Saccular potentials of the vocal plainfin midshipman fish, <Emphasis Type="Italic">Porichthys notatus</Emphasis>

The plainfin midshipman fish, Porichthys notatus, is a vocal species of teleost fish that generates acoustic signals for intraspecific communication during social and reproductive behaviors. All adult morphs (females and males) produce single short duration grunts important for agonistic encounters, but only nesting males produce trains of grunts and growls in agonistic contexts and long duration multiharmonic advertisement calls to attract gravid females for spawning. The midshipman fish uses the saccule as the main acoustic endorgan for hearing to detect and locate vocalizing conspecifics. Here, I examined the response properties of evoked potentials from the midshipman saccule to determine the frequency response and auditory threshold sensitivity of saccular hair cells to behaviorally-relevant single tone stimuli. Saccular potentials were recorded from the rostral, medial and caudal regions of the saccule while sound was presented by an underwater speaker. Saccular potentials of the midshipman, like other teleosts, were evoked greatest at a frequency that was twice the stimulus frequency. Results indicate that midshipman saccular hair cells of non-reproductive adults had a peak frequency sensitivity that ranged from 75 (lowest frequency tested) to 145 Hz and were best suited to detect the low frequency components (≤105 Hz) of midshipman vocalizations.     

Alternative Male Spawning Tactics and Acoustic Signals in the Plainfin Midshipman Fish Porichthys notatus Girard (Teleostei,Batrachoididae)

The plainfin midshipman Porichthys notatus has two male reproductive morphs, ‘Type I’ and ‘Type II’, which are distinguishable by their physical traits alone. Type I males are eight times larger in body mass than Type II males and have a six-fold larger relative sonic (vocal) muscle mass than Type II males. In contrast, the testicles of Type II males are seven times larger than those of Type I males. This study demonstrates morph-specific patterns of reproduction, including acoustic signals, for Type I and II males. Field censuses of nests showed that only Type 1 males maintained nests. Type II males and females transiently appeared in these nests in association with each other. Infra-red video and hydrophone recordings in aquaria showed that Type I males maintained nests and readily vocalized. Long-duration ‘hums’ and sequences of short-duration ‘grunts’ were produced during advertisement and agonistic contexts, respectively. Humming Type I males attracted females to their nests, pair-spawned, and then guarded egg clutches alone. By contrast, Type II males neither acoustically courted females nor maintained available nest sites, but rather ‘sneak-’ or ‘satellite-spawned’ at the nests of Type I males. Type II males infrequently produced low amplitude, short duration grunts that were similar in spectral, temporal and amplitude characteristics to the grunts of females. Type II males appear to be obligate sexual parasites of the nest-building, mate-calling, and egg-guarding Type I males. The dimorphic body and vocal muscle traits of the two male morphs in the plainfin midshipman are thus paralleled by a divergence in their reproductive tactics and the properties of their acoustic signals.     

Intrasexual and intersexual dimorphisms of the red salmon prosencephalon

Intrasexual as well as intersexual dimorphisms were found in the prosencephalon and mesencephalon of adult Oncorhynchus nerka (red/sockeye salmon). These dimorphisms are concerned with the position of the preoptic nucleus, nucleus lateralis tuberis, habenula, third ventricle, tectal ventricles, preoptic recess, recessus lateralis, horizontal commissure, posterior commissure, and toral commissure. The intrasexual dimorphism was characterized by either a rostral ("r"-pattern) or a caudal ("c"-pattern) position of the preoptic region as well as varying locations of other structures within the prosencephalon. As compared to "c"-pattern fish, the preoptic nucleus and nucleus lateralis tuberis were located more rostral, and the habenula was positioned further caudal, in "r"-type animals. The intersexual dimorphism was also characterized by different positions of the structures listed above. With the exception of the preoptic nucleus, all of these were located further rostral in "r"-pattern females than in type "r" males. In "c"-pattern females, they were positioned further caudal than in type "c" males. The number of neurons in the parvocellular and in the magnocellular portion of the preoptic region differed in the two genders with respect to "r"- as well as "c"-pattern fish. Males had more neurons than females in both the magno- and the parvocellular subdivisions of the preoptic region. In "r"- and "c"-pattern fish, the average size of magnocellular preoptic neurons was larger in females than in males. The observed intersexual variations may reflect gender-specific differences in the control of the pituitary. Functional correlates of intrasexual dimorphism are obscure.     

Otolith morphology varies between populations,sexes and male alternative reproductive tactics in a vocal toadfish Porichthys notatus

In this study, the morphology of sagittal otoliths of the plainfin midshipman fish Porichthys notatus was compared between populations, sexes and male alternative reproductive phenotypes (known as ‘type I males or guarders’ and ‘type II males or sneakers’). Sagitta size increased with P. notatus size and changes in shape were also detected with increasing body size. Porichthys notatus sagittae begin as simple rounded structures, but then elongate as they grow and take on a more triangular and complex shape with several prominent notches and indentations along the dorsal and caudal edges. Moreover, the sagittae of the two geographically and genetically distinct populations of P. notatus (northern and southern) differed in shape. Porichthys notatus from the north possessed taller sagittae with deeper caudal indentations compared to P. notatus from the south. Sagitta shape also differed between females and males of the conventional guarder tactic. Furthermore, guarder males had smaller sagittae for their body size than did sneaker males or females. These differences in sagittal otolith morphology are discussed in relation to ecological and life history differences between the sexes and male tactics of this species. This is the first study to investigate teleost otolith morphology from the perspective of alternative reproductive tactics.     

Morphological development of the swim bladder in hatchery-reared striped trumpeter Latris lineata

This study examined swim bladder morphogenesis in three cohorts of striped trumpeter (Latris lineata), a euphysoclist species with physostomous larvae. The swim bladder was first discernible 1–2 days after hatching as an evagination on the dorsal surface of the incipient digestive tract. It comprised a cluster of mesenchymal cells surrounding an inner primordium of epithelial cells. At mouth opening in larvae of 5.3 mm standard length (SL), the swim bladder was noticeably enlarged. Histologically, the swim bladder lumen was dilated and liquid filled. The pneumatic duct was first seen during the dilation stage and the rete mirabile began forming among the connective tissue surrounding the swim bladder. Initial swim bladder inflation occurred on day 11 post‐hatching in Cohort 1, at 14°C, and day 9 post‐hatching, in Cohorts 2 and 3, at 16°C. Histologically, the lumens of inflated swim bladders were ellipsoid and the epithelium was squamous, except for cuboidal gas gland cells at the anterio‐ventral and anterio‐lateral regions of the swim bladder. During the initial inflation interval the pneumatic duct was dilated in larvae both with and without swim bladder inflation. The pneumatic duct began to regress in some larvae over 7.5 mm SL. The swim bladder of striped trumpeter was similar to larvae of other altricial perciform marine fish in respect to organ derivation, tissue differentiation, luminal dilation and initial gaseous inflation. However, variations, particularly the delay in initial swim bladder inflation until after the start of feeding, were observed that could be fundamental to problems encountered during larval rearing.     

Seasonal plasticity of brain aromatase mRNA expression in glia: divergence across sex and vocal phenotypes

Although teleost fishes have the highest levels of brain aromatase (estrogen synthase) compared to other vertebrates, little is known of its regulation and function in specific brain areas. Previously, we characterized the distribution of aromatase in the brain of midshipman fish, a model system for identifying the neural and endocrine basis of vocal-acoustic communication and alternative male reproductive tactics. Here, we quantified seasonal changes in brain aromatase mRNA expression in the inter- and intrasexually dimorphic sonic motor nucleus (SMN) and in the preoptic area (POA) in males and females in relation to seasonal changes in circulating steroid hormone levels and reproductive behaviors. Aromatase mRNA expression was compared within each sex throughout non-reproductive, pre-nesting, and nesting periods as well as between sexes within each season. Intrasexual (male) differences were also compared within the nesting period. Females had higher mRNA levels in the pre-nesting period when their steroid levels peaked, while acoustically courting (type I) males had highest expression during the nesting period when their steroid levels peaked. Females had significantly higher levels of expression than type I males in all brain areas, but only during the pre-nesting period. During the nesting period, non-courting type II males had significantly higher levels of aromatase mRNA in the SMN but equivalent levels in the POA compared to type I males and females. These results demonstrate seasonal and sex differences in brain aromatase mRNA expression in a teleost fish and suggest a role for aromatase in the expression of vocal-acoustic and alternative male reproductive phenotypes.     

Differential effects of 11-ketotestosterone on dimorphic traits in a teleost with alternative male reproductive morphs

Species with dimorphic males typically have one morph (type I) associated with territoriality and courtship, and another morph (type II) associated with cuckoldry. In teleost fishes, type I males generally have higher levels of the androgen 11-ketotestosterone (KT) than type II males. Is KT causal to phenotypic differences between morphs? We investigated this question in the midshipman fish (Porichthys notatus) in which type I males have detectable levels of KT during the breeding season, whereas levels in type II males are usually undetectable. Type I midshipman will either cuckold or hold territories and court females, whereas type II males are only known to cuckold. Acoustic courtship by type I's is supported by the sonic motor nucleus, which innervates a sound-producing sonic muscle. Type I males have larger sonic motor nuclei and larger sonic muscles than type II males, consistent with the more dynamic vocal repertoire of type I's. Here, we tested whether intraperitoneal KT implants in adult type II males would induce type I male-like traits in brain, sonic muscle, and behavior. Type II's treated with KT did not differ from blank-implanted type II's in sonic motor nucleus volume. Sonic muscle mass increased in KT-implanted type II's, but did not reach the relative mass naturally observed in type I's. While neither territoriality nor courtship were induced, cuckoldry behavior intensified in KT-implanted type II's. Thus, for some but not all characters, KT exaggerated the expression of already existing type II male traits rather than inducing the type I male-like traits of territoriality, courtship, and an expansive vocal motor system.     

Early development of the motor and premotor circuitry of a sexually dimorphic vocal pathway in a teleost fish

The plainfin midshipman fish (Porichthys notatus) has a caudal hindbrain vocal motor circuit that has been proposed to share a common embryonic origin with the hindbrain vocal networks of other vertebrates. In midshipman, this vocal circuit includes three groups of neurons: sonic motor, pacemaker, and ventral medullary. Here, transneuronal transport of biocytin or neurobiotin was used to delineate the early ontogeny of the three hindbrain vocal nuclei and their pattern of connectivity. The organization of the vocal nuclei was studied in animals beginning soon after hatching until the nuclei have the adult phenotype at the time fish become free-swimming. There is a clear sequence of events whereby motoneurons establish their connections with the sonic muscle prior to establishing connections with premotor neurons; developmental milestones of the vocal pathway parallel those of the sonic muscle. The results also indicate that sexual differentiation of the vocal motor system in midshipman begins early in development, well before any evidence of sexual maturation. Embryonic males and females differ in the relationship between soma size and body length for the three hindbrain nuclei. Males are also more variable than females in body mass, volume of the sonic motor nucleus, and motoneuron cell size.     

Androgen effects on vocal muscle structure in a teleost fish with inter- and intra-sexual dimorphism

The plainfin midshipman fish Porichthys notatus has both interand intra-sexual dimorphism in the sound-producing (vocal or sonic) muscles attached to the swimbladder wall. The “Type I” and “Type II” male morphs differ in that dramatic structural changes related to sexual maturity occur in the mass, the area of mitochondria-filled sarcoplasm, and the myofiber number of the sonic muscles of Type I males, but not in those of Type II males (nor of females). Androgen implantation for 9 weeks markedly increased the relative sonic muscle size in juvenile males, juvenile females, and Type II males, whereas estradiol or cholesterol treatment did not. The principal androgen effect on myofiber structure was an increase in the area of mitochondria-filled sarcoplasm. The ratio of sarcoplasm area to myofibril area (Sr/Mf) increased by 1.4- to 2-fold in myofibers of all androgen-treated groups, with the greatest structural change occurring in juvenile males. When androgen implants were removed from juvenile males, the muscle mass and Sr/Mf ratio reverted toward the unimplanted juvenile phenotype. Total fiber number in sonic muscle increased significantly in juvenile males following androgen implantation but did not detectably change in juvenile females or Type II males. These results suggest: (1) sonic muscle in Porichthys notatus is an androgen target tissue, (2) fiber structure and fiber number are androgen-sensitive features, and (3) there exist sex- and morph-specific patterns of sonic muscle responsiveness to androgen implants. © 1993 Wiley-Liss, Inc.     

Nonsequential developmental trajectories lead to dimorphic vocal circuitry for males with alternative reproductive tactics

Midshipman fish, Porichthys notatus, have two male reproductive morphs: type I males generate long duration advertisement calls (“hums”) to attract females to a nest; type II males sneak-spawn and, like females, do not produce mate calls but generate short duration agonistic calls. A vocal pacemaker circuit includes: motoneurons in the caudal brain stem and rostral spinal cord that innervate vocal/sonic muscles; pacemaker neurons that are located ventrolateral to motoneurons and establish their fundamental discharge frequency; and a ventral medullary nucleus that couples the motoneuron-pacemaker circuit bilaterally. Transneuronal biocytin transport identified morph-specific developmental trajectories for the vocal circuit. Among nonreproductive, juvenile type I males, motoneuron soma size and motor nucleus volume increase most during a stage prior to sexual maturation. An additional increase in motoneuron size and nucleus volume is coupled to the greatest increase in pacemaker soma size at a stage coincident with the onset of sexual maturity; ventral medullary neurons show similar growth increments during both stages. Type II males (and females) mature with no or little change in cell size or motor nucleus volume. The results indicate that alternative mating tactics are paralleled by alternative developmental trajectories for the neurons that determine tactic-specific behaviors, in this case vocalizations. Together with aging data based on otolith growth, the results support the hypothesis that alternative male morphs in midshipman fish adopt nonsequential, mutually exclusive life history tactics. © 1996 John Wiley & Sons, Inc.     

Morphological and histological changes in the swim bladder during maturation of the Japanese eel

Silver eels Anguilla japonica ( I _G 1·5–3·5) had more developed rete mirabile, gas gland and submucosa than yellow eels ( I _G 0·4–1·5) and the development of swim bladder components increased with sexual maturity only in the early maturation process ( I _G 3·5). These observations indicate that the Japanese eel develops its swim bladder in either the river or shallow sea water and leaves for the open sea after the swim bladder has become adapted to a deeper-sea environment.     

Directionality and frequency tuning of primary saccular afferents of a vocal fish, the plainfin midshipman (Porichthys notatus)

While particle motion is thought to directly stimulate the inner ear of most fish species, it is difficult to measure and might not be predictable from pressure measurements in a small tank. It is therefore important to replicate experiments conducted relative to pressure measurements using stimuli of known particle motion, to ensure that unmeasured components of the stimulus field do not produce misleading frequency response profiles. The frequency sensitivity of the inner ear of the plainfin midshipman fish, Porichthys notatus, in response to isopressure stimuli has been described. This study now examines the frequency and directional response properties of midshipman saccular afferents in response to whole-body displacements simulating acoustic particle motion. Best frequencies were distributed bimodally, with peaks at 50 Hz and 100 Hz. Most units had cosinusoidally shaped directional response profiles in the horizontal and vertical planes, though some units showed slight deviations from this pattern. A few units (probably saccular efferents) had omnidirectional directional response profiles and did not phase lock to the stimulus waveform. These results are consistent with responses of the midshipman saccular nerve to isopressure stimuli, and strengthen the hypothesis that the frequency sensitivity of the midshipman ear matches the frequency content of behaviorally relevant vocalizations.     

Dimorphic male midshipman fish: reduced sexual selection or sexual selection for reduced characters?

In most taxa with male dimorphisms, some males are large inbody size with exaggerated secondary sexual characters (exaggeratedmorph), whereas other males in the same population are smalland have reduced secondary sexual characters (reduced morph).What selective pressures cause male dimorphisms? Reduced morphologiesmay result when a) some males develop a morphology that, inthe absence of sexual selection pressures for an exaggeratedmorphology, reduces energetic and developmental costs and/orb) some males opt for an alternative morphology that does wellat an alternative behavioral tactic such as cuckoldry. The 2mechanisms could act together, but each alone is theoreticallysufficient to drive dimorphisms. Here, we tested hypothesis"b" (sexual selection for reduced characters) in the plainfinmidshipman fish, Porichthys notatus. Behavioral plasticity betweenterritoriality and cuckoldry in an exaggerated male morph (typeI) allows for a direct comparison of cuckoldry by exaggeratedmorph males to cuckoldry by reduced morph (type II) males. Comparedwith type I cuckolders, type II cuckolders were able to remainnear the nest for longer periods before being chased by theterritorial type I male, suggesting that the reduced type IImorphology allows type II males to prolong the time before attackby territorial males. Combined with other studies showing arole of sexual selection in maintaining the exaggerated morph,the data support the "sexual selection for reduced characters"hypothesis and elucidate how sexual selection can act in differentways on different males to maintain 2 male morphologies withina single species.     

A proposed mechanism for the observed ontogenetic improvement in the hearing ability of hapuka (Polyprion oxygeneios)

Swim bladder extensions and hearing ability were examined in the temperate reef fish Polyprion oxygeneios (hapuka). Using the auditory evoked potential (AEP) technique, hearing thresholds were determined in four age-classes of hapuka, from larvae to juveniles. The youngest age-class had poor hearing abilities, with lowest thresholds of 132 dB re 1 μPa, and a narrow auditory bandwidth (100–800 Hz). Hearing ability improved significantly throughout the remainder of their first year, including decreases in thresholds of up to 27 dB, and an increase in auditory bandwidth (up to 1,000 Hz). Magnetic resonance imaging (MRI) was used to investigate structural mechanisms that may account for this ontogenetic improvement in hearing. These showed rostral extensions of the swim bladder developing early in the juvenile stage, and extending with increasing age closer to the otic capsule. It is suggested that this indirect connection between the swim bladder and the otic capsule could impart pressure sensitivity closer to the inner ear, accounting for the increase in sensitivity seen during development, although further investigation of older fish is required for conclusive evidence. The improvement in hearing ability in hapuka could be potentially related to a unique life history of extended pelagic durations up to 4 years.     

Dynamics and acoustical radiation of Porichthys notatus' swim bladder system

The swimbladder system of the plainfin midshipman consists of a gas-filled bladder and two intrinsic sonic muscles which are attached to the bladder at opposite sides. An experimental and analytical study was conducted to define the physical characteristics of this dynamic system, and to relate these characteristics to radiated acoustical pressure pulses. Results indicate that the system has two degrees of freedom, being comprised of two inertial, stiffness and damping components; the first and second mode components of a 23.1-centimeter midshipman are 0.002 and 0.019 kg (inertial) 2130 and 106,000 newtons per meter (stiffness) and 0.25 and 0.10 (damping) respectively. This system is excited by the sonic muscle forcing function which equals \documentclass{article}\pagestyle{empty}\begin{document}$ 0.00236{\rm}\sin \frac{{2\pi {\rm t}}}{{0.0045{\rm}\sec}}{\rm newtons}. $\end{document} Two system frequency response peaks were observed; the first was 110 hertz, at the flat section next to the sonic muscle, and was very near the repetition frequency of the sonic muscle pulses; the second was 350 hertz, at the hemispherical section, which was the frequency of the acoustical pressure pulse. These phenomena describe a dynamical system closely “tuned” to its forcing function, and a system which is highly responsive to acoustical pressure pulses radiated by neighboring midshipmen. The acoustical pressure pulse coincides in wave form with the hemispherical bladder wall acceleration.     

Relationship between Swim Bladder Morphology and Hearing Abilities-A Case Study on Asian and African Cichlids

Background

Several teleost species have evolved anterior extensions of the swim bladder which come close to or directly contact the inner ears. A few comparative studies have shown that these morphological specializations may enhance hearing abilities. This study investigates the diversity of swim bladder morphology in four Asian and African cichlid species and analyzes how this diversity affects their hearing sensitivity.

Methodology/Principal Findings

We studied swim bladder morphology by dissections and by making 3D reconstructions from high-resolution microCT scans. The auditory sensitivity was determined in terms of sound pressure levels (SPL) and particle acceleration levels (PAL) using the auditory evoked potential (AEP) recording technique. The swim bladders in Hemichromis guttatus and Steatocranus tinanti lacked anterior extensions and the swim bladder was considerably small in the latter species. In contrast, Paratilapia polleni and especially Etroplus maculatus possessed anterior extensions bringing the swim bladder close to the inner ears. All species were able to detect frequencies up to 3 kHz (SPL) except S. tinanti which only responded to frequencies up to 0.7 kHz. P. polleni and E. maculatus showed significantly higher auditory sensitivities at 0.5 and 1 kHz than the two species lacking anterior swim bladder extensions. The highest auditory sensitivities were found in E. maculatus, which possessed the most intimate swim bladder-inner ear relationship (maximum sensitivity 66 dB re 1 µPa at 0.5 kHz).

Conclusions

Our results indicate that anterior swim bladder extensions seem to improve mean absolute auditory sensitivities by 21–42 dB (SPLs) and 21–36 dB (PALs) between 0.5 and 1 kHz. Besides anterior extensions, the size of the swim bladder appears to be an important factor for extending the detectable frequency range (up to 3 kHz).     

Aromatase activity in hindbrain vocal control region of a teleost fish: divergence amoung males with alternative reproductive tactics

Type I male midshipman fish acoustically court females, whereas type II males do not but instead sneak or satellite spawn to compete with type I males for fertilizations. ''Singing'' type I males diverge from type II males and females in the organization of an expansive hindbrain pacemaker–motoneuron circuit that establishes the physical attributes of vocalizations. Here, levels of aromatase activity were determined in homogenates of brain by measuring the conversion of ³H-androstenedione (AE) to ³H-oestrone (E₁) and ³H-oestradiol (E₂). Levels were highest in the telencephalon–preoptic area and similar for all morphs. Lower levels were in a region including the diencephalon, midbrain and cerebellum, although levels were significantly higher in females compared with type I males. In the vocal hindbrain region, aromatase levels were three- to five-fold higher in type II males and females than in type I males, and in castrated type II males than in castrated type I males. Conversion of testosterone to oestrogen in type II males and females may effectively prevent testosterone-induced maturation of the vocal system that characterizes type I males. Aromatase may thus be a key enzyme regulating the expression of individual-specific brain circuitry and behaviours among members of one sex.     

Catecholaminergic Innervation of Central and Peripheral Auditory Circuitry Varies with Reproductive State in Female Midshipman Fish,Porichthys notatus

In seasonal breeding vertebrates, hormone regulation of catecholamines, which include dopamine and noradrenaline, may function, in part, to modulate behavioral responses to conspecific vocalizations. However, natural seasonal changes in catecholamine innervation of auditory nuclei is largely unexplored, especially in the peripheral auditory system, where encoding of social acoustic stimuli is initiated. The plainfin midshipman fish, Porichthys notatus, has proven to be an excellent model to explore mechanisms underlying seasonal peripheral auditory plasticity related to reproductive social behavior. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the auditory system in midshipman. Most notably, dopaminergic neurons in the diencephalon have widespread projections to auditory circuitry including direct innervation of the saccule, the main endorgan of hearing, and the cholinergic octavolateralis efferent nucleus (OE) which also projects to the inner ear. Here, we tested the hypothesis that gravid, reproductive summer females show differential CA innervation of the auditory system compared to non-reproductive winter females. We utilized quantitative immunofluorescence to measure tyrosine hydroxylase immunoreactive (TH-ir) fiber density throughout central auditory nuclei and the sensory epithelium of the saccule. Reproductive females exhibited greater density of TH-ir innervation in two forebrain areas including the auditory thalamus and greater density of TH-ir on somata and dendrites of the OE. In contrast, non-reproductive females had greater numbers of TH-ir terminals in the saccule and greater TH-ir fiber density in a region of the auditory hindbrain as well as greater numbers of TH-ir neurons in the preoptic area. These data provide evidence that catecholamines may function, in part, to seasonally modulate the sensitivity of the inner ear and, in turn, the appropriate behavioral response to reproductive acoustic signals.     

Coding of concurrent vocal signals by the auditory midbrain: effects of duration

Neural selectivity to signal duration within the auditory midbrain has been observed in several species and is thought to play a role in signal recognition. Here we examine the effects of signal duration on the coding of individual and concurrent vocal signals in a teleost fish with exceptionally long duration vocalizations, the plainfin midshipman, Porichthys notatus. Nesting males produce long-duration, multi-harmonic signals known as hums to attract females to their nests; overlapping hums produce acoustic beats at the difference frequency of their spectral components. Our data show that all midbrain neurons have sustained responses to long-duration hum-like tones and beats. Overall spike counts increase linearly with signal duration, although spike rates decrease dramatically. Neurons show varying degrees of spike rate decline and hence, differential changes in spike rate across the neuron population may code signal duration. Spike synchronization to beat difference frequency progressively increases throughout long-duration beats such that significant difference frequency coding is maintained in most neurons. The significance level of difference frequency synchronization coding increases by an order of magnitude when integrated over the entirety of long-duration signals. Thus, spike synchronization remains a reliable difference frequency code and improves with integration over longer time spans.