Duration selective neurons in the inferior colliculus of the rat: topographic distribution and relation of duration sensitivity to other response properties

1 Duration selective neurons in the inferior colliculus of the rat: topographic distribution and relation of duration sensitivity to other response properties. D. Pérez-González1,2, M. S. Malmierca1, J. M. Moore1, O. Hernández1, and E. Covey1,2 (1) Auditory Neurophysiology Unit. Laboratory for the Neurobiology of Hearing. The Institute of Neuroscience of 'Castilla y León' (INCyL) and Faculty of Medicine. University of Salamanca Campus 'Miguel de Unamuno' 37007 Salamanca - Spain (2) Department of Psychology Box 351525 University of Washington Seattle, WA 98195 USA Keywords: Duration tuning; sound duration; auditory midbrain; filtering properties; Corresponding author: Ellen Covey; email: [email protected] 2 ABSTRACT Many animals use duration to help them identify the source and meaning of a sound. Duration sensitive neurons have been found in the auditory midbrain of mammals and amphibians, where their selectivity appears to correspond to the lengths of species-specific vocalizations. In this study, single neurons in the rat inferior colliculus (IC) were tested for sensitivity to sound duration. About half (54%) of the units sampled showed some form of duration selectivity. The majority of these (76%) were long pass neurons that responded to sounds exceeding some duration threshold (range: 5 to 60 ms). Band pass neurons, which only responded to a restricted range of durations, made up 13% of duration sensitive neurons (best durations: 15 to 120 ms). Other units displayed short pass (2%), or mixed (9%) response patterns. The majority of duration sensitive neurons were localized outside the central nucleus of the IC, especially in the dorsal cortex, where over half of the neurons sampled had long pass selectivity for duration. Band pass duration tuned neurons were only found outside the central nucleus. Characteristics of duration sensitive neurons in the rat support the idea that this filtering arises through an interaction of excitatory and inhibitory inputs that converge in the IC. Band pass neurons typically responded at sound offset, suggesting that their tuning is created through the same mechanisms that have been described in echolocating bats. The finding that the first spike latencies of all long pass neurons were longer than the shortest duration to which they responded supports the idea that they receive transient inhibition prior to, or simultaneously with a sustained excitatory input. The ranges of selectivity in rat IC neurons are within the range of durations of rat vocalizations. These data suggest that a population of neurons in the rat IC have evolved to transmit 3 information about behaviorally relevant sound durations using mechanisms that are common to all mammals, with an emphasis on long pass tuning characteristics. INTRODUCTION Neurons that are selective for the duration of a sound have been described in the central auditory system of several vertebrates including frogs (Potter 1965; Narins and Capranica 1980), bats (Pinheiro et al. 1991; Casseday et al. 1994, 2000; Ehrlich et al. 1997; Fuzessery 1994; Mora and Kössl 2004), cats (He et al. 1997), chinchillas (Chen 1998) and mice (Brand et al. 2000). Duration selective neurons have only been found at or above the level of the inferior colliculus (IC) or its homolog in any of the species studied, so this form of neural filtering appears to be an emergent property that results from circuitry operating within the midbrain. The IC is innervated by multiple lower brainstem structures with excitatory or inhibitory inputs that vary in latency, temporal pattern, and sensitivity to multiple sound parameters including frequency and amplitude (e.g., Casseday et al. 2002). Previous studies have suggested that duration selectivity is generated through the interaction of such inputs, in particular, the combination of convergent excitatory and inhibitory inputs with specific temporal relationships (Ehrlich et al. 1997; Casseday et al. 1994, 2002; Brand et al. 2000; Faure et al. 2003). In certain species with specialized auditory behavior, the ranges of sound durations to which neurons are tuned correspond closely to the range of durations found in behaviorally important sounds. This matching of neural duration sensitivity to the duration of vocalizations has been described in frogs, where neurons’ duration tuning matches the durations of communication calls (Narins and Capranica 1980) and in bats, where neurons’ duration tuning matches the durations of echolocation signals (Pinheiro et al. 1991; Ehrlich et al. 1997; Faure et 4 al. 2003). More recently, duration sensitive neurons have been found in animals that lack obvious auditory specializations. Although the advantages of duration sensitivity in more generalized species have not been thoroughly investigated, it potentially serves to identify specific sounds. For example, the IC of mice contains a high proportion of long pass neurons selective for sounds longer than several tens of ms. The few observed band pass neurons typically have best durations on the order of tens of ms (Brand et al. 2000). Both of these filtering properties are congruent with the duration of adult and pup ultrasound vocalizations (Liu et al. 2003). Many calls in rats have highly stereotyped temporal patterns (Kaltwasser 1990) in which call duration could convey important information such as individual identity, social status, or contextual situations. For example, rat ultrasonic vocalizations include long (>300 ms) calls that are emitted during a negative affective state when animals are anticipating punishment or engaging in avoidance behaviors and short (<300 ms) calls that are produced during a positive affective state when they are anticipating a reward or displaying approach behaviors (Knutson et al. 2002). Isolation calls of rat pups range from about 80 to 140 ms, decreasing in duration as a function of the pup’s age. They also differ in length between individuals and show greater similarity within than across litters (Brudzynski et al. 1999). Thus, duration could be used by receivers to recognize kin and offspring (Brudzynski et al. 1993). Rats have a rich repertoire of sounds that are produced during exploration, agonistic encounters, sexual behavior, and other social interactions for which duration could convey meaning to conspecifics. To date, there have been no studies to determine whether the rat central auditory system contains duration sensitive neurons and, if so, how these relate to durations of vocal communication signals. The aim of this study was to determine whether there are duration selective neurons in the midbrain of the rat, 5 one of the most commonly used laboratory animals and, if so, how the range of duration selectivity corresponds to the duration of their known repertoire of vocalizations. Preliminary reports have been presented in abstract form elsewhere (Pérez-González et al. 2004a,b). METHODS All experimental procedures were approved by the University of Salamanca Animal Care and Use Committee, and conformed to the guidelines of the U.S. National Institutes of Health. Sixtyone adult male rats (Rattus norvegicus; 47 Long Evans pigmented, 14 Wistar albino strain), weighing between 166 and 365 grams, were used in this study. Anesthesia was induced with an intraperitoneal injection of urethane (1.5 g/kg) and maintained with supplementary doses (0.5 g/kg) as needed. Urethane is a standard anesthetic used in the rat, and was chosen because it is known to affect inhibitory processes less than barbiturate anesthetics (e.g., Hara and Harris 2002). A tracheotomy was performed in order to assure adequate ventilation, and atropine sulfate was administered subcutaneously (0.05 mg/kg) to reduce bronchial secretions. Body temperature was maintained at 38 ± 1 ºC with a thermostatically controlled electric blanket (Malmierca et al. 2003, Hernández et al. 2005). The rat’s head was immobilized by placing it in a stereotaxic frame in which the ear bars had been replaced by hollow specula that accommodated a sound delivery system (Rees et al. 1997, Hernández et al. 2005). A craniotomy was performed to expose the tissue over the recording site, usually over the right IC, and the dura was reflected to allow entry of the electrode. The surface of the brain was irrigated regularly with saline to prevent desiccation. Acoustic stimulation and electrophysiological recording was performed inside a sound attenuated booth. A tungsten electrode (Merrill and Ainsworth 1972) was placed over the 6 exposed cortex and moved along the dorsoventral axis using a piezoelectric microdrive (Burleigh 6000, EXFO Burleigh, Quebec, Canada) that was advanced by remote control from outside the booth. Once the IC was reached, single neurons were isolated, using white noise and pure tones as search stimuli, and sound-evoked action potentials were recorded extracellularly. Stimuli were synthesized by a TDT System II workstation (Tucker-Davis Technologies, Alachua, FL, USA) using custom software, and delivered through a closed field delivery system (Rees 1990) via two electrostatic speakers (TDT EC1) controlled by an electrostatic speaker driver (TDT ED1). The output of the system at each ear was calibrated in situ using a condenser microphone (Brüel and Kjær 4134, Nærum, Denmark) and a DI-2200 spectrum analyser (Diagnostic Instruments Ltd., Livingston, Scotland, UK). The maximum output of the system was flat from 0.3-5 kHz (≈100 ± 7 dB SPL), from 5-40 kHz (90 ± 5 dB SPL) with a notch at about 22-23 kHz with a slope of approximately 15 dB/octave. Second and third harmonic components in the signal were 45 dB or more below the level of the fundamental at the highest output level. Because of the nonlinearities of the loudspeaker output, all values are expressed as dB SPL. The highest frequency produced by our system was limited to 40 kHz. Action potentials were amplified (x10000) with a Bioamp amplifier (TDT) and filtered (0.5 – 3 kHz, TDT DB4) before being processed in a spike discriminator (TDT SD1). The spike times were then stored on a computer. Once a single unit was isolated, its characteristic frequency (CF) and threshold were determined by an automated procedure that consisted of the randomized presentation of pure tones across a matrix of frequency and intensity values that extended beyond the visually estimated response area. The standard duration of these stimuli was 75 ms. For duration sensitive neurons, it was set at the neuron’s estimated best duration. For the experiments in which duration 7 was varied, stimuli were pure tones at the neuron’s CF, typically 10 to 20 dB above threshold, and a rise-fall time of 1 or 2 ms. Stimuli were presented to the ear contralateral to the IC from which recordings were obtained. Stimulus duration was varied randomly among 10 or 20 stepwise values starting at 2-5 ms and extending up to 100-200 ms. To avoid confounds associated with spectral artifacts, we did not test durations below 2 ms. Each duration was presented 10 times. The data obtained were analyzed and plotted using commercial software (Microsoft Excel, Sigmaplot, and SPSS). To determine whether a neuron was sensitive to sound duration, we plotted response probability as a function of duration. Response probability (also referred to as spike probability) was defined as the number of presentations (or trials) of a given stimulus for which there was at least one stimulus-evoked action potential, divided by the total number of presentations for that stimulus. For neurons with spontaneous activity, response probability was calculated after subtracting the spontaneous rate. This measure was used instead of spike counts in order to distinguish long pass duration sensitivity from simple systematic increases in spike count with increased duration in units with sustained discharge patterns. A neuron was considered to be duration sensitive if, at any point, the response probability function dropped below 50% of the maximum. For long pass or short pass neurons the cutoff duration was defined as the point at which the probability function dropped below 50% of the maximum; if this point was between two tested durations, it was calculated by linear interpolation. For band pass neurons, which had cutoffs at both long and short durations, the best duration was defined as that at which spike count was maximal. Because we were unable to test durations shorter than 2 ms, it is possible that those neurons that we classified as short pass were actually band pass, or that they might have had band pass characteristics at some sound levels (Fremouw et al. 2005). 8 Selected recording sites and other landmarks were marked with electrolytic lesions. These marks were used to reconstruct electrode tracks and to confirm the locations of the recorded units. For histological examination of the brain, the animal was administered a lethal dose of sodium pentobarbital (Nembutal; 60 mg/kg in saline, intraperitoneal), and perfused transcardially with Ringer’s solution followed by fixative (1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH: 7.4). The brain was immersed in a 30% sucrose solution for 2-3 days before being cut into 40 µm thick transverse sections using a freezing microtome. Sections were stained with cresyl violet. Neurons were assigned to the IC subdivisions according to the parcellation scheme of Malmierca et al. (1993). RESULTS We recorded responses of 160 neurons to sounds of different durations. Of these, 5 neurons showed some level of habituation to repeated stimuli or responded weakly to all stimuli (Covey et al. 2003; Malmierca et al. 2004) and were consequently excluded from all analyses. Of the remaining 155 neurons, 84 (54.2%) showed some form of duration sensitivity. By far the most common form of duration sensitivity was long pass filtering. Long pass neurons were defined as those with a 50% cutoff on the short duration side, but a higher probability of firing at all longer durations tested. Neurons with long pass sensitivity comprised 41.3% of all IC neurons from which we recorded and 76.2% of duration sensitive neurons. Long pass neurons’ cutoff durations ranged from 5 ms to 60 ms. An example of a long pass neuron is shown in Figure 1A and B. This neuron had no spontaneous activity. It was completely unresponsive to durations less than about 35 ms. From this point, the response probability gradually increased until reaching the 50% cutoff at about 60 ms and a plateau at about 80 ms. This neuron’s response took the form of a sparse sustained discharge. Figure 1B shows that the response probability and sound duration 9 were positively correlated until the neuron reached its maximum probability of firing. First spike latency was extremely variable across trials, but was always more than 35 ms. Figure 1C shows the response from another neuron with a sustained discharge pattern, but this neuron was not duration sensitive (all pass). Although the number of spikes per trial was positively correlated with sound duration, as would be the case if recording from an auditory nerve fiber, there was no duration tested at which the neuron failed to respond reliably. The spike probability function of this neuron (Fig. 1D) was high (>70%) and essentially flat across all of the durations tested. Figure 2A and B shows another example of a long pass duration sensitive neuron, in this case one with a robust transient response of no more than 2 spikes per trial. This neuron was completely unresponsive to 5 ms stimuli, but responded reliably to durations greater than or equal to about 15 ms. Once the duration exceeded the cutoff, the number of spikes per stimulus remained constant and response probability remained flat with further increases in duration. Response latency was consistently about 15 ms, about the same as the cutoff duration. For comparison, Figure 2C and D show the response of an onset responder that was not duration sensitive (all pass). This neuron responded with one spike on every trial, even at the shortest duration tested. The spike probability was 1.0 for all durations. Because there appeared to be a correlation between the cutoff duration and response latency for long pass neurons, we compared the first spike latencies of long pass neurons to those of non-duration sensitive neurons at 20 dB above threshold (Fig. 3). The average latency of long pass neurons with sustained discharge patterns (30.5 ms, SD 15.1) was significantly longer than that of the other classes of neurons (two-tailed t-test, P < 0.001). Non-duration sensitive neurons with sustained responses had a mean latency of just 14.0 ms (SD 5.0). Long pass neurons with transient responses had a mean latency of 16.8 ms (SD 3.9) compared with non-duration 16 echolocation calls suggests that the large number of band pass neurons in the bat represents a specialization for echolocation, whereas long pass neurons may be used for processing communication sounds, and therefore be the more primitive characteristic. In rats, the cutoff durations of short pass neurons and the best durations of band pass neurons are consistent with the longer durations of rats’ vocalizations, and fall mainly in the duration range of audible calls emitted during exploration, agonistic behavior, and sexual behavior. Some are sensitive to durations corresponding to the range of pup calls (Brudzynski et al. 1999; Kaltwasser 1990). Long pass neurons’ cutoff durations were distributed over a fairly wide range, from about 5-60 ms, indicating that a subset of these neurons would be unresponsive to short duration calls such as the “broadband” signals emitted during exploration or the “short” and “wave-like modulated” calls emitted during sexual behavior. These cells would presumably be responsive to infant calls as well as the “screams” and “frequency step” calls emitted during agonistic behavior (Kaltwasser 1990). Our finding that most band pass and long pass neurons were located outside the central nucleus of the IC also seems different from the situation in the bat, where most duration sensitive neurons, at least band pass ones, are found in the caudal half of the central nucleus (Ehrlich et al. 1997). The finding that the rat has a high proportion of long pass neurons in the dorsal part of the IC is consistent with the idea that neurons in this region have longer integration times due to the influence of cascaded intrinsic projections within the IC itself (Miller et al. 2005) and/or descending input from the cortex (Caicedo and Herbert 1993; Saldaña et al. 1996; reviewed in Malmierca 2003). Previous studies in which inhibition was blocked (Casseday et al. 1994, 2000; Fuzessery and Hall 1999) provide convincing evidence that duration sensitivity of all types can be created 17 through the interaction of excitation and inhibition within the IC. However, there is also evidence that inhibition may not be directly responsible for the duration tuning of all IC neurons (Fuzessery and Hall 1999). The fact that latencies of long pass neurons were always longer than their cutoff duration, and the average latency of long pass neurons was longer than the average latency of those that were not sensitive to duration, is consistent with the idea that the duration of a single onset-evoked inhibitory input determines both the cutoff duration and response latency of long pass neurons (Fig. 14 A,B) (Brand et al. 2000; Faure et al. 2003). For neurons with sustained responses and paradoxical latency shift (Sullivan 1982), whose cutoff durations shifted to longer values with increasing sound level, this clearly seems to be the case. For these neurons, the relative strength and duration of a transient, onset-evoked inhibitory input apparently grew at a faster rate than that of a sustained, onset-evoked excitatory input (Fig. 14C). Previous studies have shown that in many IC neurons, inhibition precedes excitation (e.g., Covey et al. 1996; Kuwada et al. 1997), and that such inhibition can cause paradoxical latency shift (Covey et al. 1996). For those neurons whose cutoffs shifted to shorter durations with increases in amplitude, this shift could be explained if the situation were reversed so that the strength and duration of inhibition grew at a slower rate than that of excitation (Fig. 14D). Consistent with this idea is the finding that inhibition can have a lower threshold than excitation (Covey et al 1996; Kuwada et al. 1997) and that blocking inhibition causes a lowering of threshold in some IC neurons (Vater et al. 1992, LeBeau et al. 2001). An equally plausible alternative explanation would be that these neurons were integrating subthreshold excitatory inputs over a period as long as 90 ms, possibly through projections from a cascaded system of delay lines within the IC (Miller et al. 2005) or convergence of ascending inputs with descending excitatory projections from outside the IC. In 18 this case, increases in sound amplitude would result in larger, longer lasting EPSPs, allowing temporal summation to occur. The finding that blocking inhibition in some IC neurons in the pallid bat did not abolish their duration tuning is consistent with this hypothesis, and suggests that duration sensitivity may arise through multiple mechanisms. Moreover, the relative prevalence of each mechanism may vary across species. For long pass neurons with transient responses, a transient onset-evoked inhibition could not easily create a long pass response, because it would presumably have the same effect regardless of stimulus duration. However, if transient inhibition were evoked by the offset of the stimulus, it could eliminate responses to sounds with durations shorter than the latency of the excitatory input, while permitting responses to sounds at all longer durations. The latency of an offset response (relative to the onset of the stimulus) is equal to the duration of the stimulus plus the latency of the inhibition relative to sound offset. Based on the data in Fig. 6, the average value for the theoretical 50% cutoff duration at which the latency of the offset inhibition becomes long enough to permit a response to onset-evoked transient excitation is 7.3 ms (SD 3.1), a perfectly plausible value for latency in the rat IC. The observation that band pass neurons were typically offset responders is consistent with previous studies showing that transient onset-evoked excitation and sustained inhibition followed by an excitatory rebound at sound offset is responsible for this response pattern (Casseday et al. 1994, 2000; Faure et al. 2003). To further support this view, all of the offset responders in this study showed some kind of duration sensitivity. Although the number of offset neurons in our sample is relatively small (7), the same finding has been published in other species, including the mouse (Brand et al. 2000) and chinchilla (Chen 1998). In the bat, up to 80% of duration sensitive neurons are offset responders (Fremouw et al. 2005), a finding that is 19 consistent with the observation that the majority of duration sensitive neurons in the bat have band pass tuning (Ehrlich et al. 1994). It seems this relationship also holds in regions outside the IC. For example, in the dorsal zone of the auditory cortex of the cat, 24/28 offset neurons were described as duration sensitive (He et al. 1997). Therefore, it seems to be a highly consistent finding, both across species and across sound levels, that a majority of offset responders are sensitive to stimulus duration. The conclusion to be drawn from considering all of these data together is that duration sensitivity in the IC takes many forms, and may arise through diverse mechanisms, but that these mechanisms are consistent across species. Although it is tempting to think of duration sensitivity as an independent filtering mechanism that creates “feature detectors” selective for biologically relevant sound durations, we must also consider the possibility that it is but one component of a larger scale mechanism underlying the filtering and analysis of complex temporal sequences of natural sounds. The fact that duration tuned neurons in the bat experience suppression analogous to forward and backward masking when presented with sounds of different durations (Faure et al. 2003) suggests that their responses are also determined by the temporal context in which a given sound occurs. In the rat, we found that most duration sensitive neurons were located in the cortical areas of the IC. This finding was surprising because in the bat they have been reported only in the CNIC, mainly in the caudal portion (Ehrlich et al. 1997). In the other studies, either the recordings were performed only in the CNIC (Ehrlich et al. 1997; Faure et al. 2003; Fremouw et al. 2005) or there was no precise indication of the location (Pinheiro et al. 1991; Chen 1998; Fuzessery and Hall, 1999; Brand et al. 2000; Mora and Kössl 2004), so the opportunities for comparison are limited. There are some anatomical differences that may explain the different 20 distribution of the duration sensitive neurons in the rat and bat. In Eptesicus fuscus, the bat in which duration tuned neurons were localized, the “cortical” areas surrounding the CNIC are not nearly as prominent as they are in rats. In Eptesicus the “pericentral area” is quite small (less than 200 µm thickness) and includes regions that would presumably correspond to both the ECIC and DCIC in rats (Casseday and Covey 1992; Covey and Carr 2005). Even though we cannot rule out a sampling bias due to the relatively larger size of the cortical areas in the rat, it is still significant that only 20% of the CNIC units in our sample had any kind of duration sensitivity while the percentage was much higher in the cortical areas. This finding suggests that duration sensitive neurons in the rat may be important for orienting to sounds of specific durations. The finding that all of the different types of duration sensitive neurons that have been described in other species are present in the rat IC reinforces the idea that duration tuning is an emergent property in the midbrain of all vertebrate species, and that it arises through multiple mechanisms and takes multiple forms. The selectivity of rat IC neurons for relatively long durations that correspond to those within the rat’s vocalization repertoire suggests that there are species-specific adjustments in latencies, discharge patterns, and synaptic strengths that optimize the range of sound duration sensitivity in each species to produce filtering properties that are important for behaviors that depend on analyzing the temporal pattern of sound. Acknowledgments: Research supported by the Spanish JCyL-UE (SA0400/04, MSM), DGES (BFI-2003-09147-02-01, MSM) and NIH (DC00607, EC). Present address of J.M. 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Sounds were pure tones presented at 10 dB above threshold. In this 32 33 34 35 36 37 38 39 40 41