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Inhibitory mechanisms in the rabbit olfactory bulb: dendrodendritic mechanisms
BRAIN RESEARCH
157
INHIBITORY MECHANISMS IN THE RABBIT OLFACTORY BULB: DENDRODENDRITIC MECHANISMS
R. A. NICOLL*
Laboratory of Neuropharmacology, Division of Special Mental Health Research Programs, IR, NIMH, Saint Elizabeths Hospital, Washington, D. C. (U.S.A.) (Accepted December 12th, 1968)
INTRODUCTION
Cessation of mitral cell activity occurs following stimulation of the major pathways of the olfactory bulb. The discharge of the mitral cells, elicited by stimulation of the olfactory nerves, is followed by inhibition2a. Similarly, antidromic invasion of mitral cells evoked by stimulation of the lateral olfactory tract results in a prolonged inhibitionla,ls,19, 2s. Moreover, stimulation of the anterior limb of the anterior commissure also produces a shorter duration inhibition of mitral cellslS,26,28. By analogy with the Renshaw cell inhibitory system of the spinal cord 9, the inhibition produced by stimulation of the olfactory nerves and lateral olfactory tract was thought to be mediated by the recurrent collaterals of mitral cell axons ending either directly on mitral cellsla or activating granule cells as inhibitory interneurons24, ~s. The anatomical and physiological evidence offered strong support for a recurrent pathway and in particular implicated a pathway involving granule cells. This evidence can be summarized as follows: (a) collaterals from mitral axons end in the granular cell layer; (b) the granule cells send their processes radially to end on the dendrites of mitral cells; (c) the onset of inhibition in mitral cells has a latency suggesting at least two synaptic delaysla,2s; (d) cells in the granular layer respond repetitively to lateral olfactory tract stimulation24, zs, some discharging with a latency less than that for the onset of inhibition. However, recent evidence by Rail et al. z2 suggests that the pathway for this inhibition may involve a dendrodendritic pathway, rather than a recurrent one, with mitral to granule cell excitatory synapses and granule to mitral cell inhibitory synapses. If a recurrent collateral inhibitory system is present, it is quantitatively much less important. The purpose of the present study is to re-analyze the antidromic inhibition of mitral cells and to determine what role the granule layer cell responses might play in this inhibition. The results obtained are consistent with the view that a dendrodendritic pathway is involved in the inhibition of mitral ceils evoked by stimulation of the lateral olfactory tract. * Present address: University of Chicago Hospitals and Clinics, Chicago, Ill. 60637.
Brain Research, 14 (1969) 157-172
158
R.A. NICOLI.
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Fig. 1. A sagittal section through a portion of the left bulb showing the relationship of the cells and fibers, and the experimental arrangement of the stimulating and recording electrodes. The olfactory peduncle (not shown) connects the bulb to the rest of the brain. The olfactory bulb is divided into 6 layers: (1) olfactory nerve layer, (2) glomerular layer, (3) external plexiform layer, (4) mitral cell layer, (5) internal plexiform layer, and (6) granular layer. Examples are given of each of the major cell types: tufted cell (T), mitral cell (M), and granule cell (G). Impulses leave the olfactory bulb by way of the lateral olfactory tract (LOT) and the anterior limb of the anterior commissure (AAC). The 3 electrodes on the surface of the bulb are: the surface stimulating electrode (SURF.), the microelectrode (ME), and the surface recording electrode (SRE). The location of the chronic section is indicated by a broken line just caudal to the LOT and AAC electrodes. A metal plate was routinely placed in the frontal lobes caudal to the chronic section. In the upper right hand corner is a diagram of the 'bipolar' synaptic arrangement between mitral cell dendrite (M) and granule cell dendrite (G).
ANATOMY The olfactory b u l b is a spherical structure c o m p o s e d o f concentric layers o f n e u r o n a l elements. The m a i n a n a t o m i c a l features o f the olfactory bulb as described by Cajal 5 a n d A l l i s o n 1 are s u m m a r i z e d in Fig. 1. The olfactory nerves f o r m the superficial layer o f the olfactory b u l b a n d t e r m i n a t e in large spherical masses called glomeruli. In a d d i t i o n to the olfactory nerves, the glomeruli receive the dendritic tufts o f the olfactory system's s e c o n d a r y neurons (mitral a n d tufted cells). The cell bodies o f mitral cells are confined to a n a r r o w layer a b o u t 700-900/~ below the surface, while the tufted cells are scattered between the g l o m e r u l a r a n d the m i t r a l cell layers. B o t h m i t r a l and tufted cells possess two types o f d e n d r i t i c processes - - a m a i n dendrite, which t e r m i n a t e s in the g l o m e r u l u s a n d two or m o r e accessory dendrites which extend laterally f o r m i n g a dense plexus o f fibers in the external plexiform layer. Brain Research, 14 (1969) 157-172
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
159
The mitral cell axons form the lateral olfactory tract (LOT), while the tufted cell axons quite likely end in the anterior olfactory nucleusln, 21. The axons of neurons in this nucleus are believed to form the anterior limb of the anterior commissure (AAC)ln,21, 25. Before forming discrete tracts, the axons of mitral and tufted cells emit collaterals, some of which end below the mitral cell layer, while others double back to end in the external plexiform layer. The deepest layer of the bulb, the granular layer, contains granule cells and a number of short axon-type cells 5. The granule cell dendrites branch and extend into the external plexiform layer where they form numerous synapses with the accessory dendrites of mitral and tufted cells. These synapses are similar to Type II synapses 12 and are thought to have an inhibitory function 8. In addition, synapses oriented in the opposite direction, i.e., from mitral dendrite to granular cell dendrites have been described and are thought to be excitatory 22 (see diagram in upper right of Fig. 1). In addition to the centripetal fiber systems, consisting of the LOT and AAC, the olfactory bulb receives centrifugal fibers of two types - - the thick and thin fiber system 5. The thin fibers arise from the contralateral bulb and/or peduncle and travel in the AAC. The origin of the thick fibers, many of which run in the LOT 20, is uncertain, although the olfactory tubercle has been strongly implicated 21. METHODS
The experiments were performed on 46 New Zealand white rabbits weighing between 2 and 3 kg. The animals were anesthetized with ether. Following cannulation of the femoral vein the ether was discontinued and the animals maintained on intravenous pentobarbital for the remainder of the experiment. A heat lamp was used to keep rectal temperature between 37 and 39°C. The trachea was cannulated and the bone overlying the frontal lobes and left olfactory bulb removed. To reduce pulsation a metal plate was inserted into the frontal lobes separating the bulb from the rest of the brain 2s. As judged by examination of the surface blood vessels this procedure had little effect on the blood supply to the olfactory bulb. In 12 animals the olfactory bulb and peduncle were isolated from the rest of the brain by a transection of the caudal portion of the bulb, 2-3 months prior to the experiment to allow for sufficient time for the degeneration of most centrifugal fibers. However, it is possible that some of these fibers remained functional owing to the presence of part of the olfactory tubercle and some prepiriform cortex distal to the transection. Complete transection of the LOT in these chronic preparations was routinely verified by histological examination. These preparations will be referred to as 'chronically decentralized preparations'. To expose the LOT, the left eye was enucleated, the bone overlying the LOT removed and the dura reflected. The diagram to the bottom right of Fig. 1 illustrates the technique used for the placement of the electrodes. The distal portion of the LOT was peeled from the surface of the prepiriform cortex for a distance of about 1.5 mm and a bipolar silver electrode was placed on the LOT anterior to the reflected portion. Brain Research, 14 (1969) 157-172
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R . A . NICOLL
A bipolar concentric electrode with an outside diameter of 1 mm was used to stimulate the AAC. This electrode was inserted into the prepiriform cortex in the region exposed by the reflected LOT to a depth of 1-2 mm until the maximum AAC field potential was elicited in the granular layer of the olfactory bulb. In addition, a concentric electrode was also placed on the surface of the bulb to stimulate the olfactory nerves. Glass capillary microelectrodes (1-3 Mg2), filled with 4 M NaCI, were used to record field potentials. High-resistance microelectrodes (40-100 M[2), filled with 3 M KC1, were used for unit recording. Surface potentials were routinely monitored with a silver ball electrode on the surface of the bulb. The extracellular potentials were recorded with a capaeitatively coupled system (time constant, 1 sec). lntracellular potentials were directly coupled through a Bak cathode follower 4 to a DC amplifier. After the electrodes had been positioned on the surface of the bulb, the entire area was covered with 3 % solution ofagar made with Ringer-Locke solution. For the purposes of reproduction a number of the illustrations have been retouched. RESULTS
Inhibition of mitral cells by LOT stimulation Evidence was sought that the inhibition of mitral cells by LOT stimulation is due to the occurrence of an inhibitory postsynaptic potential (IPSP). The effects of pairing conditioning and testing stimuli to the LOT on antidromic invasion of mitral cells was studied (Fig. 2). With a test stimulus set at twice threshold and a conditioning stimulus at 3.5 V (Fig. 2), the A-B inflection n of the antidromic spike was progressively exaggerated as the conditioning-test interval was increased. At greater intervals, the B spike and finally the A spike was abolished. This suppression of antidromic invasion lasted for approximately 100 msec. As the strength of the conditioning stimulus was increased to 4.5 V, the test response was suppressed at a shorter interval (6 msec). When the conditioning stimulus was raised to 6.5 V (just over threshold for this cell) the test volley was ineffective at short intervals, while at slightly longer intervals the cell responded with an A spike which was inhibited at 4.5 msec. The results obtained from this cell are similar to those of Phillips et al.lL This is illustrated graphically in the lower part of Fig. 2; the length of the bar represents the interval within which a test spike was obtained; the end of the bar indicates the onset of the inhibition production by the conditioning stimulus. It should be noted that suppression of the test response began before the occurrence of complete inhibition. For example, for the cell illustrated in Fig. 2, when the conditioning volley was 4.5 V, and A-B inflection was observed at 2.8 msec (not shown in Fig. 2). Direct evidence for the occurrence of an IPSP consequent to LOT stimulation was obtained from intracellular recordings from mitral cells, both in acutely and chronically decentralized animals. Fig. 3A shows the occurrence of a prolonged hyperpolarization following antidromic invasion of a mitral cell in a chronically decentralized preparation. The possibility that this hyperpolarizing potential represented a Brain Research, 14 (1969) 157-172
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Fig. 2. Giant extracellular spike potentials of a mitral cell evoked by LOT stimulation. The strength of the test stimulus was set at 12 V (twice threshold) for all of the records. In the left hand column the conditioning stimulus (dot beneath each trace) was set at 3.5 V and the interval between the stimuli (numbers to the left of each trace) was increased. In the center column the conditioning stimulus was raised to 4.5 V. In the right hand column the conditioning stimulus was set at 6.5 V (just over threshold for this cell). In the graph below the unit responses, the results obtained from this mitral cell are shown. The bar represents the occurrence of any part of the test spike, the bracket at the end of each bar representing the interval that was not tested.
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Fig. 3. Antidromic responses from mitral cells recorded intracellularly from chronically decentralized animals following stimulation of the LOT. In A, the prolonged hyperpolarization which follows antidromic invasion of mitral cells is shown. In B, the effect of both threshold and subthreshold intensity stimulation on antidromic invasion and the succeeding hyperpolarization are shown. C, Same cell as in B recorded after a 30-mininterval. The time scale for B is the same as that for the left hand column of C.
162
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Fig. 4. The olfactory bulb field potential evoked by LOT stimulation. Responses to LOT stimulation are shown at different depths below the surface of the bulb. The depth is indicated beside each trace. Different waves are indicated above the trace recorded at 250 #. spike afterpotential was excluded by the use of threshold voltages where it was possible to evoke a hyperpolarizing potential in the absence of antidromic invasion (Fig. 3B). The latency for the onset of this hyperpolarization was typically about 3 msec. Further evidence indicating that this hyperpolarization is an IPSP was obtained from the following observations. The response in Fig. 3C was obtained from the same cell as Fig. 3B 30 min later. It is to be noted that the size of the hyperpolarization diminished considerably, and was almost absent, possibly due to the leakage of C1ions from the microelectrode. Furthermore, the size of a second test spike was reduced (Fig. 3C) even though there was little hyperpolarization. This reduction in the height of the test spike was not due to A-B dissociation because the reduction was graded and there was no apparent A-B inflection in this cell. This reduction of spike height suggests that the membrane conductance is increased following antidromic invasionL The fact that the hyperpolarization is present without an antidromic spike, that it may be sensitive to Cl-ions, and that there appears to be an increased conductance during the hyperpolarization, all indicate, by analogy with the motoneuron 6, that hyperpolarization of mitral cells following L O T volleys is due to an IPSP.
Pathway involved in generating IPSP in mitral cells Evidence was next sought to determine whether the inhibitory pathway is recurrent, involving mitral axon collaterals or dendrodendritic, involving mitral cell dendrites. In Fig. 4 the field potential caused by LOT stimulation is shown at varying depths in the olfactory bulb. In agreement with Rail et al. ~2, at the surface there is an initial positive wave (P1) followed by two negative waves (N1,N2) and a second long duration positive wave (P2). As the recording electrode penetrated the bulb, the P1
Brain Research, 14 (1969) 157-172
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LATENCYlmsec ) Fig. 5. The extracellular field potentials evoked by LOT stimulation. In A, records were obtained from the same experiment illustrated in Fig. 4. The 3 vertical lines indicate latencies of 1.6, 2.6, ~ d 5.0 msec, respectively, after the stimulus artifact. In B, the latency of the N1 wave is shown as a function of depth below the surface of the bulb. The latency was measured from the stimulus artifact to the beginning of the N1 wave.
wave diminished in amplitude and the latency of the N1 wave shortened. Both negative waves increased in amplitude; the N1 wave reached its maximum at the mitral cell layer (900 #), while the N2 and P2 waves attained their maxima in the external plexiform layer. As the microelectrode passed through the mitral cell layer the polarity of the Nz and P2 waves reversed. Phillips et al. 19 demonstrated that the depth at which the N2 wave inverted corresponded to the mitral cell layer. The same sequence of changes in the polarity of the waves occurred in the ventral side of the bulb. The early wave responses following L O T stimulation are shown in greater detail in Fig. 5A. At 1.6 msec (measured from the shock artifact), the N1 wave in the mitral cell layer (900/~) was synchronous with the superficial P1 wave. At 2.6 msec the superficial N1 wave was synchronous with a deep positivity seen as a shoulder on a much larger and later positive wave. The latency of the N1 wave at different depths below the surface of the olfactory bulb is shown in Fig. 5B. At depths below 900 # the latency of the N~ wave remained relatively constant. As the microelectrode was withdrawn the latency was progressively longer, until the olfactory nerve layer (250/z) was reached. Between 900 and 250/z below the surface of the bulb, the N~ wave had an approximate slope or velocity of 0.7 m/sec. Brain Research, 14 (1969) 157-172
164
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From their analysis Rail et al. 22 concluded that the 'membrane depolarization of mitral dendrites activates excitatory synapses which depolarize granule cell dendrites; the synaptic depolarization of granule dendritic membrane then activates granule to mitral inhibitory synapses'. The depolarization of the granule dendritic membrane generates the negativity (signified by the N~ wave) in the external plexiform layer. If a dendrodendritic pathway is responsible for the generation of the IPSP in mitral cells following LOT stimulation, then the inhibitory pathway would be blocked by a conditioning stimulus to the LOT since the pathway through mitral cell dendrites would be inhibited (Rail, personal communication). Alternatively, if the IPSP in mitral cells is generated by a recurrent collateral pathway, then the inhibitory pathway should not be blocked by a conditioning stimulus to the LOT as the recurrent collateral pathway by-passes the mitral cell dendrites. Therefore, the effect of conditioning LOT volleys on intracellular responses and field potentials was examined, in order to choose between these two hypotheses. Paired stimuli to the LOT cause a reduction in the test IPSP recorded intracellularly from mitral cells. As illustrated in Fig. 6, when the strength of the conditioning stimulus was increased the size of the conditioning IPSP increased, while the test IPSP was reduced. This reduction was not due to the IPSP reaching its equilibrium potential, because the combined hyperpolarization of the conditioning and test response was less than the control IPSP. It cannot be excluded, however, that this reduction might be due to alterations in synapses of the inhibitory pathway. It was found, moreover, that the N2 and P2 waves of the field potential recorded in the external plexiform layer were routinely inhibited by a conditioning LOT stimulus as illustrated in Fig. 7A from a chronically decentralized preparation. Both waves were completely suppressed at short intervals, the N~ wave actually being replaced by a slight positivity. As the interval increased, the N2 wave recovered; but only at the longest interval did the P2 wave begin to recover. This inhibitory effect is Brain Research,
14 (1969) 157-172
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Fig. 7. Extracellular responses evoked by paired LOT stimuli in a chronically decentralized preparation. In A, the recording electrode was located in the external plexiform layer and the interval between the 2 LOT stimuli was varied. The voltage gain is the same for all traces except the conditioning control (COND. CON). In B, the inhibition of the test N1 wave (percent of control) is shown as a function of the conditioning-test interval (msec). A tracing of the P~ wave evoked by the conditioning stimulus is shown above the graph to provide comparison of its time course with that of the inhibitory effect.
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"30msec Fig. 8. Inhibitory action of olfactory nerve (SURF.) and LOT volleys on the response evoked in granular layer cells following surface stimulation. In A, the inhibition of granular layer cell response evoked by a surface stimulus by a preceding surface stimulus applied to an adjacent surface electrode. In B, the inhibition of granular layer cell response evoked by a surface stimulus by a preceding LOT volley. The control response (CON) is shown in the top trace in each series. The stimulus strength to LOT is expressed in arbitrary units and is shown to the left of each trace. Voltage gain is the same for all traces.
shown graphically in Fig. 7B. It should be noted that there is a correlation between the time course of the P2 wave shown above the graph and the inhibition of the N2 wave. Brain Research, 14 (1969) 157-172
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Fig. 9. Inhibitory and facilitatory effect of an olfactory nerve volley on the response of granular layer cells evoked by a LOT volley. In A, inhibition of the LOT response by a preceding surface stimulus (given in arbitrary units). In B, facilitation of the LOT response by a preceding surface stimulus at short intervals. The number at the left of each trace in B is the interval (msec) between the surface and LOT stimulus. The control responses (CON) are shown at the top of each column. Voltage gain is the same for all traces. In C, histogram showing facilitation of surface stimulus on the LOT response of a granular layer cell at different conditioning-test intervals. The broken line indicates the number of spikes evoked by a control LOT stimulus.
The results support the hypothesis of a dendrodendritic inhibitory pathway. The reduction of a test IPSP suggests that the pathway mediating the IPSP, i.e., mitral cell dendrites is blocked by the conditioning volley. Similarly, the conditioning IPSP in mitral cells would block the N2 wave if it were due to the depolarization of granule cell dendrites by mitral cell dendrites, because the excitatory pathway through the mitral cell would be inhibited. Further evidence in support of a dendrodendritic inhibitory pathway was obtained from analyzing the responses obtained in the granular cell layer.
Unit responses recorded in the granular layer A conditioning stimulus applied to the surface of the olfactory bulb routinely inhibited the test response of granular layer cells evoked by the same or an adjacent surface electrode. In Fig. 8A two adjacent surface electrodes evoked cell discharge. However, a conditioning stimulus applied through one electrode reduced the response evoked by the other electrode from three to one spikes. A conditioning LOT stimulus also inhibited the response produced by a surface stimulus (Fig. 8B). Four spikes were elicited from the granular layer cell following suprathreshold stimulation of the surface. Even when the conditioning L O T stimulation was too weak to discharge the cell (Fig. 8B, 0.4 V), it completely suppressed the surface response. Since the LOT exerted its
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INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
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Fig. 10. Inhibition of the LOT response evoked in granular layer cells by preceding LOT or AAC stimulus, In A, inhibitory action of an LOT volley on the response evoked by an LOT stimulus in a chronically decentralized preparation. In B and C, inhibition of the LOT response by a preceding AAC volley; the response shown in C was from a chronically decentralized preparation. The dot below the traces shown in B and C indicates the AAC stimulus artifact. The control response (CON) is the first trace in each series. effect without discharging the cell, this suppression of discharge cannot be attributed to postspike refractoriness. As illustrated in Fig. 9A surface stimulation also inhibited the response of granular layer neurons to L O T stimulation. Separately, a strong surface stimulus or a L O T stimulus produced 3 spikes. The test L O T stimulus, however, was ineffective until a very weak surface stimulus was used. Strong surface stimuli could suppress an L O T evoked response for intervals greater than 100 msec. However, at short intervals the surface volley often facilitated the LOT response (Fig. 9B). Both these findings are illustrated graphically in Fig. 9C. The surface stimulus was adjusted so that it never discharged this cell. The L O T stimulus was set so that it elicited one spike once every 10 trials. At intervals between 6 and 9 msec cell firing increased to about 6 spikes in 10 trials while at longer intervals the firing decreased to zero. When stimuli were paired to the L O T at different intervals the test response of granular layer cells was often completely suppressed for 100-200 msec. In Fig. 10A the test volley elicited a spike only after the test field potential returned to its control configuration. The inhibitory effect of a conditioning LOT volley was present even when the volley failed to discharge the cell. L O T activation of granular layer cells was often inhibited by volleys to the AAC. In Fig. 10B the L O T control response consisted of two spikes. At short intervals the intact AAC had no effect while at longer intervals the L O T response was reduced to one spike and then was ineffective. The effect was also observed in chronically decentralized preparations. As illustrated in Fig. 10C the L O T response was completely removed by a preceding A A C volley. DISCUSSION
In agreement with previous results19, 28, intracellular records from mitral cells revealed that following antidromic invasion there is a prolonged hyperpolarization. Brain Research, 14 (1969) 157-172
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This hyperpolarizing potential is present in both intact and chronically decentralized preparations. Since this hyperpolarization was present even when antidromic invasion did not occur, seemed sensitive to Cl-ions, and was associated with an increased conductance, it is suggested that this hyperpolarization represents a synaptically induced inhibitory potential (IPSP). The latency for the IPSP was approximating 3 msec. This latency allows sufficient time for at least two or more synaptic delays. It should be noted that these values were obtained with subthreshold or threshold intensity stimulation. With suprathreshold stimuli the latency would be somewhat shorter. The dendrodendritic inhibitory p a t h w a y
From an analysis of the LOT field potential, Rail et al. zz postulated that the inhibition of mitral cells was mediated through their dendrites. The finding of bipolar synaptic connections between the mitral cell dendrites and granule cell dendrites 3,15,2~ provided anatomical support for this hypothesis. The results obtained in the present study add further support to the dendrodendritic hypothesisZL The short latency of the N1 wave and the finding that antidromic unit responses occur during this wave suggests that the N1 wave results from the synchronous antidromic invasion of mitral cells and their dendrites. The apparent rate at which the antidromic impulses invade the dendrites of mitral cells was calculated from the latency of the N1 wave to be approximately 0.7 m/sec. This value is in good agreement with the slow velocities obtained previously for mitral cells is and other neurons in the CNS 2,10,14,17. The results presented here do not pertain to the question of passive vs. active invasion of dendrites. As illustrated in Fig. 5A at 1.6 msec the deep N~ wave is synchronous with the superficial P~ wave. At this latency, the superficial dendrites of mitral cells are not invaded and supply current to their depolarizing cell bodies (Fig. 11A). At 2.6 msec the superficial N1 wave is synchronous with a deep positivity. During this period the superficial dendrites are invaded and the repolarizing cell bodies supply current to their depolarizing dendrites (Fig. 11B). At 5 msec, the Nz wave which is largest in the external plexiform layer is coupled with a large positive wave in the granular layer. Ochi is and Von Baumgarten et al. 27 correlated this wave with synaptically activated secondary neurons (presumably via a direct recurrent collateral pathway). This recurrent activation may contribute to the formation of the N2 wave, but by itself cannot account for the large current flow across the mitral cell layer. Rail et al. 2~ suggested that 'the population of cells which generate this current must possess a substantial intracellular pathway for the return flow of current from external plexiform layer, through the mitral cell layer, into the depths of the granule layer. This:requirement is satisfied by the large population of granule cells, but not by the mitral cells.' In their hypothesis, depolarization of mitral cell dendrites provides a synaptic excitatory input which depolarizes granule cell dendrites in the external plexiform layer. The depolarization of granule cell dendrites then activates granule to mitral cell inhibitory Brain Research, 14 (1969) 157-172
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Fig. 11. Schematic diagram of mitral and granule cells showing current flow at various latencies after a LOT volley. In A, the current flow at 1.6 msec, in B at 2.6 msec, in C at 5.0 msec, and in D at approximately 25 msec after the stimulus artifact. synapses. During this period the deep lying granule cell bodies are predominantly passive and supply current to their superficially depolarizing dendrites (Fig. l lC). The P2 wave which follows the Nz wave was not considered in the analysis by Rail et al.22. This wave was largest in the external plexiform layer and reversed polarity at the mitral cell layer. Since the time course of the P2 wave was correlated with the time course of the IPSP recorded from mitral cells and the time course of inhibition of a test N2 wave, it is proposed that the P2 wave results from the inhibitory synaptic currents generated in the dendrites of mitral cells. The finding that the P2 wave reversed polarity at the mitral cell layer suggests that the current flows from the mitral cell dendrites (active source) to passive sinks in the granular cell layer (mitral cell axons and collaterals) (Fig. 11D). In the postulated dendrodendritic inhibitory pathway, in contrast to a recurrent inhibitory pathway, the inhibition is fed back onto a portion of the inhibitory pathway, i.e., mitral cell dendrites. Accordingly, a conditioning stimulus to the lateral olfactory tract should, as observed, suppress the test L O T IPSP in mitral cells. While possible alterations in synaptic behavior (e.g., receptor desensitization or decrease in available transmitter by a preceding volley) can not be excluded, these factors appear less likely in view of several lines of evidence suggesting that the pathway mediating the IPSP is
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R.A. NICOH~
blocl~ed by the conditioning volley. The inhibition of the N2 and P2 waves of the field potential by a preceding LOT volley is consistent with a dendrodendritic hypothesis. The conditioning IPSP in mitral cells prevents the test volley from depolarizing the granule cell dendrites because the excitatory pathway through the mitral cell has been blocked. Since the generation of the P2 wave is dependent upon the depolarization of granule cell dendrites, both the N2 and P2 waves should be suppressed by a conditioning LOT volley. Responses in the granular layer
Cells in the granular layer are synaptically discharged by both surface (olfactory nerve) and LOT stimulation. The inhibitory effect of a conditioning surface (Fig. 8A) and LOT volley (Fig. 8B) on a test surface response of granule layer cells is to be expected, because the only anatomical pathway through which impulses in the olfactory nerves can excite neurons in the granular layer is through secondary neurons. The prolonged inhibition of secondary neurons following conditioning surface or LOT volleys would suppress the response of granular layer cells to surface volleys by blocking the excitatory pathway. Conversely, the observed inhibition of the test LOT response of some granular layer cells (Figs. 9A and 10A) would not be predicted if, as has been assumed by previous investigators, the granular layer cells are activated by a simple recurrent pathway, since inhibition of the mitral cell soma should not block conduction through the recurrent collaterals. The fact that the cell in Fig. 10A failed to respond until the LOT field potential returned to its control configuration suggests that the inhibition might be related to the dendrodendritic pathway. Yamamoto et al. 2s found that a conditioning stimulus to the AAC not only hyperpolarized mitral cells, but also reduced the amplitude of a test IPSP evoked by LOT stimulation. Since AAC stimulation also suppressed granular layer cells activated by LOT stimulation, they proposed that stimulation of the AAC had two independent effects: (1) inhibition of mitral cells and (2) suppression of the LOT evoked IPSP by inhibition of interneurons in the recurrent collateral pathway. The alternative hypothesis for these effects is that the AAC evoked hyperpolarization of mitral cells suppresses the LOT evoked IPSP by blocking the dendrodendritic pathway, thereby preventing mitral to granule cell excitation. At short intervals a surface volley was found to facilitate the LOT activation of granule cells (Figs. 9B and C). It is tempting to use this finding as support for the proposed dendritic pathway. The surface volley depolarizing the mitral cell dendrites would facilitate the dendritic invasion of antidromic impulses and this in turn would lead to a larger depolarization in the granule cell dendrite. However, field potential studies consistently failed to show any facilitation of antidromic invasion by a preceding surface volley (unpublished observation). It is therefore concluded that the surface facilitatory effect is due largely to its asynchronous activation of mitral cells. Since the conduction velocity in the olfactory nerves is very slow (unpublished observation), the activation of mitral cells will be dispersed in time. This in turn will produce a temporal dispersion in the EPSP generated in the granule cells. Brain Research, 14 (1969) 157-172
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
171
In conclusion, it is proposed that cells in the granular cell layer whose test response is inhibited by conditioning volleys evoked by stimulation of the surface, LOT and AAC are granule cells activated by the dendrodendritic excitatory synaptic pathway between mitral and granule cell dendrites. Since all three inputs hyperpolarized the mitral cell, a conditioning volley in any one of these three pathways should block the test response in granule cells evoked in the same or different pathway. Although the granule cell responses are the most numerous of the responses recorded in the granular layer, only a small fraction of the total population of granule cell bodies must be activated because the LOT field potentials indicate that the granule cell bodies are predominantly passive. While alternative hypotheses may not be completely excluded, the dendrodendritic hypothesis proposed by Rail et al.22 has the advantage of providing the most unified explanation of data concerning inhibition of responses in the granular layer. SUMMARY
The unitary responses of mitral and granular layer cells and olfactory bulb field potentials following stimulation of the lateral olfactory tract (LOT), olfactory nerves and anterior limb of the anterior commissure (AAC) were analyzed. The hyperpolarization that followed antidromic invasion of mitral cells was shown to result from a synaptically evoked inhibitory potential. The analysis of the LOT evoked field potential indicated that the initial negative deflection represented antidromic invasion of mitral cell somata and dendrites. The results suggest that the second negative deflection of the field potential represented a synaptically evoked depolarization of granule cell dendrites, while the succeeding positive deflection represented the synaptically evoked inhibition of mitral cell dendrites and somata. A conditioning LOT stimulus inhibited the intracellularly recorded hyperpolarization of mitral cells and abolished the field potential evoked by a test LOT stimulus. In addition, cells in the granular layer that responded to LOT stimulation were inhibited by conditioning stimuli supplied to the LOT, AAC or olfactory nerves. The results are consistent with and lend support to the dendrodendritic hypothesis advanced by Rail et al. 22 and suggest that the cells in the granular layer which are activated by the dendrites of mitral cells are granule cells. ACKNOWLEDGEMENTS
I wish to thank Drs. G. C. Salmoiraghi and F. Weight for their constant encouragement and suggestions, Drs. A. Gorman, W. Rail and P. Nelson for criticizing the manuscript and Mr. H. Poole for his help in preparing the illustrations.
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REFERENCES 1 ALLISON,A. C., The structure of the olfactory bulb and its relationship to the olfactory pathways in the rabbit and rat, J. comp. Neurol., 98 (1953) 309-353. 2 ANDERSON,P., Interhippocampal impulses. II. Apical dendritic activation of CAI neurons, Acta physiol, scand., 48 (1960) 178-208. 3 ANDRES,K. H., Der Feinbau des Bulbus Olfactorius der Ratte unter besonderer Beriicksichtigung der synaptischen Verbindungen, Z. Zellforsch., 65 (t965) 350-361. 4 BAK, A. F., A unity gain cathode follower, Electroenceph. clin. Neurophysiol., 10 (1958) 745-748. 5 CAJAL, S. RAM6N Y, Studies on the Cerebral Cortex (Limbic Structures), (transl. by L. M. Kraft), Lloyd-Luke, London, 1955. 6 COOMBS,J. S., CURTIS, D. R., AND ECCLES, J. C., The interpretation of spike potentials of motoneurones, J. Physiol. (Lond.), 139 (1957) 198-231. 7 ECCLES,J. C., The Physiology of Nerve Cells, Johns Hopkins, Baltimore, 1957, 270 pp. 8 ECCLES,J. C., The Physiology of Synapses, Springer, Berlin, 1964, 316 pp. 9 ECCLES,J. C., FATT, P., AND KOKETSU,K., Cholinergic and inhibitory synapse in a pathway from motor-axon collaterals to motoneurones, J. Physiol. (Lond.), 126 (1954) 524-562. 10 FATT, P., Electric potentials occurring around a neurone during its antidromic activation, J. Neurophysiol., 20 (1957) 27-60. 11 FUORTES, M. G. F., FRANK, K., AND nECKER, M. C., Steps in the production of motoneuron spikes, J. gen. Physiol., 40 (1957) 735-752. 12 GRAY, E. G., Ultrastructure of synapses of the cerebral cortex and of certain specializations of neuronal membranes. In J. D. BOYD, F. R. JOHNSONAND J. D. LEVER(Eds.), Electron Microscopy in Anatomy, Arnold, London, 1961, pp. 54-73. 13 GREEN,J. D., MANCIA,M., AND VON BAUMGARTEN,R., Recurrent inhibition in the olfactory bulb. I. Effects of antidromic stimulation of the lateral olfactory tract, J. Neurophysiol., 25 (1962) 461~,88. 14 HILD, W., AND TASAKI,I., Morphological and physiological properties of neurons and glial cells in tissue culture, J. Neurophysiol., 25 (1962) 277-304. 15 HIRATA, Y., Some observations on the fine structure of the synapses in the olfactory bulb of the mouse, with particular reference to the atypical synaptic configuration, Arch. Okayama (Saibo Kaku Byorigaki Zasski), 24 (1964) 293-302. 16 LOHMAN,A. H. M., The anterior olfactory lobe of the guinea pig. A descriptive and experimental anatomical study, Acta anat. (Basel), 53, Suppl. 49 (1963) 1-109. 17 LORENTEDE N6, R., Action potential of the motoneurons of the hypoglossus nucleus, J. cell. comp. Physiol., 29 (1947) 207-288. 18 OCHI, J., Olfactory bulb response to antidromic olfactory tract stimulation in the rabbit, .lap. J. Physiol., 13 (1963) 111-128. 19 PHILLIPS,C. G., POWELL, T. P. S., AND SHEPHERD,G. M., Responses of mitral cells to stimulation of the lateral tract in the rabbit, J. Physiol. (Lond.), 168 (1963) 65-88. 20 POWELL, T. P. S., AND COWAN, W. M., Centrifugal fibers in the lateral olfactory tract, Nature (Lond.), 199 (1963) 1296-1297. 21 POWELL,T. P. S., COWAN, W. M., AND RAISMAN,G., The central olfactory connections, J. Anat. (Lond.), 99 (1965) 791-813. 22 RALL, W., SHEPHERD, G. M., RESSE, T. S., AND BRIGHTMAN, M. W., Dendrodendritic synaptic pathway for inhibition in the olfactory bulb, Exp. Neurol., 14 (1966) 44-56. 23 SHEPHERD, G. M., Responses of mitral cells to olfactory nerve volley in the rabbit, J. Physiol. (Lond.), 168 (1963) 89-100. 24 SHEPHERD, G. M., Neuronal systems controlling mitral cell excitability, J. Physiol. (Lond.), 168 (1963) 101-117. 25 VALVERDE,F., Studies on the Piriform Lobe, Harvard Univ. Press, Cambridge, Mass., 1965, 131 pp. 26 VON BAUMGARTEN,R., GREEN, J. D., AND MANCIA, M., Recurrent inhibition in the olfactory bulb. II. Effects of antidromic stimulation of commissural fibers, J. Neurophysiol., 25 (1962) 489-500. 27 VON BAUMGARTEN,R., GREEN, J. D., AND MANCIA,M., Slow waves in the olfactory bulb and their relation to unitary discharges, Electroenceph. clin. Neurophysiol., 14 (1962) 631-634. 28 YAMAMOTO,C., YAMAMOTO,T., AND IWAMA, K., The inhibitory systems in the olfactory bulb studied by intracellular recording, J. Neurophysiol., 26 (1963) 403~,15.
Brain Research, 14 (1969) 157-172
157
INHIBITORY MECHANISMS IN THE RABBIT OLFACTORY BULB: DENDRODENDRITIC MECHANISMS
R. A. NICOLL*
Laboratory of Neuropharmacology, Division of Special Mental Health Research Programs, IR, NIMH, Saint Elizabeths Hospital, Washington, D. C. (U.S.A.) (Accepted December 12th, 1968)
INTRODUCTION
Cessation of mitral cell activity occurs following stimulation of the major pathways of the olfactory bulb. The discharge of the mitral cells, elicited by stimulation of the olfactory nerves, is followed by inhibition2a. Similarly, antidromic invasion of mitral cells evoked by stimulation of the lateral olfactory tract results in a prolonged inhibitionla,ls,19, 2s. Moreover, stimulation of the anterior limb of the anterior commissure also produces a shorter duration inhibition of mitral cellslS,26,28. By analogy with the Renshaw cell inhibitory system of the spinal cord 9, the inhibition produced by stimulation of the olfactory nerves and lateral olfactory tract was thought to be mediated by the recurrent collaterals of mitral cell axons ending either directly on mitral cellsla or activating granule cells as inhibitory interneurons24, ~s. The anatomical and physiological evidence offered strong support for a recurrent pathway and in particular implicated a pathway involving granule cells. This evidence can be summarized as follows: (a) collaterals from mitral axons end in the granular cell layer; (b) the granule cells send their processes radially to end on the dendrites of mitral cells; (c) the onset of inhibition in mitral cells has a latency suggesting at least two synaptic delaysla,2s; (d) cells in the granular layer respond repetitively to lateral olfactory tract stimulation24, zs, some discharging with a latency less than that for the onset of inhibition. However, recent evidence by Rail et al. z2 suggests that the pathway for this inhibition may involve a dendrodendritic pathway, rather than a recurrent one, with mitral to granule cell excitatory synapses and granule to mitral cell inhibitory synapses. If a recurrent collateral inhibitory system is present, it is quantitatively much less important. The purpose of the present study is to re-analyze the antidromic inhibition of mitral cells and to determine what role the granule layer cell responses might play in this inhibition. The results obtained are consistent with the view that a dendrodendritic pathway is involved in the inhibition of mitral ceils evoked by stimulation of the lateral olfactory tract. * Present address: University of Chicago Hospitals and Clinics, Chicago, Ill. 60637.
Brain Research, 14 (1969) 157-172
158
R.A. NICOLI.
I
3
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Fig. 1. A sagittal section through a portion of the left bulb showing the relationship of the cells and fibers, and the experimental arrangement of the stimulating and recording electrodes. The olfactory peduncle (not shown) connects the bulb to the rest of the brain. The olfactory bulb is divided into 6 layers: (1) olfactory nerve layer, (2) glomerular layer, (3) external plexiform layer, (4) mitral cell layer, (5) internal plexiform layer, and (6) granular layer. Examples are given of each of the major cell types: tufted cell (T), mitral cell (M), and granule cell (G). Impulses leave the olfactory bulb by way of the lateral olfactory tract (LOT) and the anterior limb of the anterior commissure (AAC). The 3 electrodes on the surface of the bulb are: the surface stimulating electrode (SURF.), the microelectrode (ME), and the surface recording electrode (SRE). The location of the chronic section is indicated by a broken line just caudal to the LOT and AAC electrodes. A metal plate was routinely placed in the frontal lobes caudal to the chronic section. In the upper right hand corner is a diagram of the 'bipolar' synaptic arrangement between mitral cell dendrite (M) and granule cell dendrite (G).
ANATOMY The olfactory b u l b is a spherical structure c o m p o s e d o f concentric layers o f n e u r o n a l elements. The m a i n a n a t o m i c a l features o f the olfactory bulb as described by Cajal 5 a n d A l l i s o n 1 are s u m m a r i z e d in Fig. 1. The olfactory nerves f o r m the superficial layer o f the olfactory b u l b a n d t e r m i n a t e in large spherical masses called glomeruli. In a d d i t i o n to the olfactory nerves, the glomeruli receive the dendritic tufts o f the olfactory system's s e c o n d a r y neurons (mitral a n d tufted cells). The cell bodies o f mitral cells are confined to a n a r r o w layer a b o u t 700-900/~ below the surface, while the tufted cells are scattered between the g l o m e r u l a r a n d the m i t r a l cell layers. B o t h m i t r a l and tufted cells possess two types o f d e n d r i t i c processes - - a m a i n dendrite, which t e r m i n a t e s in the g l o m e r u l u s a n d two or m o r e accessory dendrites which extend laterally f o r m i n g a dense plexus o f fibers in the external plexiform layer. Brain Research, 14 (1969) 157-172
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
159
The mitral cell axons form the lateral olfactory tract (LOT), while the tufted cell axons quite likely end in the anterior olfactory nucleusln, 21. The axons of neurons in this nucleus are believed to form the anterior limb of the anterior commissure (AAC)ln,21, 25. Before forming discrete tracts, the axons of mitral and tufted cells emit collaterals, some of which end below the mitral cell layer, while others double back to end in the external plexiform layer. The deepest layer of the bulb, the granular layer, contains granule cells and a number of short axon-type cells 5. The granule cell dendrites branch and extend into the external plexiform layer where they form numerous synapses with the accessory dendrites of mitral and tufted cells. These synapses are similar to Type II synapses 12 and are thought to have an inhibitory function 8. In addition, synapses oriented in the opposite direction, i.e., from mitral dendrite to granular cell dendrites have been described and are thought to be excitatory 22 (see diagram in upper right of Fig. 1). In addition to the centripetal fiber systems, consisting of the LOT and AAC, the olfactory bulb receives centrifugal fibers of two types - - the thick and thin fiber system 5. The thin fibers arise from the contralateral bulb and/or peduncle and travel in the AAC. The origin of the thick fibers, many of which run in the LOT 20, is uncertain, although the olfactory tubercle has been strongly implicated 21. METHODS
The experiments were performed on 46 New Zealand white rabbits weighing between 2 and 3 kg. The animals were anesthetized with ether. Following cannulation of the femoral vein the ether was discontinued and the animals maintained on intravenous pentobarbital for the remainder of the experiment. A heat lamp was used to keep rectal temperature between 37 and 39°C. The trachea was cannulated and the bone overlying the frontal lobes and left olfactory bulb removed. To reduce pulsation a metal plate was inserted into the frontal lobes separating the bulb from the rest of the brain 2s. As judged by examination of the surface blood vessels this procedure had little effect on the blood supply to the olfactory bulb. In 12 animals the olfactory bulb and peduncle were isolated from the rest of the brain by a transection of the caudal portion of the bulb, 2-3 months prior to the experiment to allow for sufficient time for the degeneration of most centrifugal fibers. However, it is possible that some of these fibers remained functional owing to the presence of part of the olfactory tubercle and some prepiriform cortex distal to the transection. Complete transection of the LOT in these chronic preparations was routinely verified by histological examination. These preparations will be referred to as 'chronically decentralized preparations'. To expose the LOT, the left eye was enucleated, the bone overlying the LOT removed and the dura reflected. The diagram to the bottom right of Fig. 1 illustrates the technique used for the placement of the electrodes. The distal portion of the LOT was peeled from the surface of the prepiriform cortex for a distance of about 1.5 mm and a bipolar silver electrode was placed on the LOT anterior to the reflected portion. Brain Research, 14 (1969) 157-172
160
R . A . NICOLL
A bipolar concentric electrode with an outside diameter of 1 mm was used to stimulate the AAC. This electrode was inserted into the prepiriform cortex in the region exposed by the reflected LOT to a depth of 1-2 mm until the maximum AAC field potential was elicited in the granular layer of the olfactory bulb. In addition, a concentric electrode was also placed on the surface of the bulb to stimulate the olfactory nerves. Glass capillary microelectrodes (1-3 Mg2), filled with 4 M NaCI, were used to record field potentials. High-resistance microelectrodes (40-100 M[2), filled with 3 M KC1, were used for unit recording. Surface potentials were routinely monitored with a silver ball electrode on the surface of the bulb. The extracellular potentials were recorded with a capaeitatively coupled system (time constant, 1 sec). lntracellular potentials were directly coupled through a Bak cathode follower 4 to a DC amplifier. After the electrodes had been positioned on the surface of the bulb, the entire area was covered with 3 % solution ofagar made with Ringer-Locke solution. For the purposes of reproduction a number of the illustrations have been retouched. RESULTS
Inhibition of mitral cells by LOT stimulation Evidence was sought that the inhibition of mitral cells by LOT stimulation is due to the occurrence of an inhibitory postsynaptic potential (IPSP). The effects of pairing conditioning and testing stimuli to the LOT on antidromic invasion of mitral cells was studied (Fig. 2). With a test stimulus set at twice threshold and a conditioning stimulus at 3.5 V (Fig. 2), the A-B inflection n of the antidromic spike was progressively exaggerated as the conditioning-test interval was increased. At greater intervals, the B spike and finally the A spike was abolished. This suppression of antidromic invasion lasted for approximately 100 msec. As the strength of the conditioning stimulus was increased to 4.5 V, the test response was suppressed at a shorter interval (6 msec). When the conditioning stimulus was raised to 6.5 V (just over threshold for this cell) the test volley was ineffective at short intervals, while at slightly longer intervals the cell responded with an A spike which was inhibited at 4.5 msec. The results obtained from this cell are similar to those of Phillips et al.lL This is illustrated graphically in the lower part of Fig. 2; the length of the bar represents the interval within which a test spike was obtained; the end of the bar indicates the onset of the inhibition production by the conditioning stimulus. It should be noted that suppression of the test response began before the occurrence of complete inhibition. For example, for the cell illustrated in Fig. 2, when the conditioning volley was 4.5 V, and A-B inflection was observed at 2.8 msec (not shown in Fig. 2). Direct evidence for the occurrence of an IPSP consequent to LOT stimulation was obtained from intracellular recordings from mitral cells, both in acutely and chronically decentralized animals. Fig. 3A shows the occurrence of a prolonged hyperpolarization following antidromic invasion of a mitral cell in a chronically decentralized preparation. The possibility that this hyperpolarizing potential represented a Brain Research, 14 (1969) 157-172
161
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
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Fig. 2. Giant extracellular spike potentials of a mitral cell evoked by LOT stimulation. The strength of the test stimulus was set at 12 V (twice threshold) for all of the records. In the left hand column the conditioning stimulus (dot beneath each trace) was set at 3.5 V and the interval between the stimuli (numbers to the left of each trace) was increased. In the center column the conditioning stimulus was raised to 4.5 V. In the right hand column the conditioning stimulus was set at 6.5 V (just over threshold for this cell). In the graph below the unit responses, the results obtained from this mitral cell are shown. The bar represents the occurrence of any part of the test spike, the bracket at the end of each bar representing the interval that was not tested.
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Fig. 3. Antidromic responses from mitral cells recorded intracellularly from chronically decentralized animals following stimulation of the LOT. In A, the prolonged hyperpolarization which follows antidromic invasion of mitral cells is shown. In B, the effect of both threshold and subthreshold intensity stimulation on antidromic invasion and the succeeding hyperpolarization are shown. C, Same cell as in B recorded after a 30-mininterval. The time scale for B is the same as that for the left hand column of C.
162
R.A. NICOLL
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Fig. 4. The olfactory bulb field potential evoked by LOT stimulation. Responses to LOT stimulation are shown at different depths below the surface of the bulb. The depth is indicated beside each trace. Different waves are indicated above the trace recorded at 250 #. spike afterpotential was excluded by the use of threshold voltages where it was possible to evoke a hyperpolarizing potential in the absence of antidromic invasion (Fig. 3B). The latency for the onset of this hyperpolarization was typically about 3 msec. Further evidence indicating that this hyperpolarization is an IPSP was obtained from the following observations. The response in Fig. 3C was obtained from the same cell as Fig. 3B 30 min later. It is to be noted that the size of the hyperpolarization diminished considerably, and was almost absent, possibly due to the leakage of C1ions from the microelectrode. Furthermore, the size of a second test spike was reduced (Fig. 3C) even though there was little hyperpolarization. This reduction in the height of the test spike was not due to A-B dissociation because the reduction was graded and there was no apparent A-B inflection in this cell. This reduction of spike height suggests that the membrane conductance is increased following antidromic invasionL The fact that the hyperpolarization is present without an antidromic spike, that it may be sensitive to Cl-ions, and that there appears to be an increased conductance during the hyperpolarization, all indicate, by analogy with the motoneuron 6, that hyperpolarization of mitral cells following L O T volleys is due to an IPSP.
Pathway involved in generating IPSP in mitral cells Evidence was next sought to determine whether the inhibitory pathway is recurrent, involving mitral axon collaterals or dendrodendritic, involving mitral cell dendrites. In Fig. 4 the field potential caused by LOT stimulation is shown at varying depths in the olfactory bulb. In agreement with Rail et al. ~2, at the surface there is an initial positive wave (P1) followed by two negative waves (N1,N2) and a second long duration positive wave (P2). As the recording electrode penetrated the bulb, the P1
Brain Research, 14 (1969) 157-172
INHIBITORYMECHANISMSIN RABBITOLFACTORYBULB
163
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LATENCYlmsec ) Fig. 5. The extracellular field potentials evoked by LOT stimulation. In A, records were obtained from the same experiment illustrated in Fig. 4. The 3 vertical lines indicate latencies of 1.6, 2.6, ~ d 5.0 msec, respectively, after the stimulus artifact. In B, the latency of the N1 wave is shown as a function of depth below the surface of the bulb. The latency was measured from the stimulus artifact to the beginning of the N1 wave.
wave diminished in amplitude and the latency of the N1 wave shortened. Both negative waves increased in amplitude; the N1 wave reached its maximum at the mitral cell layer (900 #), while the N2 and P2 waves attained their maxima in the external plexiform layer. As the microelectrode passed through the mitral cell layer the polarity of the Nz and P2 waves reversed. Phillips et al. 19 demonstrated that the depth at which the N2 wave inverted corresponded to the mitral cell layer. The same sequence of changes in the polarity of the waves occurred in the ventral side of the bulb. The early wave responses following L O T stimulation are shown in greater detail in Fig. 5A. At 1.6 msec (measured from the shock artifact), the N1 wave in the mitral cell layer (900/~) was synchronous with the superficial P1 wave. At 2.6 msec the superficial N1 wave was synchronous with a deep positivity seen as a shoulder on a much larger and later positive wave. The latency of the N1 wave at different depths below the surface of the olfactory bulb is shown in Fig. 5B. At depths below 900 # the latency of the N~ wave remained relatively constant. As the microelectrode was withdrawn the latency was progressively longer, until the olfactory nerve layer (250/z) was reached. Between 900 and 250/z below the surface of the bulb, the N~ wave had an approximate slope or velocity of 0.7 m/sec. Brain Research, 14 (1969) 157-172
164
LOT
R. A. N I C O L L
," LOT
1T
].5 2.0 ~ " - - ' - - - ' - - ~ 2.5 ~ " - - - ~ +JlOmV Fig. 6. Intracellular IPSPs recorded from mitral cells following stimulation of the LOT. The interval between the conditioning and test stimulus to the LOT was held constant, while the strength of the conditioning stimulus was increased. The bottom trace is the response obtained just outside the mitral cell at the highest stimulus intensity used.
From their analysis Rail et al. 22 concluded that the 'membrane depolarization of mitral dendrites activates excitatory synapses which depolarize granule cell dendrites; the synaptic depolarization of granule dendritic membrane then activates granule to mitral inhibitory synapses'. The depolarization of the granule dendritic membrane generates the negativity (signified by the N~ wave) in the external plexiform layer. If a dendrodendritic pathway is responsible for the generation of the IPSP in mitral cells following LOT stimulation, then the inhibitory pathway would be blocked by a conditioning stimulus to the LOT since the pathway through mitral cell dendrites would be inhibited (Rail, personal communication). Alternatively, if the IPSP in mitral cells is generated by a recurrent collateral pathway, then the inhibitory pathway should not be blocked by a conditioning stimulus to the LOT as the recurrent collateral pathway by-passes the mitral cell dendrites. Therefore, the effect of conditioning LOT volleys on intracellular responses and field potentials was examined, in order to choose between these two hypotheses. Paired stimuli to the LOT cause a reduction in the test IPSP recorded intracellularly from mitral cells. As illustrated in Fig. 6, when the strength of the conditioning stimulus was increased the size of the conditioning IPSP increased, while the test IPSP was reduced. This reduction was not due to the IPSP reaching its equilibrium potential, because the combined hyperpolarization of the conditioning and test response was less than the control IPSP. It cannot be excluded, however, that this reduction might be due to alterations in synapses of the inhibitory pathway. It was found, moreover, that the N2 and P2 waves of the field potential recorded in the external plexiform layer were routinely inhibited by a conditioning LOT stimulus as illustrated in Fig. 7A from a chronically decentralized preparation. Both waves were completely suppressed at short intervals, the N~ wave actually being replaced by a slight positivity. As the interval increased, the N2 wave recovered; but only at the longest interval did the P2 wave begin to recover. This inhibitory effect is Brain Research,
14 (1969) 157-172
165
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
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Fig. 7. Extracellular responses evoked by paired LOT stimuli in a chronically decentralized preparation. In A, the recording electrode was located in the external plexiform layer and the interval between the 2 LOT stimuli was varied. The voltage gain is the same for all traces except the conditioning control (COND. CON). In B, the inhibition of the test N1 wave (percent of control) is shown as a function of the conditioning-test interval (msec). A tracing of the P~ wave evoked by the conditioning stimulus is shown above the graph to provide comparison of its time course with that of the inhibitory effect.
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"30msec Fig. 8. Inhibitory action of olfactory nerve (SURF.) and LOT volleys on the response evoked in granular layer cells following surface stimulation. In A, the inhibition of granular layer cell response evoked by a surface stimulus by a preceding surface stimulus applied to an adjacent surface electrode. In B, the inhibition of granular layer cell response evoked by a surface stimulus by a preceding LOT volley. The control response (CON) is shown in the top trace in each series. The stimulus strength to LOT is expressed in arbitrary units and is shown to the left of each trace. Voltage gain is the same for all traces.
shown graphically in Fig. 7B. It should be noted that there is a correlation between the time course of the P2 wave shown above the graph and the inhibition of the N2 wave. Brain Research, 14 (1969) 157-172
R.A. NI(OLI,
166
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Fig. 9. Inhibitory and facilitatory effect of an olfactory nerve volley on the response of granular layer cells evoked by a LOT volley. In A, inhibition of the LOT response by a preceding surface stimulus (given in arbitrary units). In B, facilitation of the LOT response by a preceding surface stimulus at short intervals. The number at the left of each trace in B is the interval (msec) between the surface and LOT stimulus. The control responses (CON) are shown at the top of each column. Voltage gain is the same for all traces. In C, histogram showing facilitation of surface stimulus on the LOT response of a granular layer cell at different conditioning-test intervals. The broken line indicates the number of spikes evoked by a control LOT stimulus.
The results support the hypothesis of a dendrodendritic inhibitory pathway. The reduction of a test IPSP suggests that the pathway mediating the IPSP, i.e., mitral cell dendrites is blocked by the conditioning volley. Similarly, the conditioning IPSP in mitral cells would block the N2 wave if it were due to the depolarization of granule cell dendrites by mitral cell dendrites, because the excitatory pathway through the mitral cell would be inhibited. Further evidence in support of a dendrodendritic inhibitory pathway was obtained from analyzing the responses obtained in the granular cell layer.
Unit responses recorded in the granular layer A conditioning stimulus applied to the surface of the olfactory bulb routinely inhibited the test response of granular layer cells evoked by the same or an adjacent surface electrode. In Fig. 8A two adjacent surface electrodes evoked cell discharge. However, a conditioning stimulus applied through one electrode reduced the response evoked by the other electrode from three to one spikes. A conditioning LOT stimulus also inhibited the response produced by a surface stimulus (Fig. 8B). Four spikes were elicited from the granular layer cell following suprathreshold stimulation of the surface. Even when the conditioning L O T stimulation was too weak to discharge the cell (Fig. 8B, 0.4 V), it completely suppressed the surface response. Since the LOT exerted its
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INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
A LOT
~ LOT
B AAC
~- LOT
15
msec
Fig. 10. Inhibition of the LOT response evoked in granular layer cells by preceding LOT or AAC stimulus, In A, inhibitory action of an LOT volley on the response evoked by an LOT stimulus in a chronically decentralized preparation. In B and C, inhibition of the LOT response by a preceding AAC volley; the response shown in C was from a chronically decentralized preparation. The dot below the traces shown in B and C indicates the AAC stimulus artifact. The control response (CON) is the first trace in each series. effect without discharging the cell, this suppression of discharge cannot be attributed to postspike refractoriness. As illustrated in Fig. 9A surface stimulation also inhibited the response of granular layer neurons to L O T stimulation. Separately, a strong surface stimulus or a L O T stimulus produced 3 spikes. The test L O T stimulus, however, was ineffective until a very weak surface stimulus was used. Strong surface stimuli could suppress an L O T evoked response for intervals greater than 100 msec. However, at short intervals the surface volley often facilitated the LOT response (Fig. 9B). Both these findings are illustrated graphically in Fig. 9C. The surface stimulus was adjusted so that it never discharged this cell. The L O T stimulus was set so that it elicited one spike once every 10 trials. At intervals between 6 and 9 msec cell firing increased to about 6 spikes in 10 trials while at longer intervals the firing decreased to zero. When stimuli were paired to the L O T at different intervals the test response of granular layer cells was often completely suppressed for 100-200 msec. In Fig. 10A the test volley elicited a spike only after the test field potential returned to its control configuration. The inhibitory effect of a conditioning LOT volley was present even when the volley failed to discharge the cell. L O T activation of granular layer cells was often inhibited by volleys to the AAC. In Fig. 10B the L O T control response consisted of two spikes. At short intervals the intact AAC had no effect while at longer intervals the L O T response was reduced to one spike and then was ineffective. The effect was also observed in chronically decentralized preparations. As illustrated in Fig. 10C the L O T response was completely removed by a preceding A A C volley. DISCUSSION
In agreement with previous results19, 28, intracellular records from mitral cells revealed that following antidromic invasion there is a prolonged hyperpolarization. Brain Research, 14 (1969) 157-172
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This hyperpolarizing potential is present in both intact and chronically decentralized preparations. Since this hyperpolarization was present even when antidromic invasion did not occur, seemed sensitive to Cl-ions, and was associated with an increased conductance, it is suggested that this hyperpolarization represents a synaptically induced inhibitory potential (IPSP). The latency for the IPSP was approximating 3 msec. This latency allows sufficient time for at least two or more synaptic delays. It should be noted that these values were obtained with subthreshold or threshold intensity stimulation. With suprathreshold stimuli the latency would be somewhat shorter. The dendrodendritic inhibitory p a t h w a y
From an analysis of the LOT field potential, Rail et al. zz postulated that the inhibition of mitral cells was mediated through their dendrites. The finding of bipolar synaptic connections between the mitral cell dendrites and granule cell dendrites 3,15,2~ provided anatomical support for this hypothesis. The results obtained in the present study add further support to the dendrodendritic hypothesisZL The short latency of the N1 wave and the finding that antidromic unit responses occur during this wave suggests that the N1 wave results from the synchronous antidromic invasion of mitral cells and their dendrites. The apparent rate at which the antidromic impulses invade the dendrites of mitral cells was calculated from the latency of the N1 wave to be approximately 0.7 m/sec. This value is in good agreement with the slow velocities obtained previously for mitral cells is and other neurons in the CNS 2,10,14,17. The results presented here do not pertain to the question of passive vs. active invasion of dendrites. As illustrated in Fig. 5A at 1.6 msec the deep N~ wave is synchronous with the superficial P~ wave. At this latency, the superficial dendrites of mitral cells are not invaded and supply current to their depolarizing cell bodies (Fig. 11A). At 2.6 msec the superficial N1 wave is synchronous with a deep positivity. During this period the superficial dendrites are invaded and the repolarizing cell bodies supply current to their depolarizing dendrites (Fig. 11B). At 5 msec, the Nz wave which is largest in the external plexiform layer is coupled with a large positive wave in the granular layer. Ochi is and Von Baumgarten et al. 27 correlated this wave with synaptically activated secondary neurons (presumably via a direct recurrent collateral pathway). This recurrent activation may contribute to the formation of the N2 wave, but by itself cannot account for the large current flow across the mitral cell layer. Rail et al. 2~ suggested that 'the population of cells which generate this current must possess a substantial intracellular pathway for the return flow of current from external plexiform layer, through the mitral cell layer, into the depths of the granule layer. This:requirement is satisfied by the large population of granule cells, but not by the mitral cells.' In their hypothesis, depolarization of mitral cell dendrites provides a synaptic excitatory input which depolarizes granule cell dendrites in the external plexiform layer. The depolarization of granule cell dendrites then activates granule to mitral cell inhibitory Brain Research, 14 (1969) 157-172
169
INHIBITORYMECHANISMSIN RABBITOLFACTORYBULB
°E...
A
B
C
D
350 450
950 IOSO -
_j
'
Fig. 11. Schematic diagram of mitral and granule cells showing current flow at various latencies after a LOT volley. In A, the current flow at 1.6 msec, in B at 2.6 msec, in C at 5.0 msec, and in D at approximately 25 msec after the stimulus artifact. synapses. During this period the deep lying granule cell bodies are predominantly passive and supply current to their superficially depolarizing dendrites (Fig. l lC). The P2 wave which follows the Nz wave was not considered in the analysis by Rail et al.22. This wave was largest in the external plexiform layer and reversed polarity at the mitral cell layer. Since the time course of the P2 wave was correlated with the time course of the IPSP recorded from mitral cells and the time course of inhibition of a test N2 wave, it is proposed that the P2 wave results from the inhibitory synaptic currents generated in the dendrites of mitral cells. The finding that the P2 wave reversed polarity at the mitral cell layer suggests that the current flows from the mitral cell dendrites (active source) to passive sinks in the granular cell layer (mitral cell axons and collaterals) (Fig. 11D). In the postulated dendrodendritic inhibitory pathway, in contrast to a recurrent inhibitory pathway, the inhibition is fed back onto a portion of the inhibitory pathway, i.e., mitral cell dendrites. Accordingly, a conditioning stimulus to the lateral olfactory tract should, as observed, suppress the test L O T IPSP in mitral cells. While possible alterations in synaptic behavior (e.g., receptor desensitization or decrease in available transmitter by a preceding volley) can not be excluded, these factors appear less likely in view of several lines of evidence suggesting that the pathway mediating the IPSP is
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blocl~ed by the conditioning volley. The inhibition of the N2 and P2 waves of the field potential by a preceding LOT volley is consistent with a dendrodendritic hypothesis. The conditioning IPSP in mitral cells prevents the test volley from depolarizing the granule cell dendrites because the excitatory pathway through the mitral cell has been blocked. Since the generation of the P2 wave is dependent upon the depolarization of granule cell dendrites, both the N2 and P2 waves should be suppressed by a conditioning LOT volley. Responses in the granular layer
Cells in the granular layer are synaptically discharged by both surface (olfactory nerve) and LOT stimulation. The inhibitory effect of a conditioning surface (Fig. 8A) and LOT volley (Fig. 8B) on a test surface response of granule layer cells is to be expected, because the only anatomical pathway through which impulses in the olfactory nerves can excite neurons in the granular layer is through secondary neurons. The prolonged inhibition of secondary neurons following conditioning surface or LOT volleys would suppress the response of granular layer cells to surface volleys by blocking the excitatory pathway. Conversely, the observed inhibition of the test LOT response of some granular layer cells (Figs. 9A and 10A) would not be predicted if, as has been assumed by previous investigators, the granular layer cells are activated by a simple recurrent pathway, since inhibition of the mitral cell soma should not block conduction through the recurrent collaterals. The fact that the cell in Fig. 10A failed to respond until the LOT field potential returned to its control configuration suggests that the inhibition might be related to the dendrodendritic pathway. Yamamoto et al. 2s found that a conditioning stimulus to the AAC not only hyperpolarized mitral cells, but also reduced the amplitude of a test IPSP evoked by LOT stimulation. Since AAC stimulation also suppressed granular layer cells activated by LOT stimulation, they proposed that stimulation of the AAC had two independent effects: (1) inhibition of mitral cells and (2) suppression of the LOT evoked IPSP by inhibition of interneurons in the recurrent collateral pathway. The alternative hypothesis for these effects is that the AAC evoked hyperpolarization of mitral cells suppresses the LOT evoked IPSP by blocking the dendrodendritic pathway, thereby preventing mitral to granule cell excitation. At short intervals a surface volley was found to facilitate the LOT activation of granule cells (Figs. 9B and C). It is tempting to use this finding as support for the proposed dendritic pathway. The surface volley depolarizing the mitral cell dendrites would facilitate the dendritic invasion of antidromic impulses and this in turn would lead to a larger depolarization in the granule cell dendrite. However, field potential studies consistently failed to show any facilitation of antidromic invasion by a preceding surface volley (unpublished observation). It is therefore concluded that the surface facilitatory effect is due largely to its asynchronous activation of mitral cells. Since the conduction velocity in the olfactory nerves is very slow (unpublished observation), the activation of mitral cells will be dispersed in time. This in turn will produce a temporal dispersion in the EPSP generated in the granule cells. Brain Research, 14 (1969) 157-172
INHIBITORY MECHANISMS IN RABBIT OLFACTORY BULB
171
In conclusion, it is proposed that cells in the granular cell layer whose test response is inhibited by conditioning volleys evoked by stimulation of the surface, LOT and AAC are granule cells activated by the dendrodendritic excitatory synaptic pathway between mitral and granule cell dendrites. Since all three inputs hyperpolarized the mitral cell, a conditioning volley in any one of these three pathways should block the test response in granule cells evoked in the same or different pathway. Although the granule cell responses are the most numerous of the responses recorded in the granular layer, only a small fraction of the total population of granule cell bodies must be activated because the LOT field potentials indicate that the granule cell bodies are predominantly passive. While alternative hypotheses may not be completely excluded, the dendrodendritic hypothesis proposed by Rail et al.22 has the advantage of providing the most unified explanation of data concerning inhibition of responses in the granular layer. SUMMARY
The unitary responses of mitral and granular layer cells and olfactory bulb field potentials following stimulation of the lateral olfactory tract (LOT), olfactory nerves and anterior limb of the anterior commissure (AAC) were analyzed. The hyperpolarization that followed antidromic invasion of mitral cells was shown to result from a synaptically evoked inhibitory potential. The analysis of the LOT evoked field potential indicated that the initial negative deflection represented antidromic invasion of mitral cell somata and dendrites. The results suggest that the second negative deflection of the field potential represented a synaptically evoked depolarization of granule cell dendrites, while the succeeding positive deflection represented the synaptically evoked inhibition of mitral cell dendrites and somata. A conditioning LOT stimulus inhibited the intracellularly recorded hyperpolarization of mitral cells and abolished the field potential evoked by a test LOT stimulus. In addition, cells in the granular layer that responded to LOT stimulation were inhibited by conditioning stimuli supplied to the LOT, AAC or olfactory nerves. The results are consistent with and lend support to the dendrodendritic hypothesis advanced by Rail et al. 22 and suggest that the cells in the granular layer which are activated by the dendrites of mitral cells are granule cells. ACKNOWLEDGEMENTS
I wish to thank Drs. G. C. Salmoiraghi and F. Weight for their constant encouragement and suggestions, Drs. A. Gorman, W. Rail and P. Nelson for criticizing the manuscript and Mr. H. Poole for his help in preparing the illustrations.
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