Evidence for a command neuron controlling calling song in the cricket Gryllus assimilis Chu-Cheng Lin , Berthold Hedwig * Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom A R T I C L E I N F O Keywords: Intracellular recording Command neuron Calling song Cricket A B S T R A C T In invertebrates stereotypical behaviours may be controlled by command neurons. In the field cricket Gryllus bimaculatus a command neuron descending from the brain controls the generation of rhythmic wing movements underlying calling song. We analysed if a corresponding command neuron also exists in other cricket species. Our intracellular recording and stimulation experiments point to a putative command neuron for calling song in G. assimilis, although the structure of the neuron could not be revealed. When this neuron was depolarised to generate 50 AP/s the cricket raised its forewings into singing position, it started rhythmic wing movements and produced the typical species-specific calling song. Further enhancing the spike rate of the neuron increased the chirp repetition rate but not the pulse repetition rate. Blocking the spike activity of the neuron by hyper- polarizing current injection reduced the chirp repetition rate and could terminate singing activity. Our evidence indicates that the two species, G. assimilis and G. bimaculatus, have homologous neurons for controlling calling song, which may be a conserved phenotype across cricket species. 1. Introduction For invertebrates the concept of command neurons controlling spe- cific fixed action patterns has been appealing ever since the term was coined by Wiersma and Ikeda (1964) describing fibres in crayfish thoracic-abdominal connectives, which initiate rhythmic swimmeret movements when stimulated. To overcome pure operational character- isations, which dominated the field (Atwood and Wiersma, 1967; Bowerman and Larimer, 1974; Davis and Kennedy 1972), the criteria of sufficiency and necessity were introduced to identify command neurons (Kupfermann and Weiss, 1978). The activity of a command neuron should reliably elicit the motor pattern underlying a specific behaviour (suffi- ciency), the execution of the motor pattern should be prevented if the neuron’s activity is inhibited (necessity), and the command neuron should not be rhythmically active but should activate the motor pattern by tonic spike activity only. Based on intracellular recordings few com- mand neurons meeting these criteria have been reported for different behaviours in invertebrates, like escape swimming in the crayfish (Edwards et al., 1999) and the mollusc Tritonia diomedea (Frost and Katz, 1996), the control of the pedal wave motor program in Aplysia (Fredman and Jahan Parwar, 1983), the control of calling song stridulation in the grasshopper Omocestus viridulus (Hedwig, 1994) and in the cricket Gryllus bimaculatus (Hedwig, 2000a). Using neurogenetic techniques command neurons for courtship song (von Philipsborn et al., 2011), feeding (Flood et al., 2013) and backward walking (Bidaye et al., 2014) were identified in transgenic Drosophila flies. In the nematode C. elegans, the hermaphrodite specific neurons (HSNs) are command neurons for egg- laying (Brewer et al., 2019). In fish, the reticulospinal Mauthner cell drives short-latency escape behaviour in response to auditory and vibratory stimuli, and is the only example for a command neuron in vertebrates (Eaton et al., 1988; Kohashi and Oda, 2008; Weiss et al., 2006). Numerous neurons which initiate a particular behaviour but fail to demonstrate necessity are described as putative command neurons (Balaban 1979) or command-like neurons (see Gillette, 1987, and intro- duction by Frost and Katz, 1996). Here we focus on cricket singing behaviour, a classical model to study the neural organisation and control of a rhythmic motor pattern (Kutsch and Huber, 1989). Male crickets sing by rhythmically rubbing their front wings together, with the motor machinery of muscles and motoneurons located in the mesothoracic segment. In Gryllus campestris electrical stimulation of different neuropil regions in the brain elicited calling, courtship, and rivalry song, and implied the existence of com- mand neurons for singing (Huber, 1963, 1960), which would carry forward the command to sing from the brain to the singing central * Corresponding author at: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom. E-mail address: bh202@cam.ac.uk (B. Hedwig). Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys https://doi.org/10.1016/j.jinsphys.2025.104798 Received 18 November 2024; Received in revised form 25 March 2025; Accepted 25 March 2025 Journal of Insect Physiology 162 (2025) 104798 Available online 1 April 2025 0022-1910/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). https://orcid.org/0000-0002-1132-0056 https://orcid.org/0000-0002-1132-0056 mailto:bh202@cam.ac.uk www.sciencedirect.com/science/journal/00221910 https://www.elsevier.com/locate/jinsphys https://doi.org/10.1016/j.jinsphys.2025.104798 https://doi.org/10.1016/j.jinsphys.2025.104798 http://crossmark.crossref.org/dialog/?doi=10.1016/j.jinsphys.2025.104798&domain=pdf http://creativecommons.org/licenses/by/4.0/ pattern generator (CPG). Intracellular recordings revealed a descending command neuron for cricket calling song in the brain of G. bimaculatus (Hedwig, 2000a, 1996). Main dendrites of the calling song command neuron are located next to the mushroom body, while its axon descends towards the thoracic ganglia. Using intracellular recording and stimulation, the current study provides evidence for a command neuron in the brain of G. assimilis controlling calling song stridulation. Comparing the physiological properties of command neurons in G. assimilis and G. bimaculatus in- dicates that cricket species with different song patterns share conserved command neurons for the control of singing. 2. Material and methods 2.1. Experimental animals G. assimilis and G. bimaculatus were bred in boxes (52.5 x 36.5 x 28 cm) at the Department of Zoology, University of Cambridge under a 12:12hr light:dark cycle at 26-28◦C. Fish food, muesli, and water were provided daily. Last instar male nymphs were individually isolated in boxes (17.5 x 11.5 x 13 cm), reared to adulthood, and used for experi- ments 7–14 days after eclosion. Completeness of the wings and singing of calling song were confirmed for each individual. 2.2. Intracellular recording of brain neurons Male crickets were mounted dorsal side up on a plasticine block attached to a stand, with the prothorax and six legs tethered by small staples. The stand was tilted so that the head faced upward, it was then waxed to a rigid U-shaped wire to prevent movements during dissection and intracellular recording. Access to the brain was obtained by removing a rectangular piece of cuticle between the compound eyes, careful cutting the ocellar nerves and removing the fat body around the brain. The brain was stabilized between a small metal platform at its dorsal side and a metal ring gently lowered on its ventral/frontal side. An optical fibre powered by a DC light source was built in the platform and served as light source during the intracellular recording. The brain was constantly supplied with insect saline (in mmol− 1: NaCl 140; KCl 10; CaCl2 7; NaHCO3 8; MgCl2 1; N-trismethyl-2-aminoethanesulfonic acid 5; D-trehalose dehydrate 4, pH 7.4) to prevent desiccation. Capillary glass (ID 0.58 mm, OD 1.0 mm Hilgenberg, Germany) was pulled by a DMZ universal puller (Zeitz-Instruments, Germany) to micro-capillaries; they were filled with either 2 M potassium acetate (with 30–40 Ω resistance, Alexa 568 or Alexa 555) or 1 M lithium chloride (with 80–100 Ω resistance, Lucifer Yellow; all dyes from Sigma-Aldrich). Mi- croelectrodes were inserted in an electrode holder attached to a micromanipulator (Leica Micromanipulator, Germany) for fine posi- tioning. A digital gauge (Mitutoyo, Digimatic Indicator 543; resolution, 1 μm) monitored the depth of the electrode in the brain to provide in- formation for approaching the target neuron. The microelectrode signal was amplified (SEC10–05LX amplifier; NPI, Germany) and was moni- tored as audio signal by speakers. Current injection (depolarization and hyperpolarization) was used to physiologically identify the command neuron for singing. Stable recordings of calling song command neurons lasted for 15 to 30 min with spike amplitudes above 10 mV. All data were recorded by CED Spike2 software (CED, Cambridge, UK) for off- line analysis. Experiments were first performed 98 G. bimaculatus and then on 57 G. assimilis leading to 5 and 6 recordings of the pCN respectively, allowing analysis and comparison of neuronal properties. 2.3. Wing movement and song recording A 1.5-mm diameter piece of reflective foil (Scotchlite foil type 7610, 3 M Laboratories, Germany) was stuck on the right forewing for recording its movements (Lin and Hedwig, 2021a) with an optoelec- tronic camera (Hedwig, 2000b) using a linear position-sensitive photodiode (Type 1L30-UV, Laser Components, Olching Germany). The up-down movement during singing, representing the lateral projection of the opening and closing movement of the wing, was picked up with the diode by the light reflected from the disk. The up-down wing movements during calling song stridulation were 2–3 mm. Sound pro- duced by the cricket was picked up by a microphone (Teisco Sound Research UEM-83). Both wing movement and sound signal were recor- ded simultaneously with the neuronal activity in Spike2 software at a sampling rate of 44.1 kHz. 2.4. Data analysis The spike rate of recorded neurons, sound pulse rate, chirp rate, and pulse number in chirps were analysed by NEUROLAB software (Hedwig and Knepper, 1992; Knepper and Hedwig, 1997). For calculating the spike rate, recordings were differentiated to eliminate DC changes and spikes were identified using changes in membrane potential as threshold criteria. The identification of spikes was manually checked and then used to calculate the instantaneous spike frequency. For analysing the sound pulse rate, the sound signal was processed by a NEUROLAB gliding length filter function (Hedwig and Knepper, 1992; Knepper and Hedwig, 1997), a threshold criterion was used to mark the start of a sound pulse, and the resulting start times were used to calculate the instantaneous sound pulse frequency. Chirp rate was processed in the same way based on the first pulse in the chirps as reference. Pulse number in chirps was calculated by a Peri-Stimulus-Time histogram function and manually checked. 3. Results The calling song of G. assimilis consists of chirps with a duration around 100 ms, and unusual long chirp intervals in the range of 500 ms to more than 1 s. A chirp contains 7–9 sound pulses with a pulse period of 10–15 ms (Lin and Hedwig, 2021b; Pollack and Kim, 2013), (Fig. 1A). The calling song can be distinguished from rivalry song and courtship song by the song structure. Rivalry song of G. assimilis contains chirps with a higher but variable number of sound pulses, sometimes with over 20 pulses per chirp (Alexander, 1961). Courtship song, on the other hand, contains two components described as low amplitude, low fre- quency chirps and high amplitude, high frequency ticks (Vedenina and Pollack, 2012). Microelectrodes were positioned in the anterior protocerebrum, in the region where extracellular electric stimulation elicited calling song in G. campestris (Huber, 1960) and the dendrites of a command neuron for calling song were identified in G. bimaculatus (Hedwig, 2000a). The recording region was 100 μm lateral to the mid-line (left or right) and near the root of the medial ocellar nerve (Fig. 1B). Each neuron recorded was tested by depolarizing and hyperpolarizing current injection for behavioural effects. The experimental procedures were first established in G. bimaculatus and then applied to G. assimilis. The recordings in G. bimaculatus (n = 5) were also used for comparison with neurons recorded in G. assimilis. In experiments of G. assimilis, singing-like wing movements occurred in 15 experiments during probing the specific brain region. Neurons related to calling song stridulation were stably recorded 6 times. The current injection tests were repeated in all stably recorded individuals and the same results were obtained. As reliable staining of the neuron’s morphology could not be obtained, the neuron recorded in G. assimilis is described as pCN (putative Command Neuron) throughout the text. 3.1. Calling song stridulation elicited while probing the anterior brain region in G. assimilis While probing the brain for the command neuron related to singing in G. assimilis, calling song activity was elicited when the recording electrode was at depth of 120–220 μm from the ventral surface of the C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 2 brain (Fig. 1C). The forewings vibrated, they were rapidly raised and lowered and then adjusted and elevated to a singing position where stridulation started. Initially only scratchy sound pulses were generated during the wing movements (Fig. 1C, see upper sound trace). When stridulation began, the forewings engaged in rhythmic opening-closing movements with regular pauses. Sound pulses of calling song were generated during sequential sonorous closing movements. At the beginning, the forewing position was not properly adjusted, the opening-closing movements were premature and only incomplete chirps with few sound pulses were produced. Through the course of the first 3 s the wings gradually raised to a higher position, the rhythmic movements became stable and the sound pulse number per chirp increased from 1 to 8 like in normal G. assimilis calling song. The chirp rate was about 5 Hz for the first two chirps, declined to 2 Hz over the next 3 s and then settled at about 1 Hz before singing stopped. In these experiments calling song lasted for 20 to 60 s and then ceased when the wings were lowered again. The neuron was subjected to current injection if properly penetrated. Fig. 1. Calling song elicited when probing the anterior brain region in Gryllus assimilis. (A) Recording of G. assimilis natural calling song. (B) Recording area for calling song command neuron as indicated by grey circles. (C) Wing and sound recording when calling song was elicited while probing the brain area with a microelectrode. The sonorous closing and silent opening of the forewings correlates to upward (high position) and downward (low position) movement in the wing recording. Wing movements and sound pulses are enlarged for 5 chirps. Note, the initial rapid change of wing position and scratchy sound, and the gradual rising of the wing and increase of sound pulse number during stridulation. C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 3 3.2. Testing the sufficiency criterion: depolarization of the putative command neuron for calling song in G. assimilis initiates calling song stridulation Stable intracellular recordings of the pCN in G. assimilis allowed to examine the effect of the neuron’s spike activity on singing behaviour. The neuron showed tonic spike activity with no evidence of prominent EPSPs. The sufficiency criterion was tested by stepwise increasing a depolarizing current from 0.0 to 3.0nA (Fig. 2A). Spike activity was enhanced from less than 25 APs/s to almost 75 APs/s with increasing current injection. The cricket began to stridulate calling song when the current was 2.9nA and the spike rate reached about 50 APs/s. Stridulation started and the forewings showed rhythmic opening-closing movements with typical chirp intervals. For the first few chirps the an- imal adjusted the wing position and wing movements, and the chirps came with fewer sound pulses. The wings then raised to a higher posi- tion where the forewings effectively generated sound pulses with each closing movement, and the number of sound pulses per chirp gradually increased from 1 pulse to 7 pulses (not shown in the recording). The calling song showed a chirp rate between 0.7 and 1 Hz, and varying sound pulse rate in each chirp. The sound pulse rate was fastest for the first pulses of a chirp (up to 100 Hz) and gradually decreasing within the chirp (55 Hz the lowest) as in normal chirps (Fig. 1A, see enlarged chirp). This experiment demonstrates that the activity of the pCN Fig. 2. Intracellular depolarization of the pCN elicits calling song stridulation in G. assimilis. Under two experimental settings, depolarizing current to pCN reliably elicited calling song stridulation. (A) An increasing current was applied to pCN. When the spike rate reached about 50 APs/s (2.9nA) the cricket began to generate calling song. The sound pulse number increased during the course of stridulation and wing movements became coordinated. (B) While the male was in a resting state a 1.5nA depolarizing current was applied to pCN twice. Start and end of the current pulses were linked to the generation of calling song chirps. C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 4 initiates calling song stridulation, and thus fulfils the sufficiency crite- rion as laid out by Kupfermann and Weiss (1978). In another experiment, the pCN was penetrated and the animal started to generate calling song for about a minute. After that the neuron was spiking at a rate of 30–45 AP/s without any calling song produced. This provided a chance to test the effect of activating the putative command neuron on calling song stridulation. To test the immediate effect of the neuron, 1.5nA depolarizing current was injected into pCN (Fig. 2B, two current injections for 800 ms and for 650 ms). The current increased the neural activity to an initial peak of 227 and 238 APs/s, which declined over 200 ms and stabilized at 170–190 APs/s and 180–200 APs/s. 218 ms and 87 ms after each depolarization, the male started to generate calling song. It raised its wings, and generated 2 chirps for each stimulus, with a chirp rate of 1.2–1.3 Hz, and with 3–6 sound pulses per chirp. Within a chirp the sound pulse rate was 90–100 Hz for the first pulses and 60–70 Hz for the subsequent pulses. In the two current injection trials, the second chirps were generated 300 ms and 100 ms after the end of depolarizing stimulus, respectively. After removal of depolarizing current, the spiking of the neuron stopped for 127 ms and 138 ms for the two tests, and then resumed at a rate of 25–45 APs/s. The forewings were kept in raised singing position between the two trials for 4 s. These experiments suggest the activity of the recorded neuron elicited the onset of calling song stridulation and the singing activity can be manipulated by controlling the spiking activity of this neuron. 3.3. Testing the necessity criterion: hyperpolarization of the pCN terminates singing To test the necessity criterion (Kupfermann and Weiss, 1978) hyperpolarizing current was applied to the pCN to evaluate if the neu- ron’s spike activity is necessary to maintain singing. After the neuron had been penetrated it was spiking at a rate of 75–85 APs/s and the cricket was generating the calling song. The hyperpolarizing current was gradually increased in amplitude from -1nA (Fig. 3). During hyperpo- larization the spike rate decreased from 60 APs/s to 25 APs/s and the chirp rate declined from 0.85 Hz to 0.57 Hz. When the current reached -1.15nA the spike rate was below 35 APs/s and the male stopped singing. The sound pulse rate and sound pulse number in chirps stayed the same before termination of the calling song. This demonstrates that the ongoing calling song activity can be abolished by reducing the pCN activity. Thus, pCN met the criteria of necessity for a command neuron (Kupfermann and Weiss 1978). In some attempts, the calling song was not abolished right away when the recorded neuron activity was sup- pressed, but the chirp rate slowly decreased until the song stopped, indicating that the downstream CPG activity wanes when the activity of pCN gradually decreased. 3.4. Spike rate of pCN is related to chirp rate, but not to pulse rate or pulse number in chirps To analyse the relationship between the pCN spike activity and calling song parameters (chirp rate, pulse rate, and pulse number per chirp), depolarizing and hyperpolarizing currents were applied. In one experiment, 2nA depolarizing current was injected to pCN during ongoing calling song activity (Fig. 4A). Spike rate of the neuron was enhanced from 27 to 77 APs/s to a transient burst of 240 APs/s and then stabilized at 130–150 APs/s. Immediately after the depolarizing current, the neuron stopped firing for about 600 ms and then returned to its initial spiking level. The enhanced neuronal activity increased the chirp rate from 0.5-0.7 Hz to 0.9–1.3 Hz, while the sound pulse rate was not altered. The sound pulse rate showed the same decreasing pattern dur- ing each chirp before or after depolarization, with the first pulses occurring at the highest rate (90–100 Hz) and the last pulses at the lowest rate (60–70 Hz). Pulse number per chirp stayed at 6–7 Pulses/ Chirp and did not change during the depolarization. This test demon- strated the chirp rate of the calling song was enhanced along with the increased neuronal activity, but not the pulse rate or pulse number in the chirps. In a similar experiment, -2nA hyperpolarizing current was injected when the male was already generating calling song with a chirp rate of 1.5–1.8 Hz (Fig. 4B). Spike activity of pCN was totally abolished by the hyperpolarization and the chirp rate was reduced to 0.7–0.8 Hz. Removal of the hyperpolarizing current for 1.87 s brought the neuronal activity to 224 APs/s and over the following 300 ms it declined to 80–110 APs/s. Simultaneously the chirp rate raised to 1.7–1.9 Hz, Fig. 3. Hyperpolarization of the pCN terminates ongoing singing in G. assimilis. Hyperpolarizing current with increasing amplitude from -1nA was applied to pCN. Spike rate of the neuron dropped from 75 to 85 APs/s to 60 APs/s and continued decreasing to 25 APs/s as the applied current amplitude increased. Chirp rate decreased from 0.85 Hz to 0.57 Hz during current injection and singing finally stopped when the spike rate was below 35 APs/s. C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 5 however sound pulse rate and number of pulses per chirp were not altered during the process. In a subsequent test application of hyper- polarizing current again reliably stopped the neuronal activity and the chirp rate decreased to 0.67–0.75 Hz. Removing the current for 1.65 s brought neuron spike rate to a peak of 194 Hz, it then stabilized at 80–110 APs/s while the chirp rate raised again to 1.8–1.9 Hz. The application and removal of the hyperpolarizing current reliably controlled the chirp rate, while pulse rate and pulse number in the chirps were not affected by the changing neuronal activity. As before (Fig. 2B) the singing CPG showed some autonomy and remained active, while the pCN activity was suppressed. 3.5. Correlation of pCN spike rate with chirp rate and sound pulse rate in G. assimilis and G. bimaculatus Statistical analysis of the pCN spike rate for 38 chirps of calling song revealed the relationship between spike rate and chirp rate and sound pulse rate, respectively (Fig. 5). Pulse rate was calculated from the first two pulses of each chirp as pulse rate in G. assimilis calling song varied in a wide range within chirps (60–100 Hz). The range of spike frequency between chirps was 0 to 185 AP/s, the chirp rate ranged from 0.4 to 2.25 Chirps/s, and the range of the pulse rate was 75 to 100 Pulse/s. Plotting spike frequency against chirp rate revealed a positive correlation (R = 0.8595) (Fig. 5A), while no meaningful correlation occurred between spike frequency and sound pulse rate (R = 0.1640) (Fig. 5C). This sug- gests the changing spike activity of pCN affected the chirp rate, (Fig. 4), while the sound pulse rate was not altered. The same analysis was Fig. 4. Relationship between pCN spike activity and calling song parameters in G. assimilis. In two experiments, pCN spike activity was manipulated by depolarizing or hyperpolarizing current. (A) With a 2nA depolarizing current the chirp rate increased from 0.5-0.7 Hz to 0.9–1.3 Hz, but the pulse rate and the pulse number in chirps did not change. (B) During maintained hyperpolarizing current injection, the pCN activity was abolished, and the chirp rate was reduced (start of trace), chirp rate increased upon removing the hyperpolarisation. A subsequent sequence showed the same effect. Sound pulse rate and pulse number in chirps were not altered by current injection. C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 6 applied to 182 chirps of calling song while the command neuron was recorded in G. bimaculatus. The results also show a positive correlation (R = 0.7532) between spike frequency and chirp rate, however no functional correlation between spike frequency and sound pulse rate (R = 0.1568), (Fig. 5B and 5D). Taken together, the putative command neurons recorded here show a very similar effect on controlling calling song parameters as the calling song command neuron identified in G. bimaculatus (Hedwig 2000a) and recorded fibers in G. campestris (Bentley, 1977). 4. Discussion In invertebrates, escape behaviour, which requires fast motor ac- tivity and stereotypical rhythmic behaviour generated by central pattern generators, is under the control of command neurons (Edwards et al. 1999). Also acoustic, and vibratory signalling for intraspecific commu- nication demands little variation, as the sender needs to match the species-specific recognition system of the receiver, however, little is known about the higher neural control of signalling behaviour. We recorded the putative calling song command neuron in G. assimilis and in G. bimaculatus by intracellular recording in the anterior protocerebrum. The recording site matched the brain region indicated by electrical stimulation in G. campestris (Huber, 1963) and the site where the G. bimaculatus command neuron for calling song has its major dendritic arborisation (Hedwig, 2000a). Within the searching area, dozens of neurons were penetrated and tested by current injection. Neurons in different preparations showed similar properties and behavioural re- sponses indicating the same neurons were recorded. Recordings were overall challenging and as micropipettes had a tendency to block during iontophoresis reliable stainings could not be obtained. Although the structure of the pCN was not identified, the similarity of the recording site and physiological properties is taken as evidence that the same type of neuron was recorded. The control of calling song stridulation by the pCN in G. assimilis was similar to that in G. bimaculatus. In both species no prominent synaptic activity was observed in resting males upon sensory stimulation. De- polarization of the pCN was sufficient to raise of the wings into singing position which then was followed by calling song stridulation. In both species, raising the wings into singing position is a motor activity in- dependent to rhythmic singing, and once initiated maintained over extended periods of time without any command activity. In both species higher neuronal activity of the command neuron led to a higher chirp rate as in G. campestris (Bentley, 1977), while the sound pulse rate was not affected by changing spike rates. Together with the tonic activity of the command neurons this supports the finding that the species-specific pulse pattern is controlled by the singing-CPG located in the abdominal ganglia (Jacob and Hedwig, 2020; Schöneich and Hedwig, 2012). The relationship of command neuron activity and chirp rate might imply that the command neurons, directly or indirectly connect with chirp timer neurons (Jacob and Hedwig, 2020) and drive the chirp period. Current data indicate that the pCN, once activated by presynaptic neurons in the brain, generates a descending command signal i.e. a tonic spike Fig. 5. Correlation between pCN spike frequency and calling song parameters in G. assimilis and G. bimaculatus. Correlation between pCN spike rate and calling song chirp rate, and sound pulse rate for G. assimilis (A and C) and G. bimaculatus (B and D). Positive correlation occurred between spike rate and chirp rate in both G. assimilis (R = 0.8595) and G. bimaculatus (R = 0.7532), while the spike rate showed no meaningful correlation with the sound pulse rate in G. assimilis (R = 0.1640) and G. bimaculatus (R = 0.1568). C.-C. Lin and B. Hedwig Journal of Insect Physiology 162 (2025) 104798 7 sequence, which activates and drives the downstream singing-CPG organised along the thoracic-abdominal ganglia. The spike rate of the command signal determines the frequency of chirps, however, the pulse rate and pulse number in each chirp are set by the intrinsic species- specific functional properties of the singing-CPG. The current work in G. assimilis and G. bimaculatus thus demonstrates a similar, possibly conserved central neural network organisation for singing behaviour in crickets. Calling song stridulation encountered while probing the brain could be a result of unnoticed lesions to either the pCN or to any presynaptic neurons that activated the pCN and subsequently the singing-CPG. Hy- perpolarization of the G. assimilis pCN generally abolished the ongoing calling song stridulation. Thus, the neurons recorded in G. assimilis ful- filled the criteria of sufficiency and necessity for the characterization of calling song command neurons (Kupfermann and Weiss, 1978). How- ever, even when the recorded command activity was completely sup- pressed, males could continue generating chirps for 5 to 25 s as the singing-CPG has some autonomy and can sustain singing activity for a short period of time and even without a command input (see Kutsch and Otto, 1972). It also has to be considered that there is a calling song command neuron at either side of the CNS and that a complete control of the system with a single microelectrode cannot be achieved. To further study the effect of a single command neuron, one could sever a neck connective to abort the activity of the contralateral neuron, and for a more robust testing of the necessity criterion pCN activity may be controlled during pharmacological (Wenzel and Hedwig, 1999) or sen- sory initiation of calling song (Kupfermann and Weiss, 1978). We propose that the same recording area in the brain and similar physiological properties of the neurons in these two cricket species suggest conserved command neurons for controlling the species-specific calling song in extant cricket species, but revealing the morphology of the pCN in G. assimilis will be required as well as establishing the syn- aptic connections between the pCN and the CPG elements. Especially the latter should reveal, how the tonic activity of the command neurons relates to the rhythmic activity of the CPG and should provide a deeper understanding of the temporal dynamics of command and CPG activity related to latency, build-up and decline of singing behaviour. Male crickets produce long-range calling song to attract conspecific females, courtship song while courting females, and rivalry song during confrontation with another male (Alexander, 1961). There is a gradual transition from rivalry to calling song and both depend on the same abdominal ganglia, while calling/rivalry and courtship song employ very different motor patterns and different abdominal ganglia for singing (Jacob and Hedwig, 2016; Lin and Hedwig, 2022, 2021a). Depending on the stimulus rate, transitions from courtship song to calling song and from calling song to rivalry song were reported for extracellular stimulation of connective fibres bundles in G. campestris (Otto, 1971) and T. oceanicus(Bentley, 1977), indicating a multifunc- tional role of the descending neurons depending on their discharge rate. However, in the current study, neither courtship nor rivalry song occurred while probing the brain for the pCN. Besides, in this study and in Hedwig (2000a) intracellular stimulation of a single calling song command neuron never elicited courtship song or rivalry song even when the spike rate reached over 150 APs/s. This may indicate that in G. bimaculatus and G. assimilis the calling song command neurons are labelled lines for this specific motor program. But it remains open if simultaneous activation of both the left and right calling song command neuron could elicit rivalry song or even courtship song. However, as the CPG network for courtship song involves different abdominal ganglia (Lin and Hedwig 2022), the calling song command neurons likely do not control courtship song and command neurons for courtship song may be located in other brain areas. Local electrical brain stimulation elicits the normal calling song pattern in the cicada Tympanistalna gastrica pointing also to a control of singing via descending neurons (Fonseca, 2014). In the acridid grass- hopper Omocestus viridulus three stridulatory hindleg movement patterns constitute a courtship sequence, and each movement pattern is elicited by activation of one of three different descending brain in- terneurons (Hedwig and Heinrich, 1997). In all these systems we do not yet understand how the command neurons are activated by local brain circuits, besides some evidence for a local brain neuron initiating singing in the grasshopper Chorthippus biguttulus (Hedwig, 2001). Currently the best insight comes from neurogenetic studies in Drosophila. Here local brain neurons that initiate singing as well as descending command neurons for courtship singing have been identified (von Philipsborn et al., 2011) and may lead to a deeper understanding of the decision process underlying singing behaviour. CRediT authorship contribution statement Chu-Cheng Lin: Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. Berthold Hedwig: Conceptualization, Writing – review & editing, Resources. Funding C-C Lin was supported by Cambridge Trust and Ministry of Education in Taiwan, the equipment used in this research was funded by the BBSRC "United Kingdom" (BB/G018723/1). Declaration of competing interest The authors declare the following financial interests/personal re- lationships which may be considered as potential competing interests: B. 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