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Response pattern features of mitral cells in the goldfish olfactory bulb
364
Brain Research, 405 (1987) 364 - 370 Elsevier
BRE 22081
pattern features of mitral cells in the goldfis,h olfactory bulb Detlev Schild Physiologisches lnstitut der Universitat GOttingen, Abteilung Neuro- und Sinnesphysiologie, GOttingen (F. R. G.)
(Accepted 28 October 1986) Key words: Olfactory bulb; Response pattern
Temporal discharge characteristics of goldfish olfactory bulb mitral cells are investigated. In contrast to earlier findings, it has been shown that every response is determined by only two features, namely, the mean activities of the early and:the late response (dynamic and static response component). As both activities can be lower than, indistinguishable from, or higher than prestimulus activity, the two response features correspond to 9 possible response pattern types.
Mitral cells of the olfactory bulb respond to odours with patterned discharges 1,6A°,11,16 which indicates that the ongoing stimulus response activity is not stationary 18. Responses are therefore not adequately described by categorizing them as excitatory, inhibitory, and indifferent (+, - , 0) 2'7. In quantitative analyses of experimental data, the structure of temporal response patterns has been taken into account only rarely and to date there does not appear to exist a consensus of what should be accepted as a significant response. The patterns described by Meredith and Moulton 12 and by Kauer 9 were obtained by averaging only a few responses to the same stimulus. The results of these studies, which will be compared with ours, are rather complex pattern classifications. A much simpler classification is obtained if the following points are taken into consideration. First, a stimulus response is considered to be the activity within the period of stimulation, and effects subsequent to the stimulus' offset are not considered as specific for the stimulus. Second, interspike intervals of mitral cell activities are in many animals of about the same length as some pattern structures. For example, given an activity of, say, 2 spikes/s and the usually large standard deviation of mitral cell spike activity, it is hardly possible to
decide whether or not an interspike interval of I or 2 s represents an inhibition. For a reliable estimation of patterns a number of stimulus responses are necessary. Third, it has been shown that mitral cell responses are sometimes not reproducible when the same stimulus is delivered several times 22. Meredith and Moulton 12. too, found that responses to repeated stimuli, though being consistent in the temporal pattern structure, change in response amplitude. In the case of unreproducible responses, the first response might be essentially different from a later one. Averaging such responses in order to attain an averaged PSTH (peristimulus-time histogram) does not make sense because the single run PSTHs are drawn from different statistical ensembles. It would rather lead to the definition of patterns that cannot be found in reproducible responses and thus increase the number of pattern classes. In this study, a sufficiently large number (40) of responses to the same stimulus was registered, reproducible responses were separated from unreproducible ones, and from the former pattern features were extracted: a dynamic and a static response component. Though unreproducible recordings are inappropriate for defining pattern features, these lea-
Correspondence: D. Schild, PhysioiogischesInstitut der Universitfit G6ttingen, Abteilung Neuro- und Sinnesphysiologie, Humboldtallee 23, D-3400 G6ttingen, F.R.G.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
365 tures, once defined, can be found and tracked in such recordings, too. The result of this study is a pattern classification simpler than those proposed earlier and in accordante with other pattern structures in sensory physiology. Thereby, a practical method is suggested to determine exactly whether or not a mitral cell stimulus response is significant. Animal preparation, odour application as well as data acquisition were exactly as described in a previous paper 22. We give therefore only a brief account: 70 extraceUular recordings from goldfish mitral cells were evaluated, each of them including 40 presentations of the same stimulus. In 20 experiments done by Fischer 3'4 the olfactory tract was cooled to 1 °C in order to block the efferent influence upon the bulb. Details of the cooling procedure are given elsewhere 3,4. A standard craniotomy was performed in the anaesthetized animals (MS 222, 300 mg/l). During the experiment the fish were immobilized by tubocurarin chloride (0.3 mg/kg) and perfused through the mouth with continuously flowing tap water (200 ml/min). Only two stimuli, amyl acetate 10 -4 M and tubifex food extract 10 -4 g/l were applied (this is no restriction, as far as the temporal structure of patterns are concerned, because it has been shown that particular stimulus substances are not associated with particular response patterns9'12). Each of the 40 stimulus presentations had a length of at least 30 s and was preceded by an interval of at least 30 s during which tap water was delivered to the mucosa, so that every run consisted of a water phase and a stimulus phase. There was a dead time interval of about 2 s between the switch from water to stimulus (or vice versa) on the one side and the arrival of the stimulus (or water) at the mucosa on the other. The concentration rise time of the stimulus solution at the mucosa was about 0.5 s. Further details of the olfactometer have been described earlier 2°. The spike signals were conventionally amplified and the times at which mitral cell spikes occurred were stored in a computer ( D E C / P D P 11/03). The resulting raw data as well as qualitative interpretations have been described previously 22. Data evaluation was performed as follows. First of all PSTHs were calculated for each run (bin width = 2 s), so that the outcome of an experiment was represented by an activity matrix a(r,b) whereby the in-
dices r and b indicate the r th run and the b th bin: a(1,1), a(1,2) a(2,1), a(2,2)
. . . a(1,15), a(1,16) . . . a(2,15), a(2,16)
. . . a(1,30) . . . a(2,30)
a(40,1), a(40,1) . . . a(40,15), a(40,16) . . . a(40,30) The r th line, a(r, b), r = constant, b = 1, 2 . . . . 30, of the matrix is the PSTH of the r th run; in the following it is denoted at(b), indicating that r is a constant parameter while b is the independent variable. Likewise, the b th column a(r,b), b = constant, r = 1, 2 . . . . 40 "or ab(r ) is the activity within the b th bin viewed as a function of the runs. With a(r) the mean activity of the r th run, 30
d(r)
= 1
bE1 ar(b )
the overall activity change c m across the runs can be approximated by the linear regression coefficient that results when a straight line is fitted to the function a(r). Further, it is convenient to define for every bin the binwise activity change c(b) of the activities ab(r ) in the course of the runs; this activity change can approximately be obtained as the linear regression coefficient that results when a straight line is fitted to the function ab(r). So for each bin there is a number c(b) indicating how much, in the mean, activity changes across the runs. I f c(b) does not differ significantly (P = 0.05) from c m for all bins, the changes across the runs are homogenous in all bins, i.e. the time structure of the stimulus response does not change in the course of the runs. If, however, c(b) differs significantly from c m for some bins, the activity in these bins changes across the runs in a way different from the overall change. Such stimulus responses were considered unreproducible and were discarded from further evaluation. For the purpose of feature detection only the reproducible stimulus responses (49 out of 70 total responses) were considered. The question, 'is the activity in a certain bin A different from the activity in another bin B?' was tested for all pairs of bins with the Wilcoxon-Mann-Whitney test (U-test) whereby the 40 values of bin A (aA(r), r = 1, 2 . . . . 40) were compared to those of bin B (aB(r), r = 1, 2 . . . . 40).
366 As a measure of difference, the Gaussian variable ZAa provided by the U-test was taken as criterion: at the 5% level the activities in bin A and bin B are assumed to be different if ZAB ~> Z 0 . 0 5 ~- 1.96. Given the bin width of 2 s, there were 15 bins of 2 s in both the water and the stimulus phase; so 900 U-tests per experiment were carried out. The outcome of an experiment as described above are 40 p e r i s t i m u l u s - t i m e histograms. In what follows, the general t e m p o r a l structure underlying such histograms is reported. The activities in every pair of bins of a P S T H were c o m p a r e d , respectively, for activity similarity by means of the U-test: in Fig. 1
CD E 6
T l l
16
30
I P bin k
Fig. 1. Significance of activity differences between bins of a PSTH. To every pair of bins of the PSTH. which is shown at both axes of the plot, corresponds a field of the 30 x 30-matrix. For example, the pair (bin 26, bin 23] is indicated by a point in the upper right quadrant; it is the intersection of the dotted lines at bin 26 of the bin A-axis and at bin 23 of the bin B-axis. The activities in a certain bin A and another bin B differ from each other (P = 0.05) if the corresponding field in the matrix is crossed, otherwise not (white field). In the lower left quadrant the binwise activities within the water phase are compared with each other: the activities in bin 2.3 and 4 differ from the remaining water-phase activities, which are, in turn. not different from each other. The same holds for the upper right quadrant. in which the activities during the stimulus phase are compared with each other. The lower right quadrant of the matrix shows whether the binwise activities during the stimulus-phase differs from prestimulus activity: it can clearly be seen that the activities in bins 18 and 19. and, partly, also those in bin 17 and 20 differ significantly from steady-state prestimulus activity (bin 6 to bin 15), while no differences can be found between the activities in the remaining stimulus phase and the steady-state waterphase activity. The bar at the PSTH corresponds to 10 spikes/s.
the results of one experiment are plotted as crossed or white fields of a chess board-like matrix. Every field of the matrix is associated with two bins, bin A and bin B, one in the P S T H on the vertical axis at the left of the plot and the other one in the (same) P S T H on the horizontal axis above the matrix. A field is crossed if the activities in the corresponding bins are significantly different and not crossed (white), otherwise. F o r example, the white field indicated by a circle and the intersection of two d o t t e d lines in the upper right q u a d r a n t means that the activities in bin 26 (bin A-axis) does not differ from that one in bin 23 (bin B-axis). As a whole the figure possesses an obvious structure. First of all, it is symmetrical with respect to the diagonal from lower left to upper right. All fields on this straight line are white because activity in a certain bin does not differ from itself. Second, the activities from bin 17 to bin 20 are (with a few exceptions) different from those of all o t h e r bins, i.e. there is an initial stimulus response that differs significantly from what follows during the stimulus phase. Third, a similar initial activity c o m p o n e n t can be seen at the beginning of the water phase (bin 2 to bin 4). Fourth, in the lower left and the u p p e r right quadrant, all binwise activities following the mentioned initial activities do not differ from each other, respectively. Fifth, the activity in bin 21 to bin 30 is not different from those in bins 6 to 15. Fig. 1 is typical for the t e m p o r a l structure of many, though not all, experiments; the general structure underlying all experiments we p e r f o r m e d , is as follows: (i) the activities from bins 6 to 15 (12 to 30 s) do not differ from each other; (ii) the activities from bins 21 to 30 (42 to 60 s) do not differ from each other; (iii) the activities at the beginning of the water-phase usually differ from those of the rest of the water phase; and (iv) the activities at the beginning of the stimulus phase usually differ from those of the rest o f the stimulus-phase. According to items (i) and (ii), the activities in the mentioned intervals are essentially (i.e. except for stochastic fluctuations) identical so that the m e a n activities in the intervals (12 s, 30 s) and (42 s, 60 s) can be viewed as h o m o g e n o u s blocks and thus as meaningful p a t t e r n features; in accordance with-the terminology in sensory physiology these activities corre-
367 spond to static responses. The extension of this concept to the water-phase will be discussed below. Items (iii) and (iv) suggest that the activities that precede static responses of either phase represent a second pattern feature, which should be called dynamic response. Each experiment showed, independently of: (a) the stimulus delivered; (b) the cell from which was recorded; and (c) the presence of efferent influence, the same temporal structure of response p a t t e r n s - - a dynamic and a static response. These two features can therefore be assumed to be the only relevant informaton carriers. From graphs such as in Fig. 1, not only the temporal structure of both phases (dynamic and static component), but also the stimulus response can be judged. Fig. I showed, for example, that the dynamic component of the stimulus response differed from the static activity in the interstimulus interval, while the static response component does not (see lower right quadrant). In other cases, such as the one shown in Fig. 2, both stimulus response features differ from the static interstimulus activity (see lower right quadrant). Indifferent responses were typically represented by a few irregularly scattered activity differences (Fig. 3) or none at all. In these cases,
standard deviations are often almost identical to the mean values of the averaged PSTH. It is well known that in many sensory systems stimulus responses feature dynamic and static components. The main result of this study is therefore likely to appear trivial to those not involved in electrophysiological recordings from the peripheral olfactory system. It should be emphasized that with respect to recordings from mitral cells there are almost as many definitions of a 'significant stimulus response' as authors. Further, questions such as 'what are the pattern features of mitral cell responses', 'how are they brought about', or 'what information do they carry' have as yet only rarely been addressed. The goal of this paper is to show how to extract response pattern features from extracellular recordings and how the significance of a response can adequately be tested. We found for goldfish mitral cells that there are only two features: the mean activities of the dynamic and static response components; whether or not these differ from prestimulus activity is analyzed by the U-test. Once the response pattern features are known, these can also be calculated from a small number of runs. However, in order to have a sufficient number of values for a statistical test such as the U-test it is
•
"
v;a't,~,
. . . . .
s~im;J~.~.'
'' 30
30 oO c
till t6
.E .o
T I 1
I
I
I
16 ,
bin
16 •
bin
30 A
30 A
Fig. 2. Significance of activity differences between bins of a PSTH. This plot can be interpreted as Fig. I except for the fact that the binwise activities in all stimulus phase bins differ from steady-state prestimulus activity: there is a dynamic and a static response component. Bar = 5 spikes/s.
Fig. 3. Significance of activity differences between bins of a PSTH. A few spatially irregularly distributed crosses in the significance matrix were interpreted as indifferent responses. As the error probability was P = 0.05, one expects, in the mean, Pgo0= 45 crosses in the matrix. In this plot there are 12 crossed fields. Bar = 2 spikes/s.
368 usually necessary to regard at least Nr = 3 runs as a homogenous ensemble, i.e. to regard possible activity changes across the runs as slow. As this is fulfilled in good approximation for unreproducibte recordings, response features can be observed in these recordings, too. What is surprising, at least at first glance, is that the interstimulus interval activity possesses the same structure (dynamic and static component) as the stimulus response itself. Some physiologists would interpret the dynamic activity component of the interstimulus phase simply as an 'after-burst', 'off-effect', or 'rebound effect' of the application of the stimulus. This might, however, be only half of the truth, since the 'off-effect' of a certain stimulus, say A, depends not only on the stimulus A, but also on what substance, water or another stimulus B, is applied after the stimulus A. The dynamic activity component of the interstimulus interval should thus be regarded as the transition from stimulus A to B (or water), while the following static activity would characterize exclusively the stimulus currently applied. The precise way in which the dynamic responses depend on the substances applied in either phase is being investigated presently. Due to the anatomical connections of mitral cells, the structure of their discharge patterns could be influenced by afferent or efferent signals as well as by the signal processing within the bulb. However, experiments have been carried out in which the efferent signals were blocked by cooling the olfactory tract3'4: the resulting recordings showed the same temporal pattern structure as the one reported here; the patterns themselves (i.e. excitation or inhibition of either of the two response components) were often altered by cooling. The efferent control appears thus to influence activity amplitude rather than the temporal structure of dynamic and static response components. The dynamic response has a time constant of about 1-2 s; in contrast, all time constants of cells within the bulb are shorter than 0.33 S13. It is therefore difficult to imagine that the dynamic response components are brought about by the intrabulbar dynamics. These arguments suggest that the pattern structure of goldfish mitral cell discharges is mainly caused by the afferent signals entering the bulb. Unfortunately,
so far no extracellular recordings from goldfish olfactory receptors exist. However, receptor stimulus responses in other species such as frog s and tiger salamander 5 show clear dynamic and static response components to square odour pulses, the dynamic one being of about the same duration as dynamic mitral cell responses in goldfish. Assuming a similar temporal structure of receptor activities in different species, mitral cell activity might generally have the same temporal structure as receptor activities, whereby the amplitude of mitral cell activity is evidently modulated by a number of processes (glomerular convergence, lateral and feedback inhibition, efferent control). In this way, the shape of the dynamic response can become complex (as shown in Fig. 2) rather than being a simple excitation or inhibition. Another argument would support this: from a systems theory point of view, the mitral cell granule cell circuits can be regarded as a fixed point system 2~, i.e. a system that has an asymptotically constant output (mean spike activity) if input and parameters (such as synaptical coupling coefficients) are held constant; the output of such a system is known to follow its input provided the system's time constants are small as compared to input variations. As this is the case, mitral cell and receptor activity should have the same temporal structure. Time constants that are longer than the input variations have as yet only been reported in turtle using electrical stimulation TM15. Meredith and Moulton 12 recorded from the same animal as we did and classified responses into 17 response types relating responses to the stimulus concentration profiles at the fish's input naris. These authors, too, consider the activity following stimulation as part of the stimulus response. Neglecting these response components (for the above-mentioned reasons), the response activity in the Meredith and Moulton study is described by only 3 values: the first gives the response during a fast concentration increase (2 s), the second the response during a slower concentration increase (2 s), and a third indicates the response during a plateau phase of constant stimulus concentration. The latter corresponds doubtless to the static response activity. Regarding the first two activity periods of the Meredith and Moulton study as the dynamic phase and neglecting the activity after stimulation, the patterns proposed reduce to:
369
which are in good agreement with our results. Meredith and Moulton mentioned a complex dynamic response consisting of an initial excitation and a subsequent inhibition; such a dynamic response represents a short transient oscillation and was also observed by us (Fig. 1, stimulus phase). We had, however, difficulty in reproducing the E4-pattern of Meredith and Moulton that shows no reaction at all during the stimulus phase but an after-burst at the beginning of the interstimulus phase. From Table II of their paper it becomes, however, evident that the total frequency of the patterns E3, E3a, and E 4 was quite low, so that the E4-pattern might not occur very often. With the exception of this case, the 17 patterns found by Meredith and Moulton can thus be well explained by the existence of the two pattern features reported here. That the efferent input to the bulb is blocked in their experiments confirms our result that the temporal structure of mitral cell discharges is not influenced by the efferent control. Kauer 9 recorded from mitral cells of frog and tiger salamander and classified stimulus responses into 3 different types: N (no response), E (excitatory), and S (suppressive), whereby some E and S subpatterns were also found. The classification was done in terms of the activity first seen after the onset of the stimulus. Activity patterns were related to the EOG shape as suggested by OttosonlT: rising phase of stimulus concentration (I), intermediate phase (II), falling phase (III), and a fourth period (IV) when the EOG had already reached the baseline. For long stimulus pulses, phase II has to be divided into an initial peak (IIa) and a subsequent rather constant niveau (lib). There are some differences between Kauer's and
our classification: we relate the temporal structure of responses to stimulus onset and end rather than to the EOG, since the E O G indicates a stimulus effect rather than the stimulus itself; the EOG's shape (especially rise time and initial peak amplitude) changes considerably in the course of repeated applications of the same stimulus as well as a function of the length of the interstimulus interval t9,23. In Kauer's activity patterns the stimulus seems to be washed out in the middle of E O G phase III, i.e. in our terms, the activity increase (after-burst) at the end of phase III in Kauer's patterns $2 and E 3 can equally be interpreted as initial reactions of the water-phase. If these initial reactions are not considered as a part of the specific stimulus response, Kauer's classification is simplified to the following 3 response types: (a) $1, $2 = suppression during the entire stimulus phase; (b) E 1 = dynamic excitation and static indifferent response; and (c) E 2, E 3 = dynamic excitation and static inhibitory response. In addition to these patterns we observed also the following response types: $3 = dynamic suppression (or inhibition) and static inhibitory response; $4 = dynamic suppression and static indifferent response; $5 = dynamic suppression and static excitatory response; and E 4 = dynamic excitation and static excitatory response. The fact thai we observed more response patterns than Kauer might seem surprising because we recorded from less units and applied only two stimuli. The number of stimuli, however, is not crucial (as mentioned above) and so, these discrepancies might be due to the low spontaneous discharge rate in frog and salamander and to the different internal connectivity of the bulbs of goldfish, salamander, and frog. Further, Kauer used air-borne odours, which showed much faster rise-times than those used in this study. This might explain some discrepancies. The after-bursts in Kauer's and the Meredith and Moulton classification are easily interpreted as dynamic reactions of the interstimulus phase, which probably characterize the transition from one state (stimulus) of the system to another one (water).
1 Dcving, K.B., The influence of the olfactory stimuli upon the activity of secondary neurons in the burbot (Lota lota L.), Acta Physiol. Scand., 66 (1966) 290-299. 2 Dcving, K.B., An electrophysiologicalstudy of odour simi-
larities of homologous substances, J. Physiol. (London), 186 (1966) 97-109. 3 Fischer, T., Untersuchungen zur Informationsverarbeitung in Mitralzellen des Bulbus olfactorius des Goldfisches (Car-
(a) (b)
(c)
dynamic response component
static response component
0 + +
+ o
370 assius auratus) bei reversibler Blockade der efferenten Bahnensysteme, Diss. med. G6ttingen, 1987. 4 Fischer, T., Zippel, H.P. and Schild, D., Actions of the efferent innervation on goldfish olfactory bulb units during odour stimulation, Chem. Senses, (Ecro VII) in press. 5 Getchell, T.V. and Shepherd, G.M., Adaptive properties of olfactory receptors analysed with odour pulses of varying durations, J. PhysioL (London), 282 (1978) 541-560. 6 Hamilton, K.A. and Kauer, J.S., Intracellular potentials of salamander mitral/tufted neurons in response to odour stimulation, Brain Research, 338 (1985) 181-185. 7 Higashino, S., Takeuchi, H. and Amoore, J.E., Mechanisms of olfactory discrimination in the olfactory bulb of the bullfrog. In C. Pfaffmann (Ed.), Olfaction and Taste, Rockefeller University Press. New York, 1969, pp. 192-211. 8 Holley, A., Duchamps, A., Revial, M.-F. and Juge, A., Qualitative and quantitative discrimination in the frog olfactory receptors: analysis from electrophysiological data, Ann. N. Y. Acad. Sci., 237 (1974) 102-114. 9 Kauer, J.S., Response patterns of amphibian olfactory bulb to odour stimulation, J. Physiol. (London), 243 (1974) 675-715. 10 Mair, A.G., Response properties of rat olfactory bulb neurones, J. Physiol. (London), 326 (1982) 341-359. 11 Mair, A.G., Adaptation of rat olfactory bulb neurones, J. Physiol. (London), 326 (1982) 361-369. 12 Meredith, M. and Moulton, D.G., Patterned response to odour in single neurons of goldfish olfactory bulb. Influence of odour quality and other stimulus parameters, J. Gen. Physiol., 71 (1978) 615-644. 13 Mori, K. and Takagi, S.F., An intraceilular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit
olfactory bulb, J. Physiol. (London), 279 (1978) 569-588. 14 Mori, K., Nowycky, M.C. and Shepherd, G.M., Analysis of synaptic potentials in mitral cells in the isolated turtle olfactory bulb, J. Physiol. (London), 314 (1981) 295-309. 15 Mori, K., Nowycky, M.C. and Shepherd, G.M., Analysis of long-duration inhibitory potentials in mitral cells in the isolated turtle olfactory bulb, J. Physiol. (London), 314 (1981) 311-320. 16 Nanba, R., Djahanparwar, B. and yon Baumgarten, R., Erregungsmuster einzelner Fasern des tractus olfactorius lateralis des Fisches bei Reizung mit verschiedenen Geruchsstoffen, Pfli~ger's Arch., 288 (1966) 134-150. 17 Ottoson, D., Analysis of electrical activity of the olfactory epithelium, Acta Physiol. Scand., 35 (1955) Suppl. 122. 18 Papoulis, A., Probability, Random Variables, and Stochastic Processes, McGraw Hill, Kogakusha, 1965. 19 Pittman-Oldfield, E.A., Sehild, D. and Zippel, H.P., Ver~inderungen im Unterwasser-Elektro-Olfaktogramm des Goldfisches (Carassius auratus) bei repetitiver Reizung, Neurobiologentagung (G6ttingen), (1985) 176. 20 Schild, D., A device for the application of odours to aquatic animals, J. Electrophysiol. Techn., 12 (1985) 7 I - 79. 21 Schild, D., System analysis of the goldfish olfactory bulb: spatio-temporal transfer properties of the mitral cell granule cell complex, Biol. Cybernet., 54 (1986) 9-19. 22 Schild, D. and Zippel, H.P., The influence of repeated natural stimulation upon discharge patterns of mitral cells of the goldfish olfactory bulb, J. Comp. Physiol. A. 158 (1986) 563-571. 23 Van As, W., Kauer, J.S., Menco, B.P.H.M. and Koster, E.P., Quantitative aspects of the electro-olfactogram in the tiger salamander, Chem. Senses, 10 (1985) 1-21.
Brain Research, 405 (1987) 364 - 370 Elsevier
BRE 22081
pattern features of mitral cells in the goldfis,h olfactory bulb Detlev Schild Physiologisches lnstitut der Universitat GOttingen, Abteilung Neuro- und Sinnesphysiologie, GOttingen (F. R. G.)
(Accepted 28 October 1986) Key words: Olfactory bulb; Response pattern
Temporal discharge characteristics of goldfish olfactory bulb mitral cells are investigated. In contrast to earlier findings, it has been shown that every response is determined by only two features, namely, the mean activities of the early and:the late response (dynamic and static response component). As both activities can be lower than, indistinguishable from, or higher than prestimulus activity, the two response features correspond to 9 possible response pattern types.
Mitral cells of the olfactory bulb respond to odours with patterned discharges 1,6A°,11,16 which indicates that the ongoing stimulus response activity is not stationary 18. Responses are therefore not adequately described by categorizing them as excitatory, inhibitory, and indifferent (+, - , 0) 2'7. In quantitative analyses of experimental data, the structure of temporal response patterns has been taken into account only rarely and to date there does not appear to exist a consensus of what should be accepted as a significant response. The patterns described by Meredith and Moulton 12 and by Kauer 9 were obtained by averaging only a few responses to the same stimulus. The results of these studies, which will be compared with ours, are rather complex pattern classifications. A much simpler classification is obtained if the following points are taken into consideration. First, a stimulus response is considered to be the activity within the period of stimulation, and effects subsequent to the stimulus' offset are not considered as specific for the stimulus. Second, interspike intervals of mitral cell activities are in many animals of about the same length as some pattern structures. For example, given an activity of, say, 2 spikes/s and the usually large standard deviation of mitral cell spike activity, it is hardly possible to
decide whether or not an interspike interval of I or 2 s represents an inhibition. For a reliable estimation of patterns a number of stimulus responses are necessary. Third, it has been shown that mitral cell responses are sometimes not reproducible when the same stimulus is delivered several times 22. Meredith and Moulton 12. too, found that responses to repeated stimuli, though being consistent in the temporal pattern structure, change in response amplitude. In the case of unreproducible responses, the first response might be essentially different from a later one. Averaging such responses in order to attain an averaged PSTH (peristimulus-time histogram) does not make sense because the single run PSTHs are drawn from different statistical ensembles. It would rather lead to the definition of patterns that cannot be found in reproducible responses and thus increase the number of pattern classes. In this study, a sufficiently large number (40) of responses to the same stimulus was registered, reproducible responses were separated from unreproducible ones, and from the former pattern features were extracted: a dynamic and a static response component. Though unreproducible recordings are inappropriate for defining pattern features, these lea-
Correspondence: D. Schild, PhysioiogischesInstitut der Universitfit G6ttingen, Abteilung Neuro- und Sinnesphysiologie, Humboldtallee 23, D-3400 G6ttingen, F.R.G.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
365 tures, once defined, can be found and tracked in such recordings, too. The result of this study is a pattern classification simpler than those proposed earlier and in accordante with other pattern structures in sensory physiology. Thereby, a practical method is suggested to determine exactly whether or not a mitral cell stimulus response is significant. Animal preparation, odour application as well as data acquisition were exactly as described in a previous paper 22. We give therefore only a brief account: 70 extraceUular recordings from goldfish mitral cells were evaluated, each of them including 40 presentations of the same stimulus. In 20 experiments done by Fischer 3'4 the olfactory tract was cooled to 1 °C in order to block the efferent influence upon the bulb. Details of the cooling procedure are given elsewhere 3,4. A standard craniotomy was performed in the anaesthetized animals (MS 222, 300 mg/l). During the experiment the fish were immobilized by tubocurarin chloride (0.3 mg/kg) and perfused through the mouth with continuously flowing tap water (200 ml/min). Only two stimuli, amyl acetate 10 -4 M and tubifex food extract 10 -4 g/l were applied (this is no restriction, as far as the temporal structure of patterns are concerned, because it has been shown that particular stimulus substances are not associated with particular response patterns9'12). Each of the 40 stimulus presentations had a length of at least 30 s and was preceded by an interval of at least 30 s during which tap water was delivered to the mucosa, so that every run consisted of a water phase and a stimulus phase. There was a dead time interval of about 2 s between the switch from water to stimulus (or vice versa) on the one side and the arrival of the stimulus (or water) at the mucosa on the other. The concentration rise time of the stimulus solution at the mucosa was about 0.5 s. Further details of the olfactometer have been described earlier 2°. The spike signals were conventionally amplified and the times at which mitral cell spikes occurred were stored in a computer ( D E C / P D P 11/03). The resulting raw data as well as qualitative interpretations have been described previously 22. Data evaluation was performed as follows. First of all PSTHs were calculated for each run (bin width = 2 s), so that the outcome of an experiment was represented by an activity matrix a(r,b) whereby the in-
dices r and b indicate the r th run and the b th bin: a(1,1), a(1,2) a(2,1), a(2,2)
. . . a(1,15), a(1,16) . . . a(2,15), a(2,16)
. . . a(1,30) . . . a(2,30)
a(40,1), a(40,1) . . . a(40,15), a(40,16) . . . a(40,30) The r th line, a(r, b), r = constant, b = 1, 2 . . . . 30, of the matrix is the PSTH of the r th run; in the following it is denoted at(b), indicating that r is a constant parameter while b is the independent variable. Likewise, the b th column a(r,b), b = constant, r = 1, 2 . . . . 40 "or ab(r ) is the activity within the b th bin viewed as a function of the runs. With a(r) the mean activity of the r th run, 30
d(r)
= 1
bE1 ar(b )
the overall activity change c m across the runs can be approximated by the linear regression coefficient that results when a straight line is fitted to the function a(r). Further, it is convenient to define for every bin the binwise activity change c(b) of the activities ab(r ) in the course of the runs; this activity change can approximately be obtained as the linear regression coefficient that results when a straight line is fitted to the function ab(r). So for each bin there is a number c(b) indicating how much, in the mean, activity changes across the runs. I f c(b) does not differ significantly (P = 0.05) from c m for all bins, the changes across the runs are homogenous in all bins, i.e. the time structure of the stimulus response does not change in the course of the runs. If, however, c(b) differs significantly from c m for some bins, the activity in these bins changes across the runs in a way different from the overall change. Such stimulus responses were considered unreproducible and were discarded from further evaluation. For the purpose of feature detection only the reproducible stimulus responses (49 out of 70 total responses) were considered. The question, 'is the activity in a certain bin A different from the activity in another bin B?' was tested for all pairs of bins with the Wilcoxon-Mann-Whitney test (U-test) whereby the 40 values of bin A (aA(r), r = 1, 2 . . . . 40) were compared to those of bin B (aB(r), r = 1, 2 . . . . 40).
366 As a measure of difference, the Gaussian variable ZAa provided by the U-test was taken as criterion: at the 5% level the activities in bin A and bin B are assumed to be different if ZAB ~> Z 0 . 0 5 ~- 1.96. Given the bin width of 2 s, there were 15 bins of 2 s in both the water and the stimulus phase; so 900 U-tests per experiment were carried out. The outcome of an experiment as described above are 40 p e r i s t i m u l u s - t i m e histograms. In what follows, the general t e m p o r a l structure underlying such histograms is reported. The activities in every pair of bins of a P S T H were c o m p a r e d , respectively, for activity similarity by means of the U-test: in Fig. 1
CD E 6
T l l
16
30
I P bin k
Fig. 1. Significance of activity differences between bins of a PSTH. To every pair of bins of the PSTH. which is shown at both axes of the plot, corresponds a field of the 30 x 30-matrix. For example, the pair (bin 26, bin 23] is indicated by a point in the upper right quadrant; it is the intersection of the dotted lines at bin 26 of the bin A-axis and at bin 23 of the bin B-axis. The activities in a certain bin A and another bin B differ from each other (P = 0.05) if the corresponding field in the matrix is crossed, otherwise not (white field). In the lower left quadrant the binwise activities within the water phase are compared with each other: the activities in bin 2.3 and 4 differ from the remaining water-phase activities, which are, in turn. not different from each other. The same holds for the upper right quadrant. in which the activities during the stimulus phase are compared with each other. The lower right quadrant of the matrix shows whether the binwise activities during the stimulus-phase differs from prestimulus activity: it can clearly be seen that the activities in bins 18 and 19. and, partly, also those in bin 17 and 20 differ significantly from steady-state prestimulus activity (bin 6 to bin 15), while no differences can be found between the activities in the remaining stimulus phase and the steady-state waterphase activity. The bar at the PSTH corresponds to 10 spikes/s.
the results of one experiment are plotted as crossed or white fields of a chess board-like matrix. Every field of the matrix is associated with two bins, bin A and bin B, one in the P S T H on the vertical axis at the left of the plot and the other one in the (same) P S T H on the horizontal axis above the matrix. A field is crossed if the activities in the corresponding bins are significantly different and not crossed (white), otherwise. F o r example, the white field indicated by a circle and the intersection of two d o t t e d lines in the upper right q u a d r a n t means that the activities in bin 26 (bin A-axis) does not differ from that one in bin 23 (bin B-axis). As a whole the figure possesses an obvious structure. First of all, it is symmetrical with respect to the diagonal from lower left to upper right. All fields on this straight line are white because activity in a certain bin does not differ from itself. Second, the activities from bin 17 to bin 20 are (with a few exceptions) different from those of all o t h e r bins, i.e. there is an initial stimulus response that differs significantly from what follows during the stimulus phase. Third, a similar initial activity c o m p o n e n t can be seen at the beginning of the water phase (bin 2 to bin 4). Fourth, in the lower left and the u p p e r right quadrant, all binwise activities following the mentioned initial activities do not differ from each other, respectively. Fifth, the activity in bin 21 to bin 30 is not different from those in bins 6 to 15. Fig. 1 is typical for the t e m p o r a l structure of many, though not all, experiments; the general structure underlying all experiments we p e r f o r m e d , is as follows: (i) the activities from bins 6 to 15 (12 to 30 s) do not differ from each other; (ii) the activities from bins 21 to 30 (42 to 60 s) do not differ from each other; (iii) the activities at the beginning of the water-phase usually differ from those of the rest of the water phase; and (iv) the activities at the beginning of the stimulus phase usually differ from those of the rest o f the stimulus-phase. According to items (i) and (ii), the activities in the mentioned intervals are essentially (i.e. except for stochastic fluctuations) identical so that the m e a n activities in the intervals (12 s, 30 s) and (42 s, 60 s) can be viewed as h o m o g e n o u s blocks and thus as meaningful p a t t e r n features; in accordance with-the terminology in sensory physiology these activities corre-
367 spond to static responses. The extension of this concept to the water-phase will be discussed below. Items (iii) and (iv) suggest that the activities that precede static responses of either phase represent a second pattern feature, which should be called dynamic response. Each experiment showed, independently of: (a) the stimulus delivered; (b) the cell from which was recorded; and (c) the presence of efferent influence, the same temporal structure of response p a t t e r n s - - a dynamic and a static response. These two features can therefore be assumed to be the only relevant informaton carriers. From graphs such as in Fig. 1, not only the temporal structure of both phases (dynamic and static component), but also the stimulus response can be judged. Fig. I showed, for example, that the dynamic component of the stimulus response differed from the static activity in the interstimulus interval, while the static response component does not (see lower right quadrant). In other cases, such as the one shown in Fig. 2, both stimulus response features differ from the static interstimulus activity (see lower right quadrant). Indifferent responses were typically represented by a few irregularly scattered activity differences (Fig. 3) or none at all. In these cases,
standard deviations are often almost identical to the mean values of the averaged PSTH. It is well known that in many sensory systems stimulus responses feature dynamic and static components. The main result of this study is therefore likely to appear trivial to those not involved in electrophysiological recordings from the peripheral olfactory system. It should be emphasized that with respect to recordings from mitral cells there are almost as many definitions of a 'significant stimulus response' as authors. Further, questions such as 'what are the pattern features of mitral cell responses', 'how are they brought about', or 'what information do they carry' have as yet only rarely been addressed. The goal of this paper is to show how to extract response pattern features from extracellular recordings and how the significance of a response can adequately be tested. We found for goldfish mitral cells that there are only two features: the mean activities of the dynamic and static response components; whether or not these differ from prestimulus activity is analyzed by the U-test. Once the response pattern features are known, these can also be calculated from a small number of runs. However, in order to have a sufficient number of values for a statistical test such as the U-test it is
•
"
v;a't,~,
. . . . .
s~im;J~.~.'
'' 30
30 oO c
till t6
.E .o
T I 1
I
I
I
16 ,
bin
16 •
bin
30 A
30 A
Fig. 2. Significance of activity differences between bins of a PSTH. This plot can be interpreted as Fig. I except for the fact that the binwise activities in all stimulus phase bins differ from steady-state prestimulus activity: there is a dynamic and a static response component. Bar = 5 spikes/s.
Fig. 3. Significance of activity differences between bins of a PSTH. A few spatially irregularly distributed crosses in the significance matrix were interpreted as indifferent responses. As the error probability was P = 0.05, one expects, in the mean, Pgo0= 45 crosses in the matrix. In this plot there are 12 crossed fields. Bar = 2 spikes/s.
368 usually necessary to regard at least Nr = 3 runs as a homogenous ensemble, i.e. to regard possible activity changes across the runs as slow. As this is fulfilled in good approximation for unreproducibte recordings, response features can be observed in these recordings, too. What is surprising, at least at first glance, is that the interstimulus interval activity possesses the same structure (dynamic and static component) as the stimulus response itself. Some physiologists would interpret the dynamic activity component of the interstimulus phase simply as an 'after-burst', 'off-effect', or 'rebound effect' of the application of the stimulus. This might, however, be only half of the truth, since the 'off-effect' of a certain stimulus, say A, depends not only on the stimulus A, but also on what substance, water or another stimulus B, is applied after the stimulus A. The dynamic activity component of the interstimulus interval should thus be regarded as the transition from stimulus A to B (or water), while the following static activity would characterize exclusively the stimulus currently applied. The precise way in which the dynamic responses depend on the substances applied in either phase is being investigated presently. Due to the anatomical connections of mitral cells, the structure of their discharge patterns could be influenced by afferent or efferent signals as well as by the signal processing within the bulb. However, experiments have been carried out in which the efferent signals were blocked by cooling the olfactory tract3'4: the resulting recordings showed the same temporal pattern structure as the one reported here; the patterns themselves (i.e. excitation or inhibition of either of the two response components) were often altered by cooling. The efferent control appears thus to influence activity amplitude rather than the temporal structure of dynamic and static response components. The dynamic response has a time constant of about 1-2 s; in contrast, all time constants of cells within the bulb are shorter than 0.33 S13. It is therefore difficult to imagine that the dynamic response components are brought about by the intrabulbar dynamics. These arguments suggest that the pattern structure of goldfish mitral cell discharges is mainly caused by the afferent signals entering the bulb. Unfortunately,
so far no extracellular recordings from goldfish olfactory receptors exist. However, receptor stimulus responses in other species such as frog s and tiger salamander 5 show clear dynamic and static response components to square odour pulses, the dynamic one being of about the same duration as dynamic mitral cell responses in goldfish. Assuming a similar temporal structure of receptor activities in different species, mitral cell activity might generally have the same temporal structure as receptor activities, whereby the amplitude of mitral cell activity is evidently modulated by a number of processes (glomerular convergence, lateral and feedback inhibition, efferent control). In this way, the shape of the dynamic response can become complex (as shown in Fig. 2) rather than being a simple excitation or inhibition. Another argument would support this: from a systems theory point of view, the mitral cell granule cell circuits can be regarded as a fixed point system 2~, i.e. a system that has an asymptotically constant output (mean spike activity) if input and parameters (such as synaptical coupling coefficients) are held constant; the output of such a system is known to follow its input provided the system's time constants are small as compared to input variations. As this is the case, mitral cell and receptor activity should have the same temporal structure. Time constants that are longer than the input variations have as yet only been reported in turtle using electrical stimulation TM15. Meredith and Moulton 12 recorded from the same animal as we did and classified responses into 17 response types relating responses to the stimulus concentration profiles at the fish's input naris. These authors, too, consider the activity following stimulation as part of the stimulus response. Neglecting these response components (for the above-mentioned reasons), the response activity in the Meredith and Moulton study is described by only 3 values: the first gives the response during a fast concentration increase (2 s), the second the response during a slower concentration increase (2 s), and a third indicates the response during a plateau phase of constant stimulus concentration. The latter corresponds doubtless to the static response activity. Regarding the first two activity periods of the Meredith and Moulton study as the dynamic phase and neglecting the activity after stimulation, the patterns proposed reduce to:
369
which are in good agreement with our results. Meredith and Moulton mentioned a complex dynamic response consisting of an initial excitation and a subsequent inhibition; such a dynamic response represents a short transient oscillation and was also observed by us (Fig. 1, stimulus phase). We had, however, difficulty in reproducing the E4-pattern of Meredith and Moulton that shows no reaction at all during the stimulus phase but an after-burst at the beginning of the interstimulus phase. From Table II of their paper it becomes, however, evident that the total frequency of the patterns E3, E3a, and E 4 was quite low, so that the E4-pattern might not occur very often. With the exception of this case, the 17 patterns found by Meredith and Moulton can thus be well explained by the existence of the two pattern features reported here. That the efferent input to the bulb is blocked in their experiments confirms our result that the temporal structure of mitral cell discharges is not influenced by the efferent control. Kauer 9 recorded from mitral cells of frog and tiger salamander and classified stimulus responses into 3 different types: N (no response), E (excitatory), and S (suppressive), whereby some E and S subpatterns were also found. The classification was done in terms of the activity first seen after the onset of the stimulus. Activity patterns were related to the EOG shape as suggested by OttosonlT: rising phase of stimulus concentration (I), intermediate phase (II), falling phase (III), and a fourth period (IV) when the EOG had already reached the baseline. For long stimulus pulses, phase II has to be divided into an initial peak (IIa) and a subsequent rather constant niveau (lib). There are some differences between Kauer's and
our classification: we relate the temporal structure of responses to stimulus onset and end rather than to the EOG, since the E O G indicates a stimulus effect rather than the stimulus itself; the EOG's shape (especially rise time and initial peak amplitude) changes considerably in the course of repeated applications of the same stimulus as well as a function of the length of the interstimulus interval t9,23. In Kauer's activity patterns the stimulus seems to be washed out in the middle of E O G phase III, i.e. in our terms, the activity increase (after-burst) at the end of phase III in Kauer's patterns $2 and E 3 can equally be interpreted as initial reactions of the water-phase. If these initial reactions are not considered as a part of the specific stimulus response, Kauer's classification is simplified to the following 3 response types: (a) $1, $2 = suppression during the entire stimulus phase; (b) E 1 = dynamic excitation and static indifferent response; and (c) E 2, E 3 = dynamic excitation and static inhibitory response. In addition to these patterns we observed also the following response types: $3 = dynamic suppression (or inhibition) and static inhibitory response; $4 = dynamic suppression and static indifferent response; $5 = dynamic suppression and static excitatory response; and E 4 = dynamic excitation and static excitatory response. The fact thai we observed more response patterns than Kauer might seem surprising because we recorded from less units and applied only two stimuli. The number of stimuli, however, is not crucial (as mentioned above) and so, these discrepancies might be due to the low spontaneous discharge rate in frog and salamander and to the different internal connectivity of the bulbs of goldfish, salamander, and frog. Further, Kauer used air-borne odours, which showed much faster rise-times than those used in this study. This might explain some discrepancies. The after-bursts in Kauer's and the Meredith and Moulton classification are easily interpreted as dynamic reactions of the interstimulus phase, which probably characterize the transition from one state (stimulus) of the system to another one (water).
1 Dcving, K.B., The influence of the olfactory stimuli upon the activity of secondary neurons in the burbot (Lota lota L.), Acta Physiol. Scand., 66 (1966) 290-299. 2 Dcving, K.B., An electrophysiologicalstudy of odour simi-
larities of homologous substances, J. Physiol. (London), 186 (1966) 97-109. 3 Fischer, T., Untersuchungen zur Informationsverarbeitung in Mitralzellen des Bulbus olfactorius des Goldfisches (Car-
(a) (b)
(c)
dynamic response component
static response component
0 + +
+ o
370 assius auratus) bei reversibler Blockade der efferenten Bahnensysteme, Diss. med. G6ttingen, 1987. 4 Fischer, T., Zippel, H.P. and Schild, D., Actions of the efferent innervation on goldfish olfactory bulb units during odour stimulation, Chem. Senses, (Ecro VII) in press. 5 Getchell, T.V. and Shepherd, G.M., Adaptive properties of olfactory receptors analysed with odour pulses of varying durations, J. PhysioL (London), 282 (1978) 541-560. 6 Hamilton, K.A. and Kauer, J.S., Intracellular potentials of salamander mitral/tufted neurons in response to odour stimulation, Brain Research, 338 (1985) 181-185. 7 Higashino, S., Takeuchi, H. and Amoore, J.E., Mechanisms of olfactory discrimination in the olfactory bulb of the bullfrog. In C. Pfaffmann (Ed.), Olfaction and Taste, Rockefeller University Press. New York, 1969, pp. 192-211. 8 Holley, A., Duchamps, A., Revial, M.-F. and Juge, A., Qualitative and quantitative discrimination in the frog olfactory receptors: analysis from electrophysiological data, Ann. N. Y. Acad. Sci., 237 (1974) 102-114. 9 Kauer, J.S., Response patterns of amphibian olfactory bulb to odour stimulation, J. Physiol. (London), 243 (1974) 675-715. 10 Mair, A.G., Response properties of rat olfactory bulb neurones, J. Physiol. (London), 326 (1982) 341-359. 11 Mair, A.G., Adaptation of rat olfactory bulb neurones, J. Physiol. (London), 326 (1982) 361-369. 12 Meredith, M. and Moulton, D.G., Patterned response to odour in single neurons of goldfish olfactory bulb. Influence of odour quality and other stimulus parameters, J. Gen. Physiol., 71 (1978) 615-644. 13 Mori, K. and Takagi, S.F., An intraceilular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit
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