Across-fiber patterns may contain a sensory code for stimulus intensity

Brain Research Bulletin, Vol. 26, PP. 327-331. 0 Pergamon Press plc, 1991. F’rinted in the U.S.A. 0361-9230/91 $3.00 + .OO

Across-Fiber Patterns May Contain

A Sensory Code for Stimulus Intensity

BRUCE R. JOHNSON,*?’ RAINER VOIGT,* CARL L. MERRILL* AND JELLE ATEMA*

*Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 and fsection of Neurobiology and Behavior, S. G. Mudd Hall, Cornell University, Ithaca, NY 14853

Received 7 May 1990

JOHNSON, B. R., R. VOIGT, C. L. MERRILL AND J. ATEMA. Across-$ber panerns may confain a sensory code for stimulus infer&y. BRAIN RES BULL 26(3) 327-33 1, 1991. -Various models for sensory coding have used statistical approaches based on the assumption that the stimulus intensity parameter is represented in the afferent neurons as mean firing frequency. In this paper we question the assumption that this is the only code for intensity. We show that in lobster olfactory receptors narrowly tuned to hydroxyproline, an across-fiber pattern (AFP) code distinguished more concentration levels over a 5 log step range than a response magnitude code and, unlike the latter, was unaffected by response summation time. AFP discrimination of stimulus intensity ap- pears to be based on high inter-cell response variability and low intra-cell response variability.

Across-fiber patterns Olfaction Sensory coding Stimulus intensity Chemoreception Lobster

IT is generally believed that the sensory neural code for stimulus intensity is the mean firing frequency (MFF) of afferent neurons (23,27). This concept conveniently separates intensity coding from the coding of some sensory qualities which are believed to be reflected in spatial patterns of firing across a number of afferent fibers [reviewed by (9)]. While convenient for the human mind, there is no evidence that this separation of quantity and quality coding is universally accepted by the animals’ central nervous system. In cases where stimulus-response (SR) functions are rising monotonically with reasonably steep slopes, intensity coding by fiing frequency is a logical consequence. However, in many instances SR functions of individual cells are erratic or flat in slope. The resulting inter-cell response variability limits intensity information. As long as intru-cell response variability is low, these same erratic SR functions and, in addition, the common phenomenon of range fractionation (6), form a natural basis for an across-fiber pattern (AFF) code for stimulus intensity. There- fore, we want to consider here the possibility that stimulus intensity is coded across afferent fibers.

To demonstrate this point we used a series of SR functions from lobster olfactory cells. The data show that patterns can code stimulus intensities over a larger range than average firing fre- quencies, and that, unlike MFF, AFFs are not restricted by response summation times.

We examined SR functions from hydroxyproline (Hyp) cells, the most prominent population of chemoreceptor cells on the lateral antennular filament (olfactory organ) of the American lobster, Homarus americanus. These receptor cells are very narrowly

tuned to Hyp; they have no spontaneous activity (they fired no spikes during a 10 s prestimulus period) and low maximum firing rates to excitatory stimuli [around 30 spikes/5 s; (17-19)].

METHOD

Details of the preparation chamber, dissection, extracellular recording techniques, stimulus reliability, and data analysis have been described in detail recently (18,19). Briefly, ablated lateral antennular filaments placed in the stimulating compartment of a preparation chamber were continuously flushed with a carrier flow of artificial sea water (ASW, 10 mllmin) into which a standard 50 ~1 aliquot of test stimulus was injected. Stimuli contacted the chemoreceptor area approximately 500 ms postinjection and rap- idly peaked within 1.5 s to a dilution approximately 0.03 times the injected concentration. The stimulus concentrations reported here and in the results are peak concentrations corrected for this dilution factor. The halftime of the stimulus pulse was approximately 2 s; within 15 s after the stimulus pulse injection, the stimulus concentration in the chamber was below the level of de- tectability. Test stimuli were applied at one minute intervals with vigorous flushing of the stimulation chamber between stimulus injections.

A stock solution of 10-l M Hyp was prepared in ASW and frozen (- 15°C) in 20 ml volumes until thawed for use the day of an experiment. All dilutions of this stock solution were made with ASW. Single cell SR functions for cells responding to Hyp were gathered by identifying responses from small nerve bundles to 3 x 10e6 M Hyp. Since Hyp cells are not spontaneously ac-

‘Requests for reprints should be addressed to Bruce R. Johnson at his present address: Section of Neurobiology and Behavior, S. G. Mudd Hall, Cor- nell University, Ithaca, NY 14853.

327

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tive, any spike firing after the introduction of a test concentration was considered a response. Cells were presented with an ascend- ing series of Hyp concentrations beginning with the lowest and ending with the highest test concentration. At the end of any experimental series, a cell was retested for its response to a previ- ously tested mid-range concentration. This ensured that responses to high concentrations were not limited by declining cell viabil- ity. A separate group of Hyp-sensitive cells were subjected to 3 repetitive presentations of two different Hyp concentrations to determine the single cell variability to repeated applications of the same stimuli. For these tests we alternated pulse concentrations between 3 x 10m6 and 3 x IO-’ M Hyp, starting with 3 x 10e6 M; pulses were separated by 1 minute. In order to limit the number of stimuli we did not test the response specificity of the olfactory receptors reported here. Because their response characteristics correspond to those of narrowly tuned Hyp cells identified in previous studies (17,18), we will refer to these cells as Hyp cells.

Single receptor cell responses were discriminated on the basis of spike amplitude and waveform during data analysis. Response spike counts were begun with the first spike of a response and continued for up to 5 s; most responses were over within 5 s. Statistical comparisons of mean response magnitudes were made with the Wilcoxon matched-pairs signed-ranks test. Statistical differences in MFF between test concentrations were accepted at pcO.05 because this is the significance level commonly accepted for paired comparisons in biological studies. Statistical comparisons of the AFPs of receptor activity were made using the Pear- son product-moment correlation coefficient [r; as used by (lo)]. For these comparisons we eliminated absolute response magnitude by normalizing a cell’s responses to the response at its most stimulatory concentration, and then arc-sine transformed the data. Although several different methods have been used to compare the similarity of AFPs (8), we chose the Pearson’s r because correlation methods have had wide usage in chemical senses research [reviewed by (13)] and because this is a well understood statistical procedure. Significant differences in AFPs were accepted at p<O.Ol because this significance level has been commonly used for correlation comparisons in previous studies.

RESULTS

The mean SR functions for 21 Hyp cells show that at different response summation times of 250 and 500 ms. and 1 and 5 S. this cell population had a response threshold between 3 X lo-” and 3 x 1OK’ M Hyp and that response saturation for Hyp stimulation occurred near 3 x 10e4 M (Fig. 1). Based on statistical differences in the MFF of the population. the lower part of the response function provided statistically significant discrimination on whole log step stimulus intensity differences at all response summation times. However, using MFF the two highest test concentrations could not be discriminated at the 250 and 500 ms response summation times (Fig. 1). Furthermore. at 1 and 5 s response summation times, statistical differences in MFF were not only lost between the two highest, but also lost between the second and third highest test concentrations (Fig. 1).

Figure 2 shows the response correlation profiles for Hyp cells across stimulus intensities. AFPs for each intensity correlated with themselves (r= 1) but most other stimulus intensities were not significantly correlated, indicating that the stimuli can be perceived as dissimilar (8). In contrast to the lack of resolution of stimulus intensity provided by MFF analysis at the high end of the SR function, AFP analysis resulted in a lack of discrimination of stimulus intensity at the low end. However, the latter was the case only for the 500 ms and 5 s response summation times where

JOHNSON. VOIGT, MERRILL AND ATEMA

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FIG. I. Mean stimulus-response functions from 21 Hyp-sensitive olfactory receptors at different response summation times: (A) 250 ms, (B) 500 ms, (C) 1 s, (D) 5 s. Wilcoxon matched-pairs signed-ranks tests were used to compare response magnitudes in individual cells between test concentrations. A horizontal bar connects all response magnitudes which are not significantly different (~720.05). Vertical bars represent the SEM.

the two lowest concentrations were weakly correlated with each other (Fig. 2). Weak responses to test stimuli may produce low

FIG. 2. Correlation profiles across all Hyp test concentrations for the responses of 21 Hyp-sensitive cells at response summation times of 500 ms (filled squares) and 5 s (filled circles). The r values are the Pearson’s product-moment correlation coefficients. The r value for the 0.01 level of significance is represented by the broken lines.

POPULATION CODE FOR STIMULUS INTENSlTY 329

-8 -7 -6 -5 -4 -3 -2

HYP CONCENTRATION (LOG Ml

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CELLS l- 21

PIG. 3. Stimulus-response patterns for 21 Hyp-sensitive olfactory receptors. (A) Individual stimulus-response functions for 21 Hyp cells at 500 ms response su~ation time. (B) Response patterns for 21 Hyp ceils across all Hyp test concentrations. White bar at left of each cell row in- dicates 10 action potentials.

correlations between different AFPs when using response correlation profiles simply because of large response variance relative to the mean response (13). Consequently, some of the low correlations in AFPs we observed at low Hyp concen~tions may be artifacts of the statistical procedure. This should not, however, affect the high end of the SR function where AFPs appear to dis- criminate concentrations better than the MFF. In addition, the correlation coefficients of Fig. 2 show that, again unlike stimulus discrimination based upon firing frequency, AFP correlations were not affected by the response su~ation time.

The individual SR functions for these cells (shown for example, at the 500 ms response summation time, Fig. 3) demonstrated that 1) there was variability in the form of the individual SR functions within this cell population. 2) Most individual SR functions did not show a simple monotonic increase in response with increasing stimulus concentration. 3) Most cells responded over at least a 4 log concen~tion range. And 4) many cells dis- played extremely shallow SR functions. The patterns of activity across all the individual cells in response to the stimulus concentrations are visualized in Fig. 3B.

Finally, we examined the responses of another Hyp cell population to repeated presentations of two different Hyp concentrations, 3 X 1O-6 and 3 X 10V5 M. We compared the inter-cell response variability at each stimulus concen~tion to the intra-

A 3xlC?H Hyp

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REPETITION

FIG. 4. Mean responses of 16 Hyp cells to three repetitions of two different concentrations of Hyp: 3 x 10m5 and 3 X 1Om6 M. Asterisks indi- cate that the mean responses to the different concentrations of Hyp are significantly different (Wilcoxon matched-pairs signed-ranks test, p<O.OS). The mean responses to 3 repetitions of the same Hyp concen~tio~s, however, are not sig~~candy different (Wilcoxon matched-pairs signed ranks test, p>O. 10).

cell response variability at that same concentration. Mean responses to the different concentrations of Hyp were significantly different but responses to 3 re~titions of the same Hyp concentrations were not (Fig. 4). At 3 X 10e6 M Hyp, the mean square (estimate of variance) value for inter-cell responses was 142 while the intra-cell mean square value was 8. At 3 X low5 M Hyp the mean square value for inter-cell responses was 108 while the intra-cell mean square value was 17. Thus, in contrast to the inter- cell response variability, the intra-cell response variability was relatively low.

DISCUSSION

The results show that an AFP code can, at least theoretically, convey stimulus intensity information over most of a receptor ~pulation’s response range at least as well as, and in some cases better than an MFF code. The relatively low m~imum ftig rates of the Hyp cell population impose a shallow slope on the population SR function which immediately limits intensity information by an MFF code (20,25). The high inter-cell response variability and low i&a-cell variability form the basis for dis- criminable intensity responses by an AFP code. In addition, unlike the MFF code, the disc~~nation of stimulus intensity by the AFP code was independent of response su~ation time. There- fore, we suggest that an AFP code for stimulus intensity discrimination should be considered as a viable alternative to MFF coding, The two coding principles appear to operate in different conditions; MFF, in the data set used here for example, is better in low and AFP for high stimulus intensities.

Another type of population code, range ~actionation (6), may augment an AFP code at low stimulus intensities. At 3 x lo-’ M Hyp only about half of the Hyp cells responded, the rest responded at the next highest test concentration. Receptor cells with different response thresholds allow a receptor organ to function over a wide working range. Such a mechanism, for example, may con- tribute to loudness descrimination in cochlear fibers to pure tones over a 100 dB range (1 I) and has been suggested to code for

330

stimulus intensity in other crustacean chemoreceptor populations

(73). The conditions favoring MFF coding over large stimulus in-

tensity steps are poor reliability of individual afferent neurons and

massive random convergence upon second order neurons. Ran-

dom convergence would allow signal averaging in second order neurons to extract meaningful signals from the noise caused by intra-cell variability. The conditions favoring AFP coding are inter-cell response variability, reasonable reliability of individual afferent neurons and spatially organized specificity of central pro-

jections.

The more we look into the development and structure of the nervous system, the more we are struck by the precision of its architecture. And, the more we perfect experimental procedures, the more individual neurons show their reliability in signal transduction. Thus it seems that our knowledge and experimental conditions that once strongly favored the signal averaging function of MFF coding now have shifted to make the spatially organized AFP coding more attractive. The same spatial organization of central projections necessary for quality coding could serve intensity coding. AFP coding could represent an organizational prin- ciple of the nervous system (9,24); such population codes are suggested to play roles in vertebrate nervous systems ranging from movement control in primates (12) to memory processes (9).

We do not yet know if the possibility of stimulus intensity coding by AFPs exists in other chemoreceptor cell populations that may signal stimulus qualities besides Hyp. We do know that other chemoreceptor populations contain cells with relatively shallow SR functions and/or large inter-cell SR function variability. Such examples can be found in lobster olfactory taurine cells (Johnson, Voigt and Atema, unpublished observations), lobster gustatory ammonium cells (Borroni and Atema, unpublished observations) and moth pheromone receptors (22), but these cell populations have not been analyzed for intensity coding by AFPs. AFPs do not appear to code stimulus intensity in a population of lobster chemoreceptor cells responding to mixture stimuli (14). Perhaps these cells have monotonically increasing SR functions with little inter-cell response variability. Certainly intensity cod-

JOHNSON, VOIGT, MERRILL AND ATEMA

ing could be accomplished by different means depending on the SR functions of the receptor cell populations.

Intensity and quality are not generally distinct attributes of sensory stimuli. In chemoreception, AFP codes for stimulus intensity could interact with AFP codes for stimulus quality. Inten- sity and quality interactions in chemoreception can be found in higher order processing of chemosensory information. For example, response patterns of olfactory bulb neurons can change with odor intensity as well as with odor quality (29). Interactions between intensity and quality information would be consistent with the findings in chemoreception (and other senses) that stimulus intensity differences may be perceived as quality differences (3. IS. 16).

Finally, these experimental results and coding hypotheses must be placed in a natural stimulus context of rather randomly fluctu- ating stimulus intensities encountered in odor plumes (1. 2. 21). The instantaneous intensity contrasts in odor plumes vary widely from fractions of a log step to several log steps. Self and cross- adaptation [e.g., (4. 5, 19)] and slow rates of disadaptation (28) cause severe changes in response functions. The highly artificial conditions of determining SR functions are important when study- ing mechanisms of sensory signal transduction. However, sensory neural coding has an ultimate behavioral function, where natural stimulus conditions enforce selection. If the nervous system is not using the intensity information encoded in afferent patterns we must assume that the system has become organized to exclude it because-as this study shows-the patterns are there. Until we understand better what these natural stimulus conditions are. and how the connections of the afferent sensory system are organized spatially, we cannot allow ourselves to be fixated on an MFF code for stimulus intensity. Until experimental evidence forces the rejection of either one, both codes remain theoretical constructs and their use in behavioral discriminations remains to be demonstrated.

ACKNOWLEDGEMENTS

We thank Drs. P. M. DiLorenzo and .I. H. Peck for helpful discus- sions. This work was supported by NSF grant No. BNS 8512585 to J.A.

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Across-fiber patterns may contain a sensory code for stimulus intensity