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Abrupt decrease in synaptic inhibition in the postnatal rat olfactory bulb
Developmental Brain Research, 33 (1987) 134-138 Elsevier
134 BRD 60201
Short Communications
Abrupt decrease in synaptic inhibition in the postnatal rat olfactory bulb D.A. Wilson and M. Leon Department of Psychobiology, Universityof California, Irvine, CA 92717(U.S.A.) (Accepted 2 December 1986)
Key words: Olfactory bulb development; Synaptic inhibition; Granule cell development; Inhibition development; Synaptic facilitation; Paired-pulse effect
Olfactory bulb responses to paired-pulse stimulation of the lateral olfactory tract were examined in urethane-anesthetized rats, aged 5 days to adult. Brief inter-pulse intervals resulted in a depression of test responses at all ages. The magnitude of this depression decreased dramatically between postnatal days 19 and 20 to approach adult levels. Longer inter-pulse intervals resulted in a facilitation of test response amplitude in adult animals. This facilitation was evident at adult levels by postnatal day 10. These results suggest that both inhibitory and facilitatory synaptic mechanisms appear early in the course of rat olfactory bulb development. Furthermore, presumed granule cell-mediated inhibition is present at unusually high levels in the developing bulb, decreasing sharply between days 19 and 20. The rat olfactory bulb undergoes extensive neurogenesis and synaptogenesis during the early postnatal period. In particular, neurogenesis of granule cells, the major inhibitory interneuron of the bulb 17, begins postnatally ~ and continues into adult life8. Granule cells control the activity of the primary output neurons of the bulb, mitral and tufted cells, through dendrodendritic inhibitory synapses 17. Although granule cells are few in number ~and are morphologically immature 2'1° during the first postnatal week in the rat, we have recently reported that presumed granule cell-mediated suppression of mitral cell spontaneous activity, following single shocks to the lateral olfactory tract (LOT), is present by postnatal day 5 (PN5) 19. Furthermore, the duration of this suppression decreases with age, with the major decrease coming between PN15 and PN20 (ref. 19). This early appearance of inhibition in the bulb is surprising given the late development of granule cells 1'2'1°, and the relatively late appearance of inhibition in other brain regions such as neocortex 9 and hippocampus 15.
In order to further understand why inhibition in the bulb is initially robust and subsequently decreases during a period when the number of inhibitory interneurons increases, we studied the postnatal development of granule cell-mediated inhibition in greater detail. Specifically, the present study utilized paired-pulse stimulation of the L O T and examined the evoked potentials recorded in the granule cell layer of the bulb. In the mature olfactory bulb, the granule cell layer response to stimulation of the LOT is characterized by a small negative potential followed by a large positive synaptic potential (periods I and III, respectively in the terminology of Rall and Shepherd13). Period I represents antidromic activation of mitral/tufted cells. Period III represents the subsequent depolarization of granule cells by mitral/ tufted cells via dendrodendritic synapses in the external plexiform layer (EPL) 13,17. The source for the depolarizing current sink in the EPL is in the granule cell layer 13. In urethane-anesthetized mature animals, if two stimuli of equal intensity are delivered to the LOT within 10-15 ms of each other, the second
Correspondence: D.A. Wilson, Department of Psychobiology,University of California, Irvine, CA 92717, U.S.A. 0165-3806/87/$03.50 ~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
135 stimulus evokes a depressed period III response, compared to the first 12"]8. This paired-pulse depression is believed to be due to feedback inhibition of the mitral/tufted cell dendrites from the granule cells, via reciprocal dendrodendritic synapses 7'17. At longer inter-pulse intervals (15-100 ms), the second response becomes facilitated compared to the first 16"18. The mechanism of this facilitation is unknown but may represent enhanced transmitter release from the mitral/tufted cells on to the granule cells 16. The results of the present study suggest that these two processes, inhibition and facilitation, appear early in the postnatal rat olfactory bulb. In addition, the magnitude of inhibition is high from PN5 to PN19, and then markedly decreases between PN19 and PN20 to approach adult levels. Wistar rats of both sexes, aged PN5 to adult (>PN90) were used. Date of birth was PN0. A total of 43 animals were used, with at least 4 animals from at least 2 litters for each age group. Animals were anesthetized with urethane (1.5 g/kg), which has relatively minor effects on synaptic inhibition throughout the postnatal period 2°. The rats were then mounted in a stereotaxic, and warmed with a heating pad (35 °C). A glass microelectrode filled with 2 M NaC1 was lowered into the granule cell layer of the olfactory bulb to record potentials evoked by stimulation of the lateral olfactory tract (LOT). The L O T was stimulated with a bipolar, Teflon-coated, stainless-steel electrode (0.28 mm diameter). Stimuli consisted of 100/tA pulses of 0.01-0.1 ms duration. Evoked potentials were amplified and band-pass filtered (3 H z - 3 kHz) with a Grass P-15 pre-amp. The granule cell layer was identified by electrode depth and the presence of a characteristically large, positive evoked potential 13,]7. Evoked potentials morphologically similar to those described in mature bulb 13'17could be elicited in all age groups. Fig. 1 displays the reversal of the LOT-olfactory bulb potential at PN15 as the recording electrode was lowered from the deep granule cell layer, across the ventral mitral cell layer and into the external plexiform layer. A similar reversal was noted in all age groups, and was used to indicate placement of the recording electrode in the granule cell layer. Response amplitude measurements were taken from a Tektronix storage oscilloscope. The amplitude of the evoked potential from the peak of the initial, brief negativity to the
A
a
I
Fig. 1. Representative olfactory bulb responses to LOT stimulation at PN1.5. A: responses observed as recording electrode was lowered from the granule cell layer (GRL), through the ventral mitral cell body layer (MBL) and into the external plexiform layer (EPL). B: a single conditioning pulse results in a depressed test response at 10 ms IPI and a facilitated test response at 20 ms IPI. Response amplitude was measured between arrows as displayed in bottom trace. Bar = 10 ms and 1 mV, positivityupwards.
peak of the large positivity was used as the response measure (see Fig. 1). Following stabilization of response amplitude, the experimental procedure involved applying a single conditioning pulse (producing a 50-75% maximal response), followed at 10-100 ms intervals by an equal intensity test pulse. Response amplitude to the conditioning pulse ranged from 0.5 to 3 mV across groups, increasing gradually with age. Five pulse pairs were delivered at each inter-pulse interval (IPI) and the response amplitudes were averaged. The mean conditioning response amplitude was considered as baseline, and the mean amplitude of the test response was expressed as a percentage of baseline: (Test/Conditioning) x 100. Statistical comparisons between groups were done using Mann-Whitney U-tests. The effect of a single conditioning pulse applied to the LOT on the amplitude of subsequent test responses recorded in the granule cell layer is displayed in Fig. 2, for PN20 and PN5. As previously described for mature animals 16, at PN20 a single conditioning pulse evokes a brief period of depressed test response amplitude (e.g. 10 ms IPI, Fig. 2), followed by a period of facilitated test responses (peak at 20 ms
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Fig. 2. Effects of a single conditioning pulse applied to the LOT on subsequent test responses at PN20 and PN5. Values are means from 8 and 4 pups, respectively. Note greater depression at PN5 ~ompared to PN20, and lack of facilitation at PN5.
IPI, Fig. 2, PN20), which decays to baseline within 100 ms. In contrast, at PN5 (Fig. 2), the magnitude of the initial depression is greater than at PN20 (10 ms IPI; U = 32, P < 0.01) and has a longer duration, returning to baseline by 40 ms. No period of facilitation is seen at PN5. Test responses in PN20 animals were significantly enhanced at 20 ms IPI compared to responses in PN5 animals (U = 34.5, P < 0.05). The two major points of interest in the pairedpulse curves in Fig. 2 are the 10 ms IPI (depression) and the 20 ms IPI (facilitation). The magnitude of depression and facilitation at these two time points, for each age group, are displayed in Fig. 3a and 3b, respectively. As shown in Fig. 3a, the magnitude of paired-pulse depression decreases slightly between PN5 and PN19 (test response amplitude 30-40% of baseline). This depression then demonstrates a large, abrupt decrease between PN19 (n = 6) and PN20 (n = 8; PN19 vs PN20, U = 46.5, P < 0.01), to approach adult levels (90-100% of baseline). The brief time course of this decrease can be further demonstrated by results from littermates tested at PN19 and PN20. In addition to the data presented in Fig. 3a, 4 pups from a single litter were tested, two at PN19 and two at PN20. The two PN19 pups demonstrated test response amplitudes of 30% and 50% (mean 40%) of baseline at 10 ms IPI. Their littermates, tested 24 h later, demonstrated test response amplitudes of 62% and 90% (mean 76%) of baseline. In contrast, paired-pulse facilitation, absent at PN5, attained adult levels by PN10 (110-130% of
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Fig. 3. Postnatal development of paired-pulse depression and facilitation in the olfactory bulb. A: amplitude of test response expressed as a percent of baseline, 10 ms after a single conditioning pulse (inhibition). Values in parentheses are number of animals/age group. Representative evoked potentials from PN19 and PN20 are also displayed, demonstrating greater paired-pulse depression of test response amplitude at PN19. Stimulus artifact of test pulse is superimposed on the conditioning response. Bar as in Fig. 1. B: amplitude of test response expressed as a percent of baseline, 20 ms after a single conditioning pulse (facilitation). Number of animals/age group is the same as in A.
baseline; Fig. 3b). In the mature olfactory bulb, at least two independent processes control the amplitude of test responses following a single conditioning pulse - - granule cell mediated postsynaptic inhibition and (presumably presynaptic 16) facilitation. The amplitude of the test response at any given time, therefore, is dependent on the relative strengths of these two processes. Thus, in the present case, the lack of facilitation at PN5 (Figs. 2 and 3b) may represent either immature facilitation mechanisms (e.g. low levels of neurotransmitter available for release) or a masking of facilitation by long-lasting, intense inhibition. The present results cannot distinguish between these possibilities. However, it should be noted that PN10-PN19 pups demonstrated levels of inhibition similar to PN5, yet in contrast to PN5, demonstrated significant facilitation at 20 ms IPI. Thus, these results suggest that high levels of inhibition may not account for lack of facilitation at PN5, and that mechanisms controlling facilitation at
137 this synapse develop sometime between PN5 and PN10. Our previous work has demonstrated long-lasting inhibition of mitral-cell spontaneous activity following stimulation of the L O T in the postnatal bulb 19. The present results suggest that the magnitude of this inhibition is similarly enhanced in young animals, and then dramatically decreases between PN19 and PN20 to near adult levels. The mechanism of this sudden decrease is not clear. One possibility is a sudden ingrowth and/or burst of synaptogenesis by centrifugal fibers terminating on granule cells. For example, noradrenergic fibers from the locus coeruleus are believed to have a disinhibitory effect on mitral/tufted cell activity by inhibiting granule cells 6. Levels of norepinephrine are high in the olfactory bulb by PN5 (ref. 3), but adrenergic receptor sites develop more slowly, reaching mature levels around PN22 in the rat TM. Thus, a rapid increase in noradrenergic input and/or receptor sites could result in a decrease in granule cell activity, and subsequent decrease in granule cell-mediated inhibition, as shown in this and our previous report 19. We are presently examining this possibility. Interestingly, Math and Davrainville 11, who examined the postnatal development of mitral-cell spontaneous-activity suppression in the rat olfactory bulb following high-frequency stimulation of the contralateral bulb, also noted that depression reached adult levels by PN21. In that study however, stimulation of the polysynaptic pathway from the contralateral bulb induced less inhibition in neonates that in adults. Differences in pathways stimulated and stimulation parameters may account for the differences in results obtained by Math and Davrainville 11 and those reported here and elsewhere 19. 1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol., 137 (1969) 433-458. 2 Brunjes, P.C., Schwark, H.D. and Greenough, W.T., Olfactory granule cell development in normal and hyperthyroid rats, Dev. Brain Res., 5 (1982) 149-159. 3 Brunjes, P.C., Smith-Crafts, L.K. and McCarty, R~, Unilateral odor deprivation: effects on the development of olfactory bulb catecholamines and behavior, Dev. Brain Res., 22 (1985) 1-6. 4 Galef, Jr., B.G., Development of olfactory control of feeding-site selection in rat pups, J. Cornp. Physiol. Psychol., 95 (1981) 615-622.
Why inhibition should be so effective in the bulb as young as PN5 is also not clear. Granule cell neurogenesis in the rat occurs primarily postnatally 1, and those cells present have immature dendrites into the second postnatal week 2'1°. It is possible therefore, that the paired-pulse depression reported here, and the mitral/tufted-cell spontaneous-activity suppression reported previously ~9, may not be entirely mediated by granule cells in very young animals. However, in addition to the paired-pulse inhibition seen in these young animals, the morphology of evoked potentials recorded at PN5 and older was similar to granule cell-mediated responses seen in the mature olfactory bulb 13A7. Thus, at least a subset of the granule cells present in the Wistar rat olfactory bulb during the first postnatal week must have functional synaptic contacts with mitral/tufted cells. Changes in synaptic inhibition during development might be expected to have profound consequences for olfactory coding in young rats 21. In fact, there is evidence suggesting that factors controlling olfactory-guided behaviors in L o n g - E v a n s rats change between PN19 and PN25. Rat pups exposed to an artificial odor in the nest from PN1 until testing, preferentially approached either an airstream 5 or a food source n scented with that odor during testing at PN19. However, pups trained until and tested at PN25 (ref. 4) and older 5 do not preferentially approach the scent. This change in olfactory-guided behavior could be a reflection of the developmental change in olfactory bulb physiology reported here.
This work was supported by Grants BNS 8606786 to D.A.W. and M.L. and NS 21484 from NINCDS to M.L. who holds R S D A MH 00371 from NIMH. 5 Galef, Jr., B.G. and Kaner, H.C., Establishment and maintenance of preference for natural and artificial olfactory stimuli in juvenile rats, J. Comp. Physiol. Psychol., 94 (1980) 588-595. 6 Jahr, C.E. and Nicoll, R.A., Noradrenergic modulation of dendrodendritic inhibition in the olfactory bulb, Nature (London), 297 (1982) 227-229. 7 Jahr, C.E. and Nicoll, R.A., An intracellular analysis of dendrodendritic inhibition in the turtle in vitro olfactory bulb, J. Physiol. (London), 326 (1982) 213-234. 8 Kaplan, M.S. and Hinds, J.W., Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science, 197 (1977) 1092-1094. 9 Komatsu, Y., Development of cortical inhibition in kitten striate cortex investigated by a slice preparation, Dev.
138 Brain Res., 8 (1983) 136-139. 10 Mair, R.G., Gellman, R.L. and Gesteland, R.C., Postnatal proliferation and maturation of olfactory bulb neurons in the rat, Neuroscience, 7 (1982) 3105-3116. 11 Math, F. and Davrainviile, J.L., Electrophysiological study on the postnatal development of mitral cell activity in the rat olfactory bulb, Brain Res., 190 (1980) 243-247. 12 Nicoll, R.A., The effects of anesthetics on synaptic excitation and inhibition in the olfactory bulb, J. Physiol. (London), 223 (1972) 803-814. 13 Rail, W. and Shepherd, G.M., Theoretical reconstruction of field potentials and dendrodendritic synaptic interactions in olfactory bulb, J. Neurophysiol., 31 (1968)884-915. 14 Sargent-Jones, L., Gauger, L.L., Davis, J.N., Slotkin, T.A. and Bartolome, J.V., Postnatal development of brain alpha[1]-adrenergic receptors: In vitro autoradiography with [x25I]HEAT in normal rats and rats treated with alphadifluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase, Neuroscience, 15 (1985) 1195-1202.
15 Schwartzkroin, P.A., Development of rabbit hippocampus: physiology, Dev. Brain Res., 2 (1982) 469-486. 16 Scott, J.W. and Stewart, W.B., Mechanisms of augmented field potential responses in the rat olfactory bulb, Brain Res., 163 (1979) 21-32. 17 Shepherd, G.M., Synaptic organization of the mammalian olfactory bulb, Physiol. Rev., 52 (1972) 864-917. 18 Stewart, W.B. and Scott, J.W., Anesthetic-dependent field potential interactions in the olfactory bulb, Brain Res., 103 (1976) 487-499. 19 Wilson, D.A. and Leon, M., Early appearance of inhibition in the neonatal rat olfactory bulb, Dev. Brain Res., 26 (1986) 289-292. 20 Wilson, D.A. and Racine, R.J., Barbiturate-enhanced paired-pulse depression in neonatal rats, Neurosci. Lett., 56 (1985) 101-106. 21 Wilson, D.A., Sullivan, R.M. and Leon, M., Odor familiarity alters mitral cell response in the olfactory bulb of neonatal rats, Dev. Brain Res., 22 (1985) 314-317.
134 BRD 60201
Short Communications
Abrupt decrease in synaptic inhibition in the postnatal rat olfactory bulb D.A. Wilson and M. Leon Department of Psychobiology, Universityof California, Irvine, CA 92717(U.S.A.) (Accepted 2 December 1986)
Key words: Olfactory bulb development; Synaptic inhibition; Granule cell development; Inhibition development; Synaptic facilitation; Paired-pulse effect
Olfactory bulb responses to paired-pulse stimulation of the lateral olfactory tract were examined in urethane-anesthetized rats, aged 5 days to adult. Brief inter-pulse intervals resulted in a depression of test responses at all ages. The magnitude of this depression decreased dramatically between postnatal days 19 and 20 to approach adult levels. Longer inter-pulse intervals resulted in a facilitation of test response amplitude in adult animals. This facilitation was evident at adult levels by postnatal day 10. These results suggest that both inhibitory and facilitatory synaptic mechanisms appear early in the course of rat olfactory bulb development. Furthermore, presumed granule cell-mediated inhibition is present at unusually high levels in the developing bulb, decreasing sharply between days 19 and 20. The rat olfactory bulb undergoes extensive neurogenesis and synaptogenesis during the early postnatal period. In particular, neurogenesis of granule cells, the major inhibitory interneuron of the bulb 17, begins postnatally ~ and continues into adult life8. Granule cells control the activity of the primary output neurons of the bulb, mitral and tufted cells, through dendrodendritic inhibitory synapses 17. Although granule cells are few in number ~and are morphologically immature 2'1° during the first postnatal week in the rat, we have recently reported that presumed granule cell-mediated suppression of mitral cell spontaneous activity, following single shocks to the lateral olfactory tract (LOT), is present by postnatal day 5 (PN5) 19. Furthermore, the duration of this suppression decreases with age, with the major decrease coming between PN15 and PN20 (ref. 19). This early appearance of inhibition in the bulb is surprising given the late development of granule cells 1'2'1°, and the relatively late appearance of inhibition in other brain regions such as neocortex 9 and hippocampus 15.
In order to further understand why inhibition in the bulb is initially robust and subsequently decreases during a period when the number of inhibitory interneurons increases, we studied the postnatal development of granule cell-mediated inhibition in greater detail. Specifically, the present study utilized paired-pulse stimulation of the L O T and examined the evoked potentials recorded in the granule cell layer of the bulb. In the mature olfactory bulb, the granule cell layer response to stimulation of the LOT is characterized by a small negative potential followed by a large positive synaptic potential (periods I and III, respectively in the terminology of Rall and Shepherd13). Period I represents antidromic activation of mitral/tufted cells. Period III represents the subsequent depolarization of granule cells by mitral/ tufted cells via dendrodendritic synapses in the external plexiform layer (EPL) 13,17. The source for the depolarizing current sink in the EPL is in the granule cell layer 13. In urethane-anesthetized mature animals, if two stimuli of equal intensity are delivered to the LOT within 10-15 ms of each other, the second
Correspondence: D.A. Wilson, Department of Psychobiology,University of California, Irvine, CA 92717, U.S.A. 0165-3806/87/$03.50 ~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
135 stimulus evokes a depressed period III response, compared to the first 12"]8. This paired-pulse depression is believed to be due to feedback inhibition of the mitral/tufted cell dendrites from the granule cells, via reciprocal dendrodendritic synapses 7'17. At longer inter-pulse intervals (15-100 ms), the second response becomes facilitated compared to the first 16"18. The mechanism of this facilitation is unknown but may represent enhanced transmitter release from the mitral/tufted cells on to the granule cells 16. The results of the present study suggest that these two processes, inhibition and facilitation, appear early in the postnatal rat olfactory bulb. In addition, the magnitude of inhibition is high from PN5 to PN19, and then markedly decreases between PN19 and PN20 to approach adult levels. Wistar rats of both sexes, aged PN5 to adult (>PN90) were used. Date of birth was PN0. A total of 43 animals were used, with at least 4 animals from at least 2 litters for each age group. Animals were anesthetized with urethane (1.5 g/kg), which has relatively minor effects on synaptic inhibition throughout the postnatal period 2°. The rats were then mounted in a stereotaxic, and warmed with a heating pad (35 °C). A glass microelectrode filled with 2 M NaC1 was lowered into the granule cell layer of the olfactory bulb to record potentials evoked by stimulation of the lateral olfactory tract (LOT). The L O T was stimulated with a bipolar, Teflon-coated, stainless-steel electrode (0.28 mm diameter). Stimuli consisted of 100/tA pulses of 0.01-0.1 ms duration. Evoked potentials were amplified and band-pass filtered (3 H z - 3 kHz) with a Grass P-15 pre-amp. The granule cell layer was identified by electrode depth and the presence of a characteristically large, positive evoked potential 13,]7. Evoked potentials morphologically similar to those described in mature bulb 13'17could be elicited in all age groups. Fig. 1 displays the reversal of the LOT-olfactory bulb potential at PN15 as the recording electrode was lowered from the deep granule cell layer, across the ventral mitral cell layer and into the external plexiform layer. A similar reversal was noted in all age groups, and was used to indicate placement of the recording electrode in the granule cell layer. Response amplitude measurements were taken from a Tektronix storage oscilloscope. The amplitude of the evoked potential from the peak of the initial, brief negativity to the
A
a
I
Fig. 1. Representative olfactory bulb responses to LOT stimulation at PN1.5. A: responses observed as recording electrode was lowered from the granule cell layer (GRL), through the ventral mitral cell body layer (MBL) and into the external plexiform layer (EPL). B: a single conditioning pulse results in a depressed test response at 10 ms IPI and a facilitated test response at 20 ms IPI. Response amplitude was measured between arrows as displayed in bottom trace. Bar = 10 ms and 1 mV, positivityupwards.
peak of the large positivity was used as the response measure (see Fig. 1). Following stabilization of response amplitude, the experimental procedure involved applying a single conditioning pulse (producing a 50-75% maximal response), followed at 10-100 ms intervals by an equal intensity test pulse. Response amplitude to the conditioning pulse ranged from 0.5 to 3 mV across groups, increasing gradually with age. Five pulse pairs were delivered at each inter-pulse interval (IPI) and the response amplitudes were averaged. The mean conditioning response amplitude was considered as baseline, and the mean amplitude of the test response was expressed as a percentage of baseline: (Test/Conditioning) x 100. Statistical comparisons between groups were done using Mann-Whitney U-tests. The effect of a single conditioning pulse applied to the LOT on the amplitude of subsequent test responses recorded in the granule cell layer is displayed in Fig. 2, for PN20 and PN5. As previously described for mature animals 16, at PN20 a single conditioning pulse evokes a brief period of depressed test response amplitude (e.g. 10 ms IPI, Fig. 2), followed by a period of facilitated test responses (peak at 20 ms
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Fig. 2. Effects of a single conditioning pulse applied to the LOT on subsequent test responses at PN20 and PN5. Values are means from 8 and 4 pups, respectively. Note greater depression at PN5 ~ompared to PN20, and lack of facilitation at PN5.
IPI, Fig. 2, PN20), which decays to baseline within 100 ms. In contrast, at PN5 (Fig. 2), the magnitude of the initial depression is greater than at PN20 (10 ms IPI; U = 32, P < 0.01) and has a longer duration, returning to baseline by 40 ms. No period of facilitation is seen at PN5. Test responses in PN20 animals were significantly enhanced at 20 ms IPI compared to responses in PN5 animals (U = 34.5, P < 0.05). The two major points of interest in the pairedpulse curves in Fig. 2 are the 10 ms IPI (depression) and the 20 ms IPI (facilitation). The magnitude of depression and facilitation at these two time points, for each age group, are displayed in Fig. 3a and 3b, respectively. As shown in Fig. 3a, the magnitude of paired-pulse depression decreases slightly between PN5 and PN19 (test response amplitude 30-40% of baseline). This depression then demonstrates a large, abrupt decrease between PN19 (n = 6) and PN20 (n = 8; PN19 vs PN20, U = 46.5, P < 0.01), to approach adult levels (90-100% of baseline). The brief time course of this decrease can be further demonstrated by results from littermates tested at PN19 and PN20. In addition to the data presented in Fig. 3a, 4 pups from a single litter were tested, two at PN19 and two at PN20. The two PN19 pups demonstrated test response amplitudes of 30% and 50% (mean 40%) of baseline at 10 ms IPI. Their littermates, tested 24 h later, demonstrated test response amplitudes of 62% and 90% (mean 76%) of baseline. In contrast, paired-pulse facilitation, absent at PN5, attained adult levels by PN10 (110-130% of
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Fig. 3. Postnatal development of paired-pulse depression and facilitation in the olfactory bulb. A: amplitude of test response expressed as a percent of baseline, 10 ms after a single conditioning pulse (inhibition). Values in parentheses are number of animals/age group. Representative evoked potentials from PN19 and PN20 are also displayed, demonstrating greater paired-pulse depression of test response amplitude at PN19. Stimulus artifact of test pulse is superimposed on the conditioning response. Bar as in Fig. 1. B: amplitude of test response expressed as a percent of baseline, 20 ms after a single conditioning pulse (facilitation). Number of animals/age group is the same as in A.
baseline; Fig. 3b). In the mature olfactory bulb, at least two independent processes control the amplitude of test responses following a single conditioning pulse - - granule cell mediated postsynaptic inhibition and (presumably presynaptic 16) facilitation. The amplitude of the test response at any given time, therefore, is dependent on the relative strengths of these two processes. Thus, in the present case, the lack of facilitation at PN5 (Figs. 2 and 3b) may represent either immature facilitation mechanisms (e.g. low levels of neurotransmitter available for release) or a masking of facilitation by long-lasting, intense inhibition. The present results cannot distinguish between these possibilities. However, it should be noted that PN10-PN19 pups demonstrated levels of inhibition similar to PN5, yet in contrast to PN5, demonstrated significant facilitation at 20 ms IPI. Thus, these results suggest that high levels of inhibition may not account for lack of facilitation at PN5, and that mechanisms controlling facilitation at
137 this synapse develop sometime between PN5 and PN10. Our previous work has demonstrated long-lasting inhibition of mitral-cell spontaneous activity following stimulation of the L O T in the postnatal bulb 19. The present results suggest that the magnitude of this inhibition is similarly enhanced in young animals, and then dramatically decreases between PN19 and PN20 to near adult levels. The mechanism of this sudden decrease is not clear. One possibility is a sudden ingrowth and/or burst of synaptogenesis by centrifugal fibers terminating on granule cells. For example, noradrenergic fibers from the locus coeruleus are believed to have a disinhibitory effect on mitral/tufted cell activity by inhibiting granule cells 6. Levels of norepinephrine are high in the olfactory bulb by PN5 (ref. 3), but adrenergic receptor sites develop more slowly, reaching mature levels around PN22 in the rat TM. Thus, a rapid increase in noradrenergic input and/or receptor sites could result in a decrease in granule cell activity, and subsequent decrease in granule cell-mediated inhibition, as shown in this and our previous report 19. We are presently examining this possibility. Interestingly, Math and Davrainville 11, who examined the postnatal development of mitral-cell spontaneous-activity suppression in the rat olfactory bulb following high-frequency stimulation of the contralateral bulb, also noted that depression reached adult levels by PN21. In that study however, stimulation of the polysynaptic pathway from the contralateral bulb induced less inhibition in neonates that in adults. Differences in pathways stimulated and stimulation parameters may account for the differences in results obtained by Math and Davrainville 11 and those reported here and elsewhere 19. 1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol., 137 (1969) 433-458. 2 Brunjes, P.C., Schwark, H.D. and Greenough, W.T., Olfactory granule cell development in normal and hyperthyroid rats, Dev. Brain Res., 5 (1982) 149-159. 3 Brunjes, P.C., Smith-Crafts, L.K. and McCarty, R~, Unilateral odor deprivation: effects on the development of olfactory bulb catecholamines and behavior, Dev. Brain Res., 22 (1985) 1-6. 4 Galef, Jr., B.G., Development of olfactory control of feeding-site selection in rat pups, J. Cornp. Physiol. Psychol., 95 (1981) 615-622.
Why inhibition should be so effective in the bulb as young as PN5 is also not clear. Granule cell neurogenesis in the rat occurs primarily postnatally 1, and those cells present have immature dendrites into the second postnatal week 2'1°. It is possible therefore, that the paired-pulse depression reported here, and the mitral/tufted-cell spontaneous-activity suppression reported previously ~9, may not be entirely mediated by granule cells in very young animals. However, in addition to the paired-pulse inhibition seen in these young animals, the morphology of evoked potentials recorded at PN5 and older was similar to granule cell-mediated responses seen in the mature olfactory bulb 13A7. Thus, at least a subset of the granule cells present in the Wistar rat olfactory bulb during the first postnatal week must have functional synaptic contacts with mitral/tufted cells. Changes in synaptic inhibition during development might be expected to have profound consequences for olfactory coding in young rats 21. In fact, there is evidence suggesting that factors controlling olfactory-guided behaviors in L o n g - E v a n s rats change between PN19 and PN25. Rat pups exposed to an artificial odor in the nest from PN1 until testing, preferentially approached either an airstream 5 or a food source n scented with that odor during testing at PN19. However, pups trained until and tested at PN25 (ref. 4) and older 5 do not preferentially approach the scent. This change in olfactory-guided behavior could be a reflection of the developmental change in olfactory bulb physiology reported here.
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