Filiform Hairs Research Articles

Correlation of filiform hair position with sensory afferent morphology and synaptic connections in the second instar cockroach.

An attempt is made to relate the distribution of filiform hairs on the cercus of the second instar cockroach, Periplaneta americana, to the morphology and patterns of synaptic connectivity of their afferents. We studied the most distal 25 of the 39 filiform hairs which are commonly present. Filiform afferent arborizations were stained by cobalt filling from the cell body in the cercus. Three fundamental arbor types were found, two similar to those of the first instar medial (M) and lateral (L) afferents, and a third, novel type. L-type arbors could be divided into four subtypes. The most obvious correlate of arbor type is the circumferential position of the hair on the cercus. The proximodistal position of the sensillum within each cercal segment is also a determinant of its arbor. By comparison of hair positions and afferent morphologies, we were able to ascribe homologies between the second instar hairs and members of adult longitudinal hair columns. The patterns of monosynaptic connections between afferents and giant interneurons (GIs) 1, 2, 3, 5, and 6 were determined by recording synaptic potentials in GIs evoked by direct mechanical displacement of individual filiform hairs. Latency from stimulus onset to the rise phase of the first excitatory postsynaptic potential (EPSP) was used as the criterion of monosynapticity. The EPSP amplitudes of the two original L and M afferents are halved in the second instar, in the absence of a significant decrease in GI input resistance. The other afferents can be divided into two basic classes: those which input to GI5 (M-type), and those which input to GI3 and GI6 (L-type). The former is correlated with a central or medial position, while the latter is associated with a group of afferents situated laterally on the cercus. In segments 3 and 4, input to GIs 1 and 2 also correlates with a medial cercal position, however, in the more proximal segments 5 and 6, afferents at all positions input to these interneurons. The occurrence of afferents of identical morphology and similar connectivity in equivalent positions in different segments suggests that each sensory neuron is determined by its two-dimensional position within a segment. The presence of afferents with the same morphology which display proximodistal differences in synaptic connectivity, and of other afferents which have M-type connectivity despite L-type morphology, means that anatomy is generally a poor predictor of synaptic connectivity.

Response Properties Of Interneurons Of The Cricket Cercal Sensory System Are Conserved In Spite Of Changes In Peripheral Receptors During Maturation

ABSTRACT During postembryonic development of the cricket, the total number of filiform hair sensilla in the cereal sensory system increases approximately 40-fold. In addition, individual receptor hairs grow in size, changing the transducer properties of the sensilla and, thereby, the information transmitted to the central nervous system (CNS) by the sensory neurons. Interneurons MGI and 10-3 receive monosynaptic inputs from these sensory neurons and send outputs to anterior ganglia. We show that, in spite of the changes in the periphery, the response properties of these interneurons are relatively constant during development. The two interneurons differ in their frequency response, intensity response and rate of response decrement. Their respective response properties are conserved during the postembryonic period. The results suggest that systematic rearrangement of the sensory neuron-to-interneuron synapses plays an important role in maintaining a constant output of this sensory system to higher centers of the CNS during maturation of the cricket.

Development of cricket sensory hairs: changes of dynamic mechanical properties

Mechanical oscillation properties of cricket (Acheta domesticus) filiform hair sensilla were measured at different larval stages, as an indication of larval sensory capacities and for comparison with data in the literature on central nervous changes during development. The hairs were stimulated by airborne vibration over a frequency range of 10 to 220 Hz. Best frequency, angular displacement at best frequency, slope of angular-displacement tuning curve and phase of hair deflection relative to air particle velocity were tested for correlation with hair length, which is proportional to the age of a sensillum. The ranges found for the various oscillation parameters in early larval stages were similar to or larger than those in adults. Oscillation properties changed with both the developmental stage of the hair sensilla and that of the whole animal. Four individually identifiable hair sensilla were analysed separately; the sensory neurons of two of them are known to change synaptic properties during maturation. Angular displacement at a given stimulus intensity was maximal for all hairs after differentiation, and decreased during further development. The hairs did not show clear common changes for any of the other oscillation parameters. Yet particular changes were found for individual hairs.

Positional information determines the anatomy and synaptic specificity of cockroach filiform hair afferents using independent mechanisms

Mutant first instar cockroaches (Periplaneta americana) with supernumerary filiform hair sensilla on their cerci were used to study the effects of cell body position on axonal morphology and synaptic connections. The wild-type cercus has two hairs, one lateral (L) and the other medial (M), each with an underlying sensory neuron. Silver-intensified cobalt fills show that the supernumerary lateral neuron (SIN) in the mutant has the same shape of arborization as L, and electrophysiological recording shows that it forms synaptic connections with the same subset of giant interneurons (GIs) as L in the terminal ganglion: GI3 and GI6. The supernumerary medial neuron (SuM) has the same axonal morphology as M and synapses with the same GIs as does M: ipsilateral GIs 1 and 2 and contralateral GIs 1, 2, 3, 5 and 6. In 0.1% of approximately 8000 animals screened, a supernumerary hair arose on the cercal midline (C hair). The C neuron sends its axon to the CNS in the same branch of the cercal nerve as the L and SIN, and has a similar arborization. However, the C neuron forms synapses with the same GIs as do M and SuM. Electron microscopy of horseradish peroxidase-injected neurons was used to confirm that the C afferent forms a monosynaptic connection to GI2. It was concluded that the position of the sensory neuron cell body does control its axonal morphology and synaptic connectivity, but that these characteristics are produced by independent mechanisms.

Specificity of filiform hair afferent synapses onto giant interneurons in Periplaneta americana: Anatomy is not a sufficient determinant

The synapses between the filiform hair sensory afferents and giant interneurons (GIs) 1-6 of embryonic and first instar cockroaches, Periplaneta americana, were used to investigate the role of neuronal anatomy in determining synaptic specificity. The pattern of afferent-to-GI synapses was first determined by intracellular recording of excitatory postsynaptic potentials (EPSPs). The lateral (L) axon synapses only with GIs 3, 4, and 6, while the medial (M) axon synapses with the contralateral dendrites of all six GIs but with the ipsilateral dendrites only of GIs 1, 2, and 4. The three-dimensional anatomy of the filiform afferents and GIs was determined by injection of cobalt. There is little anatomical segregation of the filiform afferents; consequently, there is no correlation between the anatomy of the GIs and their synaptic inputs. The M axon and ipsilateral GI3 were studied in more detail by light and electron microscopy. Despite the presence of an anterior M axon branch which loops around the ipsilateral GI3 neurite at a distance of 2 microns, no synapses are formed between them. This lack of synapses is not due to the presence of physical barriers. Investigation of filiform afferents and GIs in embryonic ganglia shows that at no stage are the afferents sufficiently separated for their anatomy to be an important factor in determining the specificity of the synaptic inputs of the GIs. It was postulated that two pairs of complementary cell surface labels would be sufficient to code for this specificity, and that, in GIs 3, 5, and 6, spatial differences in the expression of these labels allow the M axon to distinguish ipsilateral dendrites from contralateral.

Synaptic Connections of Different Strength Between Wind-sensitive Hairs and an Identified Projection Interneuron in the Locust.

An identified intersegmental interneuron in Locusta and Schistocerca, with its cell body in the fourth abdominal ganglion and an axon which projects to the brain is excited by mechanosensory inputs from receptors on the head and neck. The organization of its receptive field, the types of sensory receptors which contribute to it and the patterns and strengths of the afferent connections were investigated by intracellular recording from the axon of the interneuron close to a spike-initiating site in the prothoracic ganglion. The receptive field of the interneuron consists of a small patch of hairs on the head ipsilateral to the axon, and from hairs on two regions of the prosternum (a cuticular structure on the ventral surface of the prothoracic segment), first an ipsilateral, lateral region and second a medial but contralateral region. Hairs on the pronotum (dorsal neck) also contribute but were not investigated here. Each spike in the afferent from a hair with a filiform appearance and with a pigmented base on the prosternum consistently evokes an EPSP in the interneuron. These have a short and constant latency, indicating that the connection is probably direct. The head hairs also appear to make direct connections with the interneuron in the prothoracic ganglion, so that the spike-initiating site here can integrate signals evoked by wind on the head and on the prosternum. Stiff tactile hairs on the prosternum do not connect with the interneuron. The EPSPs evoked by the long filiform hairs are consistently larger than those produced by the short filiform hairs and a single spike in some of the afferents from the long filiform hairs can evoke a spike in the interneuron. The effectiveness of an afferent is therefore correlated with the length of the filiform hair it innervates. The hairs with the most powerful effects are always the longest and occur in the same position on every locust. The shape of the receptive field and the different strengths of connections are apparent even in early larval instars. The axonal branches of the interneuron are restricted to the same side of the ganglion as the axon itself. Afferents from filiform hairs on the medial region of the prosternum project contralaterally, and those from the lateral region project ipsilaterally. Afferents from some of the head hairs project ipsilaterally directly to the prothoracic ganglion. The terminals of all these afferents overlap with the branches of the interneuron. By contrast, the afferents of tactile hairs which do not connect, project to different regions of neuropile. The connections ensure that the high sensitivity of the filiform hairs is maintained at the first stage in the central processing and suggest a role for this interneuron in supplying information about small changes in air currents that may be of use in controlling steering manoeuvres during flight.

Digger wasp against crickets. II. An airborne signal produced by a running predator

Females of the digger wasp Liris niger Fabr. hunt crickets to provide food for their offspring by running with high velocity on the ground (>20–50 cm/s). Crickets are able to detect the running wasps by the air particle movement generated by the predator. We measured signals produced by running wasps using a microphone sensitive to air particle velocity. The wasps generated single air puffs with peak air particle velocities of 1–2 cm/s measured close to the running wasp. We measured frequency spectra of the signals containing only components below 50 Hz, with increasing intensities towards lower frequencies, especially below 10 Hz. We measured the air particle movement generated by artificially moved wasps, crickets or a styrofoam dummy of similar size to investigate the effect of velocity and shape of the moving object upon the composition of the signal. The velocity of movement appeared to be important for the intensity and frequency composition of the air particle movement. The shape of the moved body had an influence on the intensity but only little effect on the frequency spectrum. Measurements with a thermistor anemometer showed that a moving object caused air currents lasting longer than 100 ms after passing or approaching the probe. The air particle movements generated by hunting wasps are entirely sufficient with respect to intensity and frequency range to be registered by the filiform hair sensilla upon the cerci of crickets.

The proventriculus of the worker honeybee and its differentiation

The proventriculus (pv) of the honeybee regulates the flow of food from the crop into the midgut. It is a formation of the foregut intervening between the crop and the midgut and consists of four bulging pv folds forming a x-shaped lumen. At their ends (=lips) the folds bear filiform hairs forming a comb at their inner margins. The pv folds are lined by a sclerotized cuticular intima. Between the basal plates of the pv folds and at the outside of the organ a flexible cuticle is present. By gulping movements (inner longitudinal and outer circular muscle layer) and with the aid of the hairs it can separate liquid food from pollen grains.Studies were made of 4- and 6-7-day-old pupae and of foragers (Apis mellifera L.) Scanning electron microscopic preparations show that in 4-day-old pupae the pv is already present in its basic form. The lips have filiform hairs which are repeatedly split at their ends and spine-like hairs on the outer side of the lips.

Oscillation of cricket sensory hairs in a low-frequency sound field

1. Filiform hairs of various lengths on the cerci of adult crickets vibrate in a sound field. These movements were measured with a photodetector for sound frequencies from 10 Hz to 200 Hz in the species Acheta domestica, Gryllus bimaculatus and Phaeophilacris spectrum. 2. With low air-particle velocities, the hair shafts were deflected sinusoidally from their resting position, without bending or secondary oscillations (Figs. 2 A, 3 A). At higher velocities (from ca. 80 mm/s peak velocity, depending on the properties of the individual hairs), the shaft struck the cuticular rim of the socket in which the base of the hair is seated (Fig. 2B). This contact was made at an average angular displacement from the resting position of 5.16°±1.0°. 3. The best frequencies of the hairs were found to be between 40 Hz and 100 Hz (Fig. 5A). The slope of the amplitude curve for constant peak air-particle velocity at frequencies below the best frequencies was between 0 and 6 dB/octave. Long hairs had smaller slope values than short hairs (Fig. 5C). 4. At its best frequency the ratio of maximal tip displacement of a hair to the displacement of the air particles in the sound field was between 0.2 and 2. Only a small number of hairs (2 out of 36) showed tip displacements exceeding twice the air-particle displacement. The values of maximal angular displacement were not correlated to hair length (Fig. 5 B). 5. The angular displacement of the hairs was phase shifted with respect to the air-particle velocity by 0° to +45° (phase lead) at sound frequencies around 10 Hz and by -45° to -120° (phase lag) at 200 Hz (Figs. 3C, 4B). At a particular frequency long hairs tended to have larger phase lags than shorter hairs (Fig. 5D).

Mechanical polarization in the air-current sensory hair of a cricket

We examined the mechanical anisotropy of the basement spring which underlies the directional sensitivity of the cercal filiform hair of the cricket (Gryllus bimaculatus). Spring stiffness varied with the direction of hair deflection. The anisotropic ratios were 8 for short hairs and 4 for long ones, whereas the absolute values of spring stiffness varied 100-fold with hair length.

Synaptic specificity in the first instar cockroach: patterns of monosynaptic input from filiform hair afferents to giant interneurons.

Direct evidence for monosynaptic connections between filiform hair sensory axons and giant interneurons (GIs) in the first instar cockroach, Periplaneta americana, was obtained using intracellular recording and HRP injection followed by electron microscopy. GIs 1-6 all receive monosynaptic input from at least one filiform afferent axon. GI1, GI2 and GI5 receive input only from the medial (M) axon, while GI3, GI4 and GI6 receive input from both M and lateral (L) axons. The dendrites of GI3 and GI6 which are contralateral to the cell bodies receive input from both axons whereas the smaller ipsilateral dendritic fields have synapses only from the L axon. GI5 has M axon input only onto its contralateral dendrites. In 50% of preparations GI7 receives weak input from the ipsilateral L axon. There is no obvious relationship between the morphology of the giant interneurons and the pattern of input they receive from the filiform afferents.

Parallel inputs shape the response of a giant interneurone in the cercal system of the locust

The terminal ganglion of the CNS of the locust contains a bilateral pair of Giant Interneurone 1's (GIN 1's). GIN 1 has a lateral cell body, an axon which leaves the ganglion anteriorly via the contralateral connective, and an extensively branched, posteriorly directed dendrite. The response of a GIN 1 to wind stimuli directed at the cerci, or electrical stimulation of the cercal nerve, consists of a depolarization supporting spikes. Wind-sensitive filiform afferents on the cerci send axons via the cercal nerve into the terminal ganglion where they evoke short-latency EPSPs in GIN 1. The input from a number of filiform hairs on a cercus converges onto GIN 1. The response of GIN 1 to a single electrical stimulus applied to the cercal nerve consists of: an initial depolarization and spike, evoked by input from filiform afferents; and a delayed, sometimes suprathreshold, depolarization which is evoked by input from an interneurone. Simultaneous intracellular recordings and stainings reveal this delayed input to GIN 1 originates from a spiking local interneurone. The magnitude of the delayed depolarization in GIN 1 depends on the degree to which the local neurone is hyperpolarized by its own presynaptic input. The response of GIN 1 to cercal stimulation is therefore determined by parallel inputs, both of which are activated by cercal hairs.

Connectivity pattern of the cercal-to-giant interneuron system of the American cockroach.

1. The pattern of connectivity between identified cercal afferents and the three largest giant interneurons (GIs 1, 2, and 3) of the American cockroach was investigated by intracellular methods. These three GIs all have different directional response sensitivities and appear to be especially important in initiating the short latency escape behavior of the American cockroach. 2. One of the interneurons, GI 1 responds to wind from all four quadrants of space about the animal. However, it clearly shows a greater ipsilateral versus contralateral (relative to the GI's axon within the nerve cord) wind sensitivity. In contrast, the directional sensitivity of GI 2 is more nearly bilaterally symmetrical. Both of these interneurons receive excitatory synaptic input from the sensory cells of the nine most prominent columns (a, d, g, f, h, i, k, l, and m) of filiform hairs of the ipsilateral cercus. 3. The nine ipsilateral inputs all made roughly equivalent strength excitatory connections with GI 1. The connectivity pattern to GI 2 was the same as that to GI 1 except that the connection strength for two of the nine columns, h and i, was substantially stronger to GI 2 than to GI 1. The remaining seven sensory columns all make equivalent strength connection with GI 2. 4. Only select columns of contralateral sensory cells made synaptic connection with GIs 1 and 2. All detectable connections produced subthreshold depolarizations. 5. The response curve of GI 3 is more sharply restricted in space than that of either GI 1 or 2 and this interneuron only responds to wind stimuli originating from in front of the animal. GI 3 received excitatory synaptic input only from ipsilateral columns d, f, g, i, and k, all of which have their best excitatory directions well within the boundaries of the response curve of GI 3. Columns a and l with best excitatory directions near the edges of the response curve of GI 3 made no detectable connection. The remaining two columns (h and m) with best excitatory directions well outside the boundaries of the response curve of GI 3 provided inhibitory input. 6. GI 3 received synaptic input from contralateral columns d, f, g, h, i, k, and m.(ABSTRACT TRUNCATED AT 400 WORDS)

Ecdysial growth of the filiform hairs and sensitivity of the cercal sensory system of the cricket,Gryllus bimaculatus

1. The ecdysial growth of cercal filiform hairs was investigated in the cricketGryllus bimaculatus. The length of hairs varied from 40 to 500 μm in the 1st, from 40 to 650 μm in the 3rd and from 30 to 800 μm in the 5th instar nymphs (Fig. 1). Hemimetabolous development causes both hair growth and the appearance of new hairs at each ecdysis (Figs. 2, 3). The newly acquired hairs were shorter than 200 μm in every case (Fig. 4). 2. Velocity thresholds of cercal sensory interneurons (CSIs) to sinusoidal air-currents were measured in 3rd instar nymphs (Fig. 5 A, B, C). CSIs 8-1 (medial giant interneuron: MGI) and 9-1 (lateral giant interneuron: LGI) showed threshold curves of acceleration sensitivity similar to those in adults. The thresholds for CSIs 8-1 and 9-1 were on the average higher in nymphs than in adults. The threshold curves for the two velocity-sensitive CSIs 10-2 and 10-3 were similar for nymphs and adults. 3. Velocity thresholds of cercal filiform sensilla were measured in 3rd instar nymphs (Fig. 6). In spite of the small size of nymphal hairs, the most sensitive ones showed the same sensitivity as did the long 1000 μm hairs of the adult. 4. The filiform hairs in 3rd instar nymphs were supported by a weaker spring than in adults (Fig. 7). Relative stiffness was about 50% of that in the long hairs in adults, but not much different than that in the short hairs. 5. Based on a theoretical estimation of hair motion, the threshold angle of a filiform sensillum in the 3rd instar nymph was calculated (Fig. 9). Threshold angles of the long sensilla seemed to be unchanged throughout hemimetabolous development.

Presynaptic inhibition of identified wind-sensitive afferents in the cercal system of the locust

The paired cerci located at the tip of the locust abdomen bear a large number of wind-sensitive filiform hairs, each of which sends an axon via the cercal nerve into the terminal ganglion of the CNS. The filiform afferents fire bursts of action potentials when their hairs are displaced by wind or mechanical stimuli. Filiform axon terminals in the CNS are depolarized concomitantly with the discharge of another type of unit (a primary afferent-depolarizing, or PAD, unit) recorded in the cercal nerve. The instantaneous spike frequency of PAD unit discharges matches the evoked depolarization very closely, and during such depolarizations spike amplitudes in the filiform afferent terminals are reduced by up to 55%. Depolarizing current pulses injected into the axonal terminals of an identified filiform afferent evoke spikes that are blocked by the PAD unit, probably via an intercalated interneuron. The PAD unit makes a monosynaptic connection with only one of the 4 giant interneurons (GIN 2) on each side of the terminal ganglion, and indirect connections with 2 others. Depolarizing current pulses injected into the neuropilar segments of GINs evoke fewer spikes when the PAD unit is active, consistent with the PAD unit's mediation of conductance changes in postsynaptic cells. Iontophoretic injection of Lucifer yellow shows the PAD unit to be an afferent with axon terminals overlapping those of filiform afferents and posteriorly directed branches of interneurons such as GIN 2 in the CNS. Passive movements of a cercus, monitored with a position transducer, show that the PAD unit fires discrete bursts during cercal displacement. The PAD unit most probably has its soma and dendrites in tissue spanning the cercal base. By responding to cercal movements sufficient to also activate filiform hairs, and by mediating conductance changes in both the presynaptic terminals of filiform afferents and the postsynaptic membranes of interneurons, the PAD unit desensitizes a pathway to movement-generated afferent input, and ensures that the locust remains sensitive to external wind stimuli.

Lophotoxin Blocks Synaptic Acetylcholine Receptors in the Cockroach Periplaneta Americana

ABSTRACT The actions of lophotoxin on synaptic acetylcholine receptors were investigated, using the cholinergic synapse between the lateral filiform hair sensory neurone and giant interneurone 3 in the first-instar cockroach Periplaneta americana. Lophotoxin (1·0×10−6mol l−1) blocked transmission without affecting the passive or active membrane properties of the pre- and postsynaptic neurones, and without affecting non-cholinergic synaptic inputs. Micromolar concentrations of lophotoxin also blocked the depolarizing response of giant interneurone 3 to carbamylcholine, when applied ionophoretically to its dendritic branches. The toxin shifted the dose-response curve to the right by two orders of magnitude, suggesting that it was acting as a competitive antagonist.

Analysis of synaptic integration using the laser photoinactivation technique

Crickets (and many other insects) have two antenna-like appendages at the rear of their abdomen, each of which is covered with hundreds of ‘filiform’ hairs resembling the bristles on a bottle brush. Deflection of these filiform hairs by wind currents activates mechanosensory neurons at the base of the hairs. The axons from these sensory neurons project into the terminal abdominal ganglion to form a topographic representation (or “map”) containing information about the direction, velocity and acceleration of wind currents around the animal. Information is extracted from this map by primary sensory interneurons that are also located within the terminal abdominal ganglion. In this paper, we review the progress that has been made toward understanding the mechanisms underlying directional sensitivity of an identified sensory interneuron in the cricket,Acheta domesticus. The response properties of the cell have been found to depend to a large extent upon the structure of its dendritic branches, which determines its synaptic connectivity with the sensory afferents in the map of wind space and the relative efficacy of its different synaptic inputs.

A club-shaped sensory hair on the first instarEctropis excursaria (Lepidoptera: Geometridae) and its role in the behaviour of the caterpillars

A unique club-shaped hair on the first instars of an Australian geometrid caterpillar is described. The sensory hairs occur on the body segments and is absent on the head and the last abdominal segment. These hairs have different orientations and are replaced by filiform hairs after the first moult. Regression lines describing the movement of hairs of different orientations in relation to a force is presented. With these results the deflection in the sensory hair due to gravity when the caterpillar is on a vertical plane was calculated. These calculations suggest that the hairs may not function as gravity receptors. The possible role of these hairs in the behaviour of caterpillars is discussed.

Initiation and conduction of impulses in mechanosensory neurons: Effects of hypoxia

1. 1. Extracellular recordings were made from cercal sensilla of Periplaneta americana. In bristle hair sensilla, mechanical stimulation induced biphasic spikes showing a negative initial phase on which a small positive deflection was superimposed. 2. 2. This sequence (the opposite of that observed in most insect sensilla) was inverted by repetitive stimulation or depolarization of the apical dendrite. 3. 3. In fliliform hair sensilla, the spikes were also biophasic but showed a positive leading phase. 4. 4. In vitro, spikes initiated in the sensilla rapidly failed to propagate along the sensory nerve unless the cercus was oxygenated. 5. 5. Invasion of the sensilla by antidromic spikes was also prevented by hypoxia. 6. 6. The conduction block occurred in the sensilla themselves rather than in the axons. 7. 7. Interpretation: sensory spikes are not initiated in the axon hillock but in the basal dendrite (filiform hair sensilla) or the apical dendriate (bristle hair sensilla); in the latter case, the trigger site can move from an apical to a basal location. 8. 8. After initiation, spikes are propagated towards the axon but they can be blocked by hypoxia at the dendrite-soma boundary.

Calcium Conductance in An Identified Cholinergic Synaptic Terminal in the Central Nervous System of the Cockroach

ABSTRACT Intracellular microelectrodes were used to study a cholinergic synapse between two identified neurones: the lateral filiform hair sensory neurone (LFHSN) and giant interneurone 3 (GI 3) in the terminal ganglion of the first-instar cockroach Periplaneta americana. The presynaptic cell, LFHSN, was impaled in a region of the axon which forms large numbers of output synapses. The sign and magnitude of the LFHSN spike afterpotential were shown to depend on [Ca2+]o. l μmol l−1 tetrodotoxin (TTX) abolished LFHSN spikes but the addition of 0·l mmol l−1 4-aminopyridine (4-AP) enabled regenerative depolarizations to be evoked which were followed by large EPSPs in GI3. Addition of 20 mmol l−1 tetraethylammonium ions (TEA+) abolished the cholinergic EPSPs but resulted in long-duration LFHSN spikes. Intracellular injection of caesium ions (Cs+) into LFHSN enabled long-duration spikes to be evoked and had no effect on synaptic transmission. Long-duration LFHSN spikes were (1) increased in amplitude by increased [Caz+]o; (2) accompanied by an increase in conductance; (3) not abolished by replacement of external Na+ with Tris+ or choline+; (4) blocked by 1 mmol l−1 Cd2+ and 10 mmol l−1 Co2+; (5) not supported by substitution of Mg2+ for Ca2+; and (6) supported by Ba2+ substitution. They are thus considered to be Ca2+ spikes. The Ca2+ spikes were blocked by organic Ca2+ channel blockers at 0·5–1 mmol l−1. The putative Ca2+ spike was followed by a hyperpolarizing afterpotential (HAP), the duration of which was proportional to the amplitude and duration of the Ca2+ spike. The HAP was (1) accompanied by a conductance increase; (2) reversed at potentials 30mV more negative than resting potential; (3) not supported by substituting Ba2+ for Ca2+; and (4) partially blocked by 150 mmol l−1 TEA+. The HAP is considered to result from an increase in Ca2+-dependent K+ conductance. It is concluded that, in addition to Na+ channels and delayed rectifying K+ channels, Ca2+ channels and Ca2+-dependent K+ channels are present in the axonal membrane of LFHSN, in a region which forms many output synapses.