neural network model whose dynamic behavior is similar to that of CPGs. The theory predicts the strength of all possible connections between pairs of neurons on the basis of the outputs of the CPG. It also allows the mean operating levels of the neurons to be deduced from the measured synaptic strengths between the pairs of neurons. We apply our theory to the CPG controlling escape swimming in the mollusk Tritonia diomedea. The basic rhythmic behavior is shown to be consistent with a simplified model that approximates neurons as threshold units and slow synaptic responses as elementary time delays. The model we describe may have relevance to other fixed action behaviors, as well as to the learning, recall, and recognition of temporally ordered information.     

Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.     

Effect of selective protein kinase C inhibitor on synaptic plasticity of command neurons of defensive behaviour during sensitization in snails

Polymyxin B (proteinkinase C inhibitor) effects on nociceptive sensitization of semiintact preparation were investigated in LP11 and RP11 snail neurons. It was found that application of sensitizing stimuli to control snail head initiated neural membrane depolarization, increase its excitability as well as depression of neural responses evoked by sensory stimulation during short-term stage. Polymyxin B application suppressed neural responses evoked by sensitizing (nociceptive) stimuli. At the same time changes in neural membrane excitability as well as neural responses evoked by tactile stimulation of snail foot or chemical stimulation of snail head were similar with ones in control snails. Polymyxin Bdid does not change the depression of neural responses evoked by tactile stimulation of snail head during short-term stages of sensitization but significantly suppressed facilitation of neural responses evoked by tactile stimulation of snail head during long-term stage of sensitization. It was suggested that proteinkinase C is involved in regulation of nociceptive mechanisms as well as in plasticity selective induction mechanisms in command neuron synaptic inputs activated by tactile stimulation of snail head.     

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted,Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.     

Critical role of intracellular calcium in plasticity mechanisms of defense behavior command neurons LPl1 and PPl1 of Helix lucorum during nociceptive sensitization

The role of intracellular calcium in changes in excitability and responses of defense behavior command neurons LP11 and PP11 of Helix lucorum to sensory stimulation was investigated in semi-intact preparation of a snail during nociceptive sensitization. It was found that application of sensitizing stimuli onto the snail's head initiated membrane depolarization, increase in its excitability as well as depression of neural responses evoked by sensory stimuli in short-term period of sensitization and significant facilitation of neural responses in long-term period of sensitization. To elucidate the contribution of LP11 and PP11 neurons in plasticity rearrangements involved in the mechanisms of sensitization, we applied sensitizing stimuli during strong hyperpolarization of the neurons or after intracellular injection of calcium chelators. Application of sensitizing stimuli during hyperpolarization of the neurons suppressed the increase in membrane excitability and depressed the neural responses evoked by chemical stimulation of snail's head i.m. short- and long-term periods of sensitization. At the same time, synaptic facilitation of neural responses evoked by tactile stimulation of snail's head and foot was observed, which was similar to synaptic facilitation in the control sensitized snail. Intracellular injection of EGTA or BARTA (calcium chelators) before sensitization suppressed synaptic facilitation in neural responses evoked by sensory stimulation. Under these conditions, the increase in excitability was more pronounced then in the control snail neurons. The experimental results suggest the changes in neural responses evoked by sensory stimulation in sensitized snails involve postsynaptic calcium-dependent mechanisms of plasticity in LP11 and PP11 neurons.     

Gap junctional communication among motor and other neurons shapes patterns of neural activity and synaptic connectivity during development

We are studying the functional roles of neuronal gap junctional coupling during development, using motor neurons and their synapses with muscle fibers as a model system. At neuromuscular synapses, several studies have shown that the relative pattern of activity among motor inputs competing for innervation of the same target muscle fiber determines how patterns of innervation are sculpted during the first weeks after birth. We asked whether gap junctional coupling among motor neurons modulates the relative timing of motor neuron activity in awake, behaving neonatal mice. We found that the activity of motor neurons innervating the same muscle is temporally correlated perinatally, during the same period that gap junction-mediated electrical and dye coupling are present. In vivo blockade of gap junctions abolished temporal correlations in motor neuron activity, without changing overall motor behavior, motor neuron activity patterns or firing frequency. Together with preliminary studies in mice lacking gap junction protein Cx40, our data suggest that developmentally regulated gap junctional coupling among motor and other neurons affects the activity in nascent neural circuits and thus in turn affects synaptic connectivity. Dynamic monitoring of dye coupling can be used to explore this possibility in normal mice and in mice lacking gap junction proteins during embryonic and neonatal development.     

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons).Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.Abbreviations AL antennal lobe - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - IPSP inhibitory postsynaptic potential - LN local interneuron - MGC macroglomerular complex - OB olfactory bulb - PN projection neuron - TES N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid     

Activity in a Premotor Cortical Nucleus of Zebra Finches Is Locally Organized and Exhibits Auditory Selectivity in Neurons but Not in Glia

Motor functions are often guided by sensory experience, most convincingly illustrated by complex learned behaviors. Key to sensory guidance in motor areas may be the structural and functional organization of sensory inputs and their evoked responses. We study sensory responses in large populations of neurons and neuron-assistive cells in the songbird motor area HVC, an auditory-vocal brain area involved in sensory learning and in adult song production. HVC spike responses to auditory stimulation display remarkable preference for the bird''s own song (BOS) compared to other stimuli. Using two-photon calcium imaging in anesthetized zebra finches we measure the spatio-temporal structure of baseline activity and of auditory evoked responses in identified populations of HVC cells. We find strong correlations between calcium signal fluctuations in nearby cells of a given type, both in identified neurons and in astroglia. In identified HVC neurons only, auditory stimulation decorrelates ongoing calcium signals, less for BOS than for other sound stimuli. Overall, calcium transients show strong preference for BOS in identified HVC neurons but not in astroglia, showing diversity in local functional organization among identified neuron and astroglia populations.     

Physiological properties and response modulations of mushroom body feedback neurons during olfactory learning in the honeybee, Apis mellifera

Mushroom bodies are central brain structures and essentially involved in insect olfactory learning. Within the mushroom bodies γ-aminobutyric acid (GABA)-immunoreactive feedback neurons are the most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was investigated by analyzing modulations of odor responses of feedback neurons during olfactory learning in vivo. In the honeybee, Apis mellifera, feedback neurons were intracellularly recorded at their neurites. They produced complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials indicate that their synaptic input region lies within the lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of different neurons to the same odor were highly variable. Response modulations were determined by comparing odor responses of feedback neurons before and after one-trial olfactory conditioning or sensitisation. Shortly after pairing an odor stimulus with a sucrose reward, odor-induced spike activity of feedback neurons decreased. Repeated odor stimulations alone, equally spaced as in the conditioning experiment, did not affect the odor-induced excitation. A single sensitisation trial also did not alter odor responses. These findings indicate that the level of odor-induced inhibition within the mushroom bodies is specifically modulated by experience. Accepted: 9 September 1999     

Auditory input to motor neurons of the dorsal longitudinal flight muscles in a noctuid moth (Barathra brassicae L.)

Summary Motor neurons innervating the dorsal longitudinal muscles of a noctuid moth receive synaptic input activated by auditory stimuli. Each ear of a noctuid moth contains two auditory neurons that are sensitive to ultrasound (Fig. 1). The ears function as bat detectors. Five pairs of large motor neurons and three pairs of small motor neurons found in the pterothoracic ganglia innervate the dorsal longitudinal (depressor) muscles of the mesothorax (Figs. 2 to 5). In non-flying preparations the motor neurons receive no oscillatory synaptic input. Synaptic input to a cell resulting from ultrasonic stimulation is consistent and can be either depolarizing or hyperpolarizing (Figs. 6 to 9). Quiescent neurons only rarely fire a spike in response to auditory inputs. Motor neurons in flying preparations receive oscillatory synaptic drive from the flight pattern generator and usually fire a spike for each wingbeat cycle (Figs. 10 to 12). Ultrasonic stimulation can provide augmented synaptic drive causing a neuron to fire two spikes per wingbeat cycle thus increasing flight vigor (Fig. 11). The same stimulus presented on another occasion can also inhibit spiking in the same motor neuron, but the rhythmic drive remains (Fig. 12). Thus, when the flight oscillator is running auditory stimuli can modulate neuronal responses in different ways depending on some unknown state of the nervous system. Sound intensity is the only stimulus parameter essential for activating the auditory pathway to these motor neurons. The intensity must be sufficient to excite two or three auditory neurons. The significance of these responses in relation to avoidance behavior to bats is discussed.     

Visual response properties of neck motor neurons in the honeybee

Recent behavioural studies have demonstrated that honeybees use visual feedback to stabilize their gaze. However, little is known about the neural circuits that perform the visual motor computations that underlie this ability. We investigated the motor neurons that innervate two neck muscles (m44 and m51), which produce stabilizing yaw movements of the head. Intracellular recordings were made from five (out of eight) identified neuron types in the first cervical nerve (IK1) of honeybees. Two motor neurons that innervate muscle 51 were found to be direction-selective, with a preference for horizontal image motion from the contralateral to the ipsilateral side of the head. Three neurons that innervate muscle 44 were tuned to detect motion in the opposite direction (from ipsilateral to contralateral). These cells were binocularly sensitive and responded optimally to frontal stimulation. By combining the directional tuning of the motor neurons in an opponent manner, the neck motor system would be able to mediate reflexive optomotor head turns in the direction of image motion, thus stabilising the retinal image. When the dorsal ocelli were covered, the spontaneous activity of neck motor neurons increased and visual responses were modified, suggesting an ocellar input in addition to that from the compound eyes.     

Predicting synchronous firing of large neural populations from sequential recordings

A major goal in neuroscience is to understand how populations of neurons code for stimuli or actions. While the number of neurons that can be recorded simultaneously is increasing at a fast pace, in most cases these recordings cannot access a complete population: some neurons that carry relevant information remain unrecorded. In particular, it is hard to simultaneously record all the neurons of the same type in a given area. Recent progress have made possible to profile each recorded neuron in a given area thanks to genetic and physiological tools, and to pool together recordings from neurons of the same type across different experimental sessions. However, it is unclear how to infer the activity of a full population of neurons of the same type from these sequential recordings. Neural networks exhibit collective behaviour, e.g. noise correlations and synchronous activity, that are not directly captured by a conditionally-independent model that would just put together the spike trains from sequential recordings. Here we show that we can infer the activity of a full population of retina ganglion cells from sequential recordings, using a novel method based on copula distributions and maximum entropy modeling. From just the spiking response of each ganglion cell to a repeated stimulus, and a few pairwise recordings, we could predict the noise correlations using copulas, and then the full activity of a large population of ganglion cells of the same type using maximum entropy modeling. Remarkably, we could generalize to predict the population responses to different stimuli with similar light conditions and even to different experiments. We could therefore use our method to construct a very large population merging cells’ responses from different experiments. We predicted that synchronous activity in ganglion cell populations saturates only for patches larger than 1.5mm in radius, beyond what is today experimentally accessible.     

Effect of zif268 early gene antisense oligonucleotide on mechanisms of synapse-specific plasticity

It was found that nociceptive sensitization was followed by long-term facilitation of synaptic responses evoked by chemical sensory stimulation of the snail "head", tactile stimulation of the snail "head" and foot in LP11 command neuron of defence behavior in snail Helix lucorum. Sensitizing stimulation during the intracellular injection of antisense olygonucleotide immediate early gene zif268 resulted in a selective suppression of synaptic facilitation in LP11 neuron responses evoked by tactile and chemical stimulation of the snail "head". At the same time, development of synaptic facilitation of responses in the LP11 neuron evoked by tactile stimulation of the foot was the same as in control sensitized snails. The results suggest that immediate early gene zif268 is selectively involved in the mechanisms of specific regulation of plasticity of the synaptic "input" of LP11 neuron from sensory receptors of the snail "head".     

Synaptic responses produced in lobster abdominal postural motor neurons by mechanical stimulation of the swimmeret

1. Intracellular recordings were obtained from the somata of identified abdominal postural motor neurons in lobster to examine their subthreshold and suprathreshold responses to tactile stimulation of the swimmeret. 2. Pressure stimulation of the swimmeret surface evoked abdominal extension by producing tonic spiking in the extensor excitors and the synergistic flexor inhibitor (f5) and hyperpolarizing responses in the extensor inhibitor and antagonistic flexor excitors. These responses often continued for several seconds following the termination of the stimulus. The receptive fields of these motor responses extended over most of the swimmeret surface. 3. More localized tactile stimulation of the swimmeret surface elicited EPSPs in f5 and the extensor excitors, and IPSPs in the flexor excitors. The amplitude of these synaptic potentials decreased as the stimulus intensity was reduced. 4. Stimulation of feathered hair (both sexes) and smooth hair (female only) sensilla produced responses characteristic of extension whereas bristly spines on the male accessory lobe excited only two flexor excitors without affecting any of the other postural motor neurons. 5. Summed synaptic responses recorded from the motor neurons differed in their amplitudes and latencies according to the type of mechanoreceptor stimulated-cuticular receptors, feathered hairs or smooth hairs. Stimulation of the swimmeret cuticle produced the strongest responses (shortest latency, largest amplitude), while feathered hair stimulation initiated the weakest responses (longest latency, smallest amplitude). 6. The relatively long latencies (greater than 35 ms) and the complex form of the EPSPs and IPSPs indicate the involvement of multisynaptic interneuronal pathways in the reflex arcs.     

Presynaptic contributions to response shape in an auditory neuron of the grasshopper

Neuron 714 is morphologically one of the most prominent neurons in the central auditory pathway of the grasshopper with arborizations extending from the abdominal neuromeres of the metathoracic ganglion to the brain. The aim of this study is to explore auditory information flow involving neuron 714 at the level of the ventral nerve cord. Paired intracellular recordings were made from neuron 714 in the mesothorax on the one hand, and from candidate presynaptic auditory neurons of the metathorax on the other. Electrical stimulation of the tympanal nerves provides an estimate of the synaptic distance between these interneurons and auditory afferents. Four, including neuron 714, are monosynaptically connected to afferents, the remainder disynaptically. Current-injection and spike-triggered averaging reveal that of nine neurons examined, seven make either monosynaptic, disynaptic or polysynaptic connections onto neuron 714. All connections are excitatory. Paired recordings show that response duration and response amplitude in synaptically linked cells vary according to the frequency of the stimulus. Measurements of the latency of the first excitatory post-synaptic potential evoked in neuron 714 by afferents and by metathoracic interneurons show how the synaptic drive from these sources shapes the auditory response of neuron 714. Accepted: 14 October 1998