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Hypothalamic influences on the electrical activity of the olfactory pathway
Brain Research Bf.d&tin,
AI1 rights of reproduction
Vol. 1, pp. 263-272, 1976. Copyright 0 ANKHO International in any form reserved. Printed in the U.S.A.
Inc.
Hypothalamic Influences on the Electrical Activity of the Olfacto~ Pathway H. U. AGUILAR-BARTURONI,
R. GUEVARA-AGUILAR,
H. ARIkHIGA
AND C. ALCOCERCUAR6N
~e~arra~ent~ de Fisi~~~~~a, Fa~itad de ~edicina, Apartado Postal 70250 U~i~er~idad rational A~t~no~a de Mt!xico, Mkxico 20, D.F.
(Received 2 December
1975)
AGUILAR-BATURONI, H. U., R. GUEVARA-AGUILAR, H. ARBCHIGA AND c. ALCOCERCUARGN. Hyporhalamic jn~ue~ceS on the electrical activity of the olfactory pathway. BRAIN RES. BULL. l(3) 263-272, 1976. - By means of evoked potentials a direct efferent connection was found to run from the posterior hypothalamus and medial forebrain bundle to primary olfactory structures (olfactory bulb, olfactory tubercle and prepyriform cortex). The pathway from the hypothalamus to the olfactory bulb follows in the lateral olfactory tract at a conduction velocity S-10 m/set. The olfactory tubercle functions as a relay station for the efferent fibers from various sources, running to the olfactory bulb. In animals with electrodes chronically implanted in the olfactory structures, hypothalamic stimulation gives rise to a prolonged train of hypersynchronous bursts of activity (40-50 Hz), which resemble the arousal reaction. This response is modified by transectmg the cervical sympathetic trunk. By pathways still to be defined, potentiafs are evoked in the olfactory bulb by stimulation of the cervicai sympa~etic trunk and the te~ination of these sympathetic fibers shows a common postsynaptic neuronal pool with axons-of hypothalamic origin. Epinephrine topically applied to the olfactory mucosa induced hypersynchronous activity in olfactory structures, quite similar to that consequent to hypothalamic stimulation. These results suggest a multichanneled hypothalamic modulation of olfactory input. Modulatory influences on olfactory pathway
Afferent regulation
Hypothalamus
Olfactory pathway
[ 15 I . Gault and Coustan [ 141 postulated that the “spindling” activity of 40 Hz in the olfactory bulb of the cat during “emotional” states is mediated by changes in respiratory movements. However, respiratory effects are not necessary for the appearance of “spindling activity.” During the arousal reaction in cats Hem~ndez-Peon et al. [ 191 found no correlation between the spindles and respiratory movements. In addition, Peiialoza-Rojas and Alcocer-Cuaron [ 281 demonstrated spindling activity even in tracheotomized cats. Thus, the problem of the channeling of activity from the hypoth~amus to the olfactory system and its relation to respiration is as yet unsolved. It is the purpose of this paper to analyse some of the possible pathways involved.
EVIDENCE from various sources suggests that the hypothalamus exerts a modulatory influence on olfactory input. Yamamoto and Iwama [35] using electrical stimulation of the posterior hypothalamus in curarized rabbits found changes in spontaneous and induced electrical activity of the olfactory bulb. Degenerating fibers have been observed in the olfactory tubercle of rabbits after
mesencephalic lesions [lo]. These fibers run rostrally through the hypothalamus. More specifically, lesions of the medial forebrain bundle in the hypothalamus of the rat resulted in fiber degeneration which was traced into the olfactory tubercle and anterior olfactory nucleus, and degenerating fibers after lesions in the rostra1 hypothalamus and preoptic areas were successfully followed up to the olfactory bulb [ 29,301. All this evidence points to the existence of neural connections from the hypothalamus to the olfactory system. On the other hand, it is well known that electrical stimulation of the hypothal~us triggers behavioral patterns such as panting, sniffing and affective defense reactions, during which changes in respiratory activity are the rule [ 181. The electrical activity of the olfactory bulb in the hedgehog was reported by Adrian [ 1] to be influenced by the air flow through the nostrils and, in rabbits, changes of the pattern of electrical activity in the olfactory bulb take place upon awakening from anesthesia and can sometimes be induced by a single inspiratory movement [2]. A definite correlation of respiratory rate and frequency of induced electrical activity in the olfactory bulb of the cat was reported for various behavioral states
METHOD Animals and Procedure
The experiments were conducted on 127 adult cats of both sexes, weighing 2.5-3.0 kg. Two types of preparations were used. Acute preparations. In 58 cats, under ether and chloralose (70 mg/kg) anesthesia bipolar or tetrapolar stainless steel electrodes (20-30 Kfi resistance) with 1 mm vertical separation of their tips were implanted in various parts of the olfactory pathway: olfactory bulb (OB), prepyriform cortex CPPCf, olfactory tubercle (OT) and lateral olfactory tract (LOT), as well as the anterior commissure (AC) and neocortex (rostra1 marginal gyrus, NC). Stimulating electrodes of the same type as those used for recording were 263
264
AGUILAR-BATURONI
placed in the posterior hypothalamus (PH) within the area (A = 10.0, L = 1 .O, H = -4.5) known since Hess’ [ 18 ] studies to be related to aggressive reactions, and in the medial forebrain bundle (MFB), following the atlas by Jasper and Ajmone-Marsan [20]. Once the surgical procedure was completed, the animals were immobilized with Flaxedil (10 mg/kg) and local anesthesia was applied henceforth at 30 min intervals to the external auditory meatus, at the contact points with the stereotaxic apparatus, and to the surgical incision. Potentials were evoked by 0.5-1.5 V, 0.25 msec pulses. An AC-coupled preamplifier with a time constant of 0.12-0.16 msec was used in conjunction with a dual-beam oscilloscope (Tektronix 502) from which the responses were photographed with a Grass, C-4 kymograph camera. In 37 animals, platinum bipolar electrodes, covered with polythene except at their tips [ 131 were placed on the cervical sympathetic trunk (CS). Chronic preparations. In 32 cats, under pentobarbital anesthesia, electrodes were chronically implanted in the same regions as those for acute experiments. Recording was begun 3 days after surgery. Stimulation was most effective as trains of 0.5.-3.0 mA, 1.0 msec pulses, at 40 Hz for 5 sec. In 7 animals, electrodes of the same type as those used in acute preparations were chronically implanted around the CS. Electrical stimulation of the CS was started 8 days after electrode implantation. Activity from the olfactory areas was recorded in these preparations by a pen recorder (Grass Model 78 polygraph), EKG and respiratory move-
ments were recorded through metal pins inserted under the thoracic skin and led after preamplification to the pen recorder. Puffs of odorized air or drops of chemicals were delivered to the olfactory mucosa by means of a polythene
?OB
r.-.---_
k-T .,II,.
cannula, introduced to the nasal cavity through the frontal sinus. In both types of preparation, once the experimentation was completed, the animal was sacrificed under deep anesthesia and the position of electrodes was ascertained according to the technique of Guzmdn-Flores et al. [ 171. In
some of the chronic preparations in order to analyse the behavioral responses to hypothalamic stimulation, photographs of the cats were taken, RESULTS kjfects of Electrical Hype thalamus on the Structures
Stimulation Activity of
of the Primary
Posterior Olfactory
In cats with electrodes chronically implanted in posterior hypothalamus, the parameters of stimulation were adjusted to values such as to produce attentive behavior, as can be seen in Fig. 1. The response to stimulus trains (5 set, at 0.5 mA, 1 ms pulses at 40 Hz) was a pronounced mydriasis and contraction of ear and neck muscles. There were no changes in heart rate or in respiratory movements. The only modification apparent (Fig. 1) is an induced bursting activity in the homolateral olfactory bulb, quite similar in spindling described by Lavin et al. [22] as the arousal reaction in the olfactory bulb. The intra-burst rate was 40-45 Hz, and persisted within that range throughout the entire response. It was independent of the intensity, duration or rate of stimulation. This pattern of activity lasted for 3-5 min after a single 5 set period of stimulation. The intra-burst intervals (IBI) gradually lengthened from an initial value of 1 set during the first minute after the stimulation, to about 2 set ai the end of the
I
FIG. 1. Simulataneous recording of electrical activity in right (ROB) and left olfactory bulb (LOB). In upper traces the recording electrode was placed on the surface of the olfactory bulb; in lower traces, 6.0 mm within it. Electrocorticogram (ECG) from the marginal gyms. Pneumogram (PnG), electrocardiogram (EKG). At left, record from quiet, relaxed animal, as illustrated in the upper photograph. The traces at right begin within a few 100 msec after cessation of stimulation of the posterior hypothalamus at the locus indicated by the tip of the bar shown in the diagram at center (A, 10.0; L, 1.0; H, -4.5). Note bilateral mydriasis induced by stimulation (lower photograph). With the type of stimuli applied to hypothalamus (0.5 mA, 1.0 msec, 40 HZ for 5 set) only the ipsilateral OB shows the typical bursting response, without changes in respiratory or heart rates. Cal, ROB 100 ILV, LOB 500 CCV, FrfZ lflflIIV FKC All uV Time: 1 sec.
HYPOTHALAMIC
INFLUENCES
ON OLFACTORY
response. When the intensity of stimulation was gradually increased, the following responses were successively detected: (a) spread of bursting response to the contralateral bulb, (b) generalization to the cortex, (c) at the highest intensities (3.5 mA), the various behavioral components of the defense reaction. The effects shown in Fig. 1 for the olfactory bulb were also found in other areas of the olfactory pathway (i.e. LOT, PPC and OT) and it was possible to evoke them by stimulation of the MFB. Figure 2 shows the effect of left MFB stimulation on various olfactory structures. In this experiment, the rate and duration of stimuli, and the length of the stimulus train were the same as in Fig. 1, but the intensity was greater (1.5 mA) in order to illustrate the propagation of activity to both sides. As can be seen, both the prepyriform cortices and lateral olfactory tracts change their pattern of activity. The spindle bursts in PPC and LOT, however, differ in frequency (30-39 Hz) from those in OB. In this experiment, the neocortex also displays bursting activity after stimulation. No behavioral changes were observed aside from those described above as attentive behavior. An analysis of the temporal distribution of the spindles revealed that those in OB usually preceded by 100-200 msec those in PPC. Another primary olfactory structure where a similar change in activity can be induced by hypothalamic stimulation is the olfactory tubercle (OT). The lack of respiratory change following stimuli which modify the electrical activity of the olfactory pathway strongly suggests that these effects are central in origin, the influence of peripheral changes was further explored, however, in the tracheotomized animal, thus eliminating the effects of ventilation. The trachea was sectioned, the oral stump was tied and cannula was inserted into the bronchial end. Figure 3A illustrates the characteristic response to stimulation of the left posterior hypothalamus. At 24 hr after tracheotomy (Fig. 3B) no changes can be evoked, and it is only after 72 hr (Fig. 3C) that spindles can be induced. At 120 hr, the full pattern is elicitable, with characteristics entirely similar to those of controls (Fig. 3D). These experiments point to the existence of direct neural influences from hypothalamus to the olfactory structures, and the rest of this paper deals with possible channels for these influences. The Pathway from factory Structures
the Posterior
Hypothalamus
265
PATHWAY
to Ol-
In 58 acute preparations electrodes were placed in PH and the following structures: (a) prepyrifo~ cortices; (b) olfactory tubercles; (c) lateral olfactory tracts; (d) olfactory bulbs. Stimulation of PH with single shocks evoked potentials bilaterally at the 4 levels of the olfactory pathways. Several deflections could be detected in the evoked response at each olfactory station, and consistent differences in the waveform of the evoked potential were recognized. The waveform at any given station was nevertheless consistent from one preparation to another, and specific for that particular position. The notation followed in Table 1 to describe the latencies to the peak of the various deflections takes into account only the order to appearance, although as shown below, their origins can be quite different, e.g., N, in the response in contralateral PPC is not due to a direct hypothetic projection, and therefore its latency is comparable to that of N, at other
LPPC LLOT
RPPC
RLOT
A
ECG
ENG
PnG
LPPC
LLOT
RPPC
RLOT
B ECQ
EKG
PttG
FIG. 2. Bursting response induced in various olfactory structures by stimulation of left medial forebrain bundle. (A) control, (B) after stimulation. RPPC, Right Prepyriform Cortex, LPPC, Left Prepyriform Cortex, RLOT, Right Lateral Olfactory Tract, LLOT, Left Lateral Olfactory Tract, ECG, Electrocorticogram, EKG, Electrocardiogram, Pn G, Pneumogram. The more intense stimulation (1.5 mA, 40 Hz, 5 set) results in a bilateral bursting response. Calibration: 100 pV, 1 sec.
olfactory stations. The number of components listed in the table is the maximum recorded, although at some levels one or more components could be missing. No attempt is made in this paper to analyze the source of each component. The potentials evoked in OB by stimulation of PH showed 3 components (see Fig. 4): an initial negative wave with a latency of about 2.1 msec (N, ); a positive wave at 4.9 ms (P, ), second negative wave (N, ) at 12.0 msec and a slow negative deflection with a latency of 54.8 msec (N,). The potentials evoked in PPC were very similar, showing the same 3 components and only 2 in OT (see Table 1). The olfactory tubercle as a relay station for efferent fibers to the olfactory bulb. Since OT is a major relay
station for the fibers running from the bulbar reticular fo~ation to the olfactory pathway, the effects of blocking the activity of OT by topical application of procaine were studied. In the experiment illustrated in Fig. 4, 4S-~1 of 10% procaine, divided in 3 injections of 15 ~1 were applied to OT (at A = 18.5, L = 7.0, 5.0 and 3.0, H = -2.0, - 3.0 and -4.0). The slow components of the evoked potentials disappear, while N, remains. Yet, when procaine is applied at more medial coordinates (L = 1.0-2.0) even the fast components could be abolished. The potentials at PPC are
266
AGUILAR-BATURONI
A08
Figure 5 summarizes the connections which we surmise must exist between PH and the olfactory structures. The organization of this pathway is presently under investigation.
LOB A ROE
Sympathetic Path way
LOB
ROE LOB
B ROB LOB
ROB
C
LOB ROB LOB
ROE LO8
D ROB
LOB
6‘T A 1..
I
FIG. 3. Effect of tracheotomy on bursting responses in right (ROB) and left olfactory bulb (LOB) to hypothalamic stimulation. From A to D, upper 2 traces show activity before hypothalamic stimulation, and lower 2 traces are recordings taken immediately after stimulation with parameters specified in Fig. 1. (A) Control, (B) lack of response 24 hr after tracheotomy. (C) 72 hr after operation; only a very slight response is evoked. At 120 hr (D) the response is recovered on left. Calibration: 100 nV, 1 sec.
modified by procaine in OT regardless of the site of application, while N, persists, and the slow components are abolished (see Fig. 4). The efferent fibers to the olfactory bulb run in the lateral olfactory tract. Section of LOT results in a total
disappearance of the potential evoked in OB by hypothalamic stimulation, while those in PPC, OT and the proximal stump of LOT remain unchanged. Section of AC, on the other hand, does not affect any of the components in the homolateral bulb, but the response in the contralateral bulb is partially suppressed. The conduction velocity of efferent fibers in LOT was determined in a manner similar to that used to measure that for fibers from the bulbar reticular formation @RF). A stimulating electrode was placed at the usual coordinates in PH, and a recording electrode was positioned at various distances along LOT; the velocity, as calculated from the measure latencies, is 5-10 m/set, i.e. the same range as found for fibers from BRF [ 161.
Influences
on the Activity of the OlfhctorJ
In chronic preparations electrodes were implanted unilaterally on the cervical sympathetic trunk, and in various olfactory structures. In quiet, unrestrained animals 5 set trains of stimuli (1.0 mA, 1 msec, 15 Hz pulses) were applied to the CS. These parameters of stimulation induce bilateral mydriasis, without any other vegetative or behavioral changes being apparent, e.g., the cats frequently showed electrophysiological and behavioral evidence of sleep during this stimulation. In Fig. 6 the changes in the pattern of electrical activity in the olfactory bulb are shown. The typical response was a prolonged discharge (3-4 min) of spindles with characteristics entirely similar to those described above for hypothalamic stimulation. The electrocortical activity, however, is consistently desynchronized by the stimulation. This result suggests a possible sympathetic channel in the integration of the hypothalamic influence on the olfactory pathway. A series of experiments was consequently carried out in order to define what role the CS might play in this modulatory function. In 6 cats, electrodes were placed in the usual hypothalamic and olfactory areas, and the effects of hypothalamic stimulation were assessed. Both CS’s were then severed, and a 1 cm segment of the distal stump removed. In order to be certain that no regeneration had taken place in CS during the period of observation, the separation between the stumps was verified at necropsy. The animals were left to recover for 24 hr and the tests for hypothalamic effects on the activity of olfactory structures were resumed. The characteristics of the response are significantly affected (Fig. 7). The latency to the onset of the bursting response, the duration of response, and its intensity, measured as the rate of bursting (number of spindles/min) reveal a lessened responsiveness. It was consistently found that the latency to appearance of bursting was not greatly affected on the first day, but was greatly prolonged by the second and third days (Fig. 7). Why the prolongation of latency does not appear immediately is not understood. Even after 21 days the responsiveness has not yet attained full recovery (Fig. 7). The depression of the intensity of the bursting response and its total duration is even deeper and more prolonged. No changes in the response to hypothalamic stimulation were observed after sham operations, involving manipulations of the sympathetic trunk. Since no information is available about the projections of cervical sympathetic fibers into the olfactory bulb, some experiments were performed recording at various depths of OB while stimulating CS with single shocks. At the same level at which evoked potentials can be recorded after stimulation of PH, monophasic, bimodal potentials are evoked by CS stimulation with a latency of 6.1 + 0.2 ms. These potentials follow the stimulation rate one-to-one only up to 20 Hz. Higher rates result in loss of the response, yet such high rates resulted in post-tetanic potentiation of the evoked response in OB. In order to find whether an interaction of this pathway exists with the direct pathway from PH to the olfactory
HYPOTHALAMIC
267
INFLUENCESONOLFACTORYPATHWAY
I
2
3
FIG. 4.Potentials evoked in olfactory bulb (l), olfactory tubercle (2) and prepyriform cortex (3), by stimulation of homolateral (left) posterior hypothalamus. (A) before, (B) after local repetitive injection of 5 ~1 at different lateral deep to the total of 45 ~1 of 10% procaine in olfactory tubercle. Section at center shows area of placement of cannulae (A, 18.5; L, 7.0, 5.0 and 3.0; H, -2.0, -3.0 and -4.0). The graphs shows the effect upon the different components of the evoked potentials in the OB by administration of procaine. Note that the N, component is depressed only when the procaine is applied more medially (L 1.0). Calibration: 100 MV, 10 msec. Superimposition of 5 traces.
TABLE 1 LATENCIES (AVERAGE
AND S.E.) FOR VARIOUS
THALAMIC
NUCLEI TO DIFFERENT
COMPONENTS OF EVOKED RESPONSES LEVELS OF OLFACTORY PATHWAY
FROM HYPO-
=I 32.9*0.4
n.221
ECG
B
LOB
ECG
C
LOB LOB ECG
FIG. 5. Diagram of pathways postulated from posterior hypothalamus to olfactory structures (shaded areas), ventral view of brain. OB, olfactory bulb, OT, olfactory tubercle, PPC prepyriform cortex, PH posterior hypothalamus. Connections indicated by arrows.
bulb, different types of experiments were carried out. Repetitive stimulation of HP (1.5 mA, 0.1 ms and 50 Hz) was maintained during 1 min, while single test shocks were applied to CS (Fig. 8). The amplitudes of evoked potentials in OB are greatly reduced as compared with controls; the effect lasting 5 min. In another series of experiments, the recovery cycle of OB responses to CS stimuli was determined by application of pairs of shocks at varying intervals. When PH is stimulated, an occlusion is apparent between the two pathways, thus suggesting they share a common postsynaptic neuronal pool. Finally, another possible path for hypothalamic influences was explored. Since peripheral effects for sympathetic action on olfactory receptors have also been described [4,6], a cannula was inserted in the nasal cavity, in animals with electrodes chronically implanted in OB. Epinephrine was then topically applied to the nasal mucosa. This evoked trains of spindles (induced by nasal instillation of 0.5 ml of 10e6 g/l) in OB (Fig. 9). Again the characteristics of the bursting response were entirely similar to those resulting from PH or CS stimulation. Similar instillations of Ringer solution, or procaine failed to elicit spindles. DISCUSSION
Three influence
pathways olfactory
by which the hypothalamus might structures can be discerned from the
FIG. 6. Bursting response induced in right (ROB) and left olfactory bulb (LOB), by electrical stimulation of left cervical sympathetic trunk. Upper trace of each pair, from superficial electrode, lower trace, from electrode 6 mm deep in OB. (A) control, (B) response to sympathetic stimulation (horizontal bar 1.0 mA, 1.0 msec, 15 Hz, 5 set). (C) record continuous with that of B. ECG, electrocorticogram from marginal gyrus. Respiration (not shown) did not change. Calibration: ROB, 1 mV, LOB, 0.5 mV, ECG, 100 @V, I sec. above: (1) a direct path from posterior hypothalamus to both the olfactory cortex and tubercle, (2) a direct projection from the hypothalamus to the olfactory bulb, (3) by the sympathetic system via CS. We are presently endeavoring to determine if the hypothalamic projections are bilateral and, if so where decussation occurs. In a previous paper we described the projections of the bulbar reticular formation to the olfactory structures [ 161. The posterior hypothalamus receives fibers from the brain stem [ 101, and recent evidence suggests that the hypothalamo-olfactory pathway is in series with a reticulohypothalamic projection (Guevara-Aguilar, in preparation). Therefore, it is not surprising that the projections from the posterior hypothalamus to the olfactory areas show the same distribution as those from the brain-stem, and that the latencies, albeit shorter, bear the same relationships, and as does the conduction velocity of efferent axons in the lateral olfactory tract for the two systems. It seems likely, given the effects of MFB stimulation, that other hypothalamic areas are also connected to the olfactory pathway [21 I. The hypothalamus thus seems to be an integrative center for influences acting on the olfactory pathway: other regions connected with olfactory structures, such as the septum and amygdala, also send projections to the hypothalamus [3, 26, 301. The concept that the hypothalamus influences the olfactory system is not new. It was common in the past, on anatomical grounds, to consider the
HYPOTHALAMIC
269
INFLUENCES ONOLFACTORYPATHWAY
200
I50
f 5
2
x
200
100
8
8 8
f
50
DLIYS FIG. 7.Sympathectomy depresses bursting response to PH stimulation; mean and standard error for 6 cats. At left, latency for bursting response in olfactory bulb to ipsilateral (left) PH stimulation. Heavy arrows indicate time of sympathectomy. Latencies (average and S.E.) for successive days in samples from the 1st to the 21st. At right, duration (in set on left ordinates in light columns), and number of spindles (right ordinate, shaded columns) in responses to PH stimulation as modified by sympathetic transection. Abscissa, days after CS section.
FIG. 8. Influence of hypothalamic stimulation upon potentials evoked in OB by stimulation of cut right CS. Hypothalamic stimulation, 50 Hz, 1.5 mA, 0.1 msec pulses, applied for 1 min between 2 and 3 min on graph. Stimulation ofCS, 5 Hz: 1, control (at min); 2, 4 min (2 min after cessation of PH stimulation at arrow); 3, 5 min;4, 8 min. Calibration: 100 GV, 2 msec. Time course of the depression is shown on inset, taking amplitude of response before PH stimulation as 100%. Abscissa, time, min.
210
A LOB
ROB
LOB
FIG. 9. Effect on electrical activit of left (LOB) and right olfactory bulb (ROB) of topical application of 0.5 ml epinephrine hydrochloride (1 X lo- B gr/l) to olfactory mucosa in the alert cat. (A) activity before, (B) after instillation of epinephrine (signaled by arrow B) in the left nostril. Although the cat often sneezed or sniffed immediately upon application of the solution, the electrical effects endured long after such respiratory effects were apparent, and it can be seen that the electrical activity is initially altered only on the side homolateral to the site of application. Also note the decease in ROB during the initial, protracted burst in LOB. Calibration: 100 PV, 1 sec.
hypothalamus as an integral part of the olfactory pathway [23]. Although our results are restricted to the olfactory projections of the posterior hypothalamus and MFB, a more detailed analysis, exploring different hypothalamic regions, has shown that the olfactory areas recieve projections from at least two more hypothalamic areas in the lateral and anterior hypothalamus (Guevara-Aguilar, in preparation). The dual effect of hypothalamic stimulation on the spontaneous activity of the olfactory structures is well in line with the role commonly assigned to its modulatory action. On the one hand, in chronic preparations, the usual effect was the triggering of a hypersynchronous bursting pattern of activity, entirely similar to the arousal spindles described by Lavin et al. [22]. Unfortunately there is no solid ground to discuss the cellular basis of the various patterns of rhythmic activity recorded from the olfactory areas with gross electrodes [32], and therefore it would be premature to speculate about the role of this hypothalamic pathway on the transfer of information along the olfactory pathway. Clearly more detailed studies on this subject are necessary. The bilateral effects of unilateral stimulation of the CS in the intact animal require comment. It seems likely that the commissural connections between the olfactory bulbs could account for a bilateral effect, one bulb altering the activity of the other when it itself is excited via the CS. The bilateral mydriasis, however, must be explained as stimulation either of unknown afferents in the CS or more likely, recurrent collateral effects from antidromic stimulation. The possibility of the mydriasis being attributable to inadvertent stimulation of nonCS afferents seems to be excluded by the lack of any behavioral arousal consequent to such stimulation, and the fact that mydriasis was strictly unilateral following proximal section of CS. In other
respects stimulation of CS was comparable to that in other components of the autonomic nervous system in that the optimum rates of stimulation, although somewhat higher than the average, are within the range described for the fibers to various autonomic effecters [31]. Other evidence of sympathetic influence on olfaction is the fact that norepinephrine when applied iontophoretically [ 5,7] ; has been found to influence the activity of a number of neurons in the olfactory bulb. Monoaminergic nerve terminals have been identified in the olfactory bulb of the rabbit [ 121. Norepinephrine has also been demonstrated to be taken up at the level of periglomerular cells of the olfactory bulb of the mouse [ 24 ] and to be released in the olfactory bulb of rabbit [8 I. However, not all noradrenergic fibers in the olfactory bulb can be taken as sympathetic in origin. The central pathways from the hypothalamus to OB also contain noradrenergic elements. The medial forebrain bundle is a well known noradrenergic system [ 1 1,271, which from our results, sends projections to the olfactory pathway. It will certainly be necessary to have a differential mapping of the noradrenergic fibers ending on the OB in order to define their functional role. The indirect hypohtalamic influence through the sympathetic fibers reaching the nasal blood vessels and olfactory receptors is also known from Beidler’s description of an enhancement of electrical activity in the olfactory nerve fibers after stimulation of the sympathetic bundle in the ethmoidal nerve of the rabbit [6]. This was ascribed to a vascular effect, yet, in isolated preparations of the frog’s olfactory receptors, Ar6chiga and Alcocer [4] found an enhancement in amplitude of the electroolfactogram after topical application of epinephrine and norepinephrine to the olfactory mucosa. They suggested, therefore, a dual facilitatory effect, acting in parallel on olfactory receptors and parareceptor structures. There is an
HYPOTHALAMIC
INFLUENCES
ON OLFACTORY
211
PATHWAY
obvious similarity in this effect on electrical activity of OB described herein, when epineph~e was topically applied to the nasal mucosa, and the effect of hypothalamic or sympathetic stimulation. The fact that the response to hypothalamic stimulation is greatly modified after sympathectomy indicates a role for this peripheral pathway in the overall, normal pattern of hypothalamic influence on olfactory input. The existence of neural connections between posterior hypothalamus and the olfactory structures does not exclude the possibility of influences from other sources. The transient suppression of the bursting response to PH stimulation immediately after tracheotomy confirms the influence of respiration on this pattern of activity, as postulated by Gault and Leaton and by Gault and Coustan [ 14,15 1. On the other hand, the sensitivity of olfactory receptors to epinephrine, the induction of the typical
bursting pattern of bulbar electrical activity by this substance, suggests that a humoral channel could be activated via sympatho-adren~ mechanisms. This, since the early studies of Cannon [ 9 ] and his associates, is know to occur with the defense reaction. The posterior hypothalamus has also been related to the cycles of sleep and wakefulness (see [ 341). Hypothalamic projections to the cerebral cortex have only recently been described [ 331. The possibility has been raised in another context that the hypothalamus influences sensory input [25]. Thus, the centrifugal pathways described in this paper may well play an important role in such modulation of olfactory input. ACKNOWLEDGEMENT
The authors are grateful to Professor Miguel R. Covian and F’mfessor H6ctor BrustCarmona for his suggestions for this paper.
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17. Guzmin-Flores, C., M. V. Alcaraz and A. Fernindez-Guardiola. Rapid procedure to localize electrodes in experimental neurophysiology. Bohr. Inst. Estnd. med. Biol. Univ. nac. h&x. 16: 29-31,195s. 18. Hess, W. R. ~y~thalamus und Thalamus, translated by S. L. Sherwood and A. M. Woolsman, edited by George Thieme, Stuttgart: Springer Verlag, 1956, pp. 70. 19. HernindezPe6n, R., A. Lavin, C. AlcocerCuaron and J. P. Marcelin. Electrical activity of the olfactory bulb during wakefulness and sleep. Electroenceph. clin. Neurophysiol. 12: 41-58,196O. 20. Jasper, H. H. and C. Ajmone-Marsh. A Stereotaxic Atlas of the ~iencephalon of the Cat. Nat. Res. Council of Canada: Ottawa, 1955. 21. Kievit, J. and H. G. J. M. Kuypers. Basal forebrain and hypothalamic connections to frontal and parietal cortex in the rhesus monkey. Science 187: 660-662,197s. 22. Lavin, A., C. AlcocerCuar6n and R. HernindezPe6n. Centrifugal arousal in the arousal bulb. Science 129: 332-333,1959. 23. Le Gros Clark, W. E. Anatomical relationships between the cerebral cortex and the hypoth~amus. Br. -Med. Bull. 6: 341-345,195o.
9. Cannon, W. B. Bodily Changes in Pain, Hunger, Fear and Rage.
ExplNeurol.
16. Guevara-Aguilar, R., H. U. Agu~ar-Baturoni, H. Arechiga and C. Alcocer-Cuaron. Efferent evoked response in the olfactory pathway of the cat. Electroenceph. ciin. Neurophysiol. 34:
24. Lichtensteiger, W. Uptake of norepinephrine in glomerular cells of the olfactory bulb of the mouse. Nature 210: 955-956, 1966. 25. Marshall, J. F. and P. TeiteIbaum. Further analysis of sensory inattention following lateral hypothalamic damage in rats. i COIIID.
uhvs~oi. Psvchol. 68: 375-395.1974.
26. Nauta,‘W: J. H. Anatomical relationships between the amygdaloid complex, the dorsomedial thalamic nucleus and orbitofrontal cortex in the monkey. Anat. Rec. 136: 25 1, 1960. 27. Parent, A. and L. J. Poirier. The medial forebrain bundle (MFB) and ascending monoammergic pathways in the cat. Can. J. Physiol. Pharmac. 47: 781-785,1969.
for chronic experiments.
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Nastuk. New York: Academic Press, 1964, Vol. -V, pp. 121-122. 14. Gault, F. P. and D. R. Coustan. Nasal air flow and rhinencephalic activity. Electroenceph. clin. Neurophysiol. 18: 617-624,196s. 15. Gault, F. P. and R. N. Leaton. Electrical activity of the olfactory system. clin. 15: 299-304, 1963.
28. Peiialoza-Rojas, J. H. and C. AlcocerCuaron. The electrical activity of the olfactory bulb in cats with nasal and tracheal breathing. Electroenceph. ciin. Neurophysiol. 22: 468-472, 1967. 29. Powell, T. P. S., W. M. Cowan and G. Raisman. Olfactory relationships of the diencephalon. Nature 199: 710-712.-, 1963. 30. Powell, T. P. S., W. M. Cowan and G. Raisman. The central olfactory connexions. L Anat. 99: 791-813,196s.
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31. Rosenblueth, A. The chemical mediation of autonomic nervous impluses as evidenced by summation of responses. Am. J. Physiol. 102: 12-38, 1932. 32. Sheoherd. G. M. Svnaotic organization of the mammalian olfactory bulb. Phy&Z. &v. 52:864-917, 1972. 33. Siegel, J. and R. Y. Wang. Electroencephalographic, behavioral and single-unit effects produced by stimulation of forebrain inhibitory structures in cats. Expl Neurol. 42: 28--50, 1974.
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C‘T /I I,.
34. Swett, J. H. and J. A. Hobson. The effects of posterior hypothalamic lesions on behavioral and electroencephalographic manifestations of sleep and waking in cats. Arc& ifal. Biol. 106: 283-293.1968. 35. Yamamoto, C. and’K. Iwama. Central nervous regulation of electrical activity of the olfactory bulb in rabbits. Proc. .Jupan Acad. 36: 295-298, 1960.
AI1 rights of reproduction
Vol. 1, pp. 263-272, 1976. Copyright 0 ANKHO International in any form reserved. Printed in the U.S.A.
Inc.
Hypothalamic Influences on the Electrical Activity of the Olfacto~ Pathway H. U. AGUILAR-BARTURONI,
R. GUEVARA-AGUILAR,
H. ARIkHIGA
AND C. ALCOCERCUAR6N
~e~arra~ent~ de Fisi~~~~~a, Fa~itad de ~edicina, Apartado Postal 70250 U~i~er~idad rational A~t~no~a de Mt!xico, Mkxico 20, D.F.
(Received 2 December
1975)
AGUILAR-BATURONI, H. U., R. GUEVARA-AGUILAR, H. ARBCHIGA AND c. ALCOCERCUARGN. Hyporhalamic jn~ue~ceS on the electrical activity of the olfactory pathway. BRAIN RES. BULL. l(3) 263-272, 1976. - By means of evoked potentials a direct efferent connection was found to run from the posterior hypothalamus and medial forebrain bundle to primary olfactory structures (olfactory bulb, olfactory tubercle and prepyriform cortex). The pathway from the hypothalamus to the olfactory bulb follows in the lateral olfactory tract at a conduction velocity S-10 m/set. The olfactory tubercle functions as a relay station for the efferent fibers from various sources, running to the olfactory bulb. In animals with electrodes chronically implanted in the olfactory structures, hypothalamic stimulation gives rise to a prolonged train of hypersynchronous bursts of activity (40-50 Hz), which resemble the arousal reaction. This response is modified by transectmg the cervical sympathetic trunk. By pathways still to be defined, potentiafs are evoked in the olfactory bulb by stimulation of the cervicai sympa~etic trunk and the te~ination of these sympathetic fibers shows a common postsynaptic neuronal pool with axons-of hypothalamic origin. Epinephrine topically applied to the olfactory mucosa induced hypersynchronous activity in olfactory structures, quite similar to that consequent to hypothalamic stimulation. These results suggest a multichanneled hypothalamic modulation of olfactory input. Modulatory influences on olfactory pathway
Afferent regulation
Hypothalamus
Olfactory pathway
[ 15 I . Gault and Coustan [ 141 postulated that the “spindling” activity of 40 Hz in the olfactory bulb of the cat during “emotional” states is mediated by changes in respiratory movements. However, respiratory effects are not necessary for the appearance of “spindling activity.” During the arousal reaction in cats Hem~ndez-Peon et al. [ 191 found no correlation between the spindles and respiratory movements. In addition, Peiialoza-Rojas and Alcocer-Cuaron [ 281 demonstrated spindling activity even in tracheotomized cats. Thus, the problem of the channeling of activity from the hypoth~amus to the olfactory system and its relation to respiration is as yet unsolved. It is the purpose of this paper to analyse some of the possible pathways involved.
EVIDENCE from various sources suggests that the hypothalamus exerts a modulatory influence on olfactory input. Yamamoto and Iwama [35] using electrical stimulation of the posterior hypothalamus in curarized rabbits found changes in spontaneous and induced electrical activity of the olfactory bulb. Degenerating fibers have been observed in the olfactory tubercle of rabbits after
mesencephalic lesions [lo]. These fibers run rostrally through the hypothalamus. More specifically, lesions of the medial forebrain bundle in the hypothalamus of the rat resulted in fiber degeneration which was traced into the olfactory tubercle and anterior olfactory nucleus, and degenerating fibers after lesions in the rostra1 hypothalamus and preoptic areas were successfully followed up to the olfactory bulb [ 29,301. All this evidence points to the existence of neural connections from the hypothalamus to the olfactory system. On the other hand, it is well known that electrical stimulation of the hypothal~us triggers behavioral patterns such as panting, sniffing and affective defense reactions, during which changes in respiratory activity are the rule [ 181. The electrical activity of the olfactory bulb in the hedgehog was reported by Adrian [ 1] to be influenced by the air flow through the nostrils and, in rabbits, changes of the pattern of electrical activity in the olfactory bulb take place upon awakening from anesthesia and can sometimes be induced by a single inspiratory movement [2]. A definite correlation of respiratory rate and frequency of induced electrical activity in the olfactory bulb of the cat was reported for various behavioral states
METHOD Animals and Procedure
The experiments were conducted on 127 adult cats of both sexes, weighing 2.5-3.0 kg. Two types of preparations were used. Acute preparations. In 58 cats, under ether and chloralose (70 mg/kg) anesthesia bipolar or tetrapolar stainless steel electrodes (20-30 Kfi resistance) with 1 mm vertical separation of their tips were implanted in various parts of the olfactory pathway: olfactory bulb (OB), prepyriform cortex CPPCf, olfactory tubercle (OT) and lateral olfactory tract (LOT), as well as the anterior commissure (AC) and neocortex (rostra1 marginal gyrus, NC). Stimulating electrodes of the same type as those used for recording were 263
264
AGUILAR-BATURONI
placed in the posterior hypothalamus (PH) within the area (A = 10.0, L = 1 .O, H = -4.5) known since Hess’ [ 18 ] studies to be related to aggressive reactions, and in the medial forebrain bundle (MFB), following the atlas by Jasper and Ajmone-Marsan [20]. Once the surgical procedure was completed, the animals were immobilized with Flaxedil (10 mg/kg) and local anesthesia was applied henceforth at 30 min intervals to the external auditory meatus, at the contact points with the stereotaxic apparatus, and to the surgical incision. Potentials were evoked by 0.5-1.5 V, 0.25 msec pulses. An AC-coupled preamplifier with a time constant of 0.12-0.16 msec was used in conjunction with a dual-beam oscilloscope (Tektronix 502) from which the responses were photographed with a Grass, C-4 kymograph camera. In 37 animals, platinum bipolar electrodes, covered with polythene except at their tips [ 131 were placed on the cervical sympathetic trunk (CS). Chronic preparations. In 32 cats, under pentobarbital anesthesia, electrodes were chronically implanted in the same regions as those for acute experiments. Recording was begun 3 days after surgery. Stimulation was most effective as trains of 0.5.-3.0 mA, 1.0 msec pulses, at 40 Hz for 5 sec. In 7 animals, electrodes of the same type as those used in acute preparations were chronically implanted around the CS. Electrical stimulation of the CS was started 8 days after electrode implantation. Activity from the olfactory areas was recorded in these preparations by a pen recorder (Grass Model 78 polygraph), EKG and respiratory move-
ments were recorded through metal pins inserted under the thoracic skin and led after preamplification to the pen recorder. Puffs of odorized air or drops of chemicals were delivered to the olfactory mucosa by means of a polythene
?OB
r.-.---_
k-T .,II,.
cannula, introduced to the nasal cavity through the frontal sinus. In both types of preparation, once the experimentation was completed, the animal was sacrificed under deep anesthesia and the position of electrodes was ascertained according to the technique of Guzmdn-Flores et al. [ 171. In
some of the chronic preparations in order to analyse the behavioral responses to hypothalamic stimulation, photographs of the cats were taken, RESULTS kjfects of Electrical Hype thalamus on the Structures
Stimulation Activity of
of the Primary
Posterior Olfactory
In cats with electrodes chronically implanted in posterior hypothalamus, the parameters of stimulation were adjusted to values such as to produce attentive behavior, as can be seen in Fig. 1. The response to stimulus trains (5 set, at 0.5 mA, 1 ms pulses at 40 Hz) was a pronounced mydriasis and contraction of ear and neck muscles. There were no changes in heart rate or in respiratory movements. The only modification apparent (Fig. 1) is an induced bursting activity in the homolateral olfactory bulb, quite similar in spindling described by Lavin et al. [22] as the arousal reaction in the olfactory bulb. The intra-burst rate was 40-45 Hz, and persisted within that range throughout the entire response. It was independent of the intensity, duration or rate of stimulation. This pattern of activity lasted for 3-5 min after a single 5 set period of stimulation. The intra-burst intervals (IBI) gradually lengthened from an initial value of 1 set during the first minute after the stimulation, to about 2 set ai the end of the
I
FIG. 1. Simulataneous recording of electrical activity in right (ROB) and left olfactory bulb (LOB). In upper traces the recording electrode was placed on the surface of the olfactory bulb; in lower traces, 6.0 mm within it. Electrocorticogram (ECG) from the marginal gyms. Pneumogram (PnG), electrocardiogram (EKG). At left, record from quiet, relaxed animal, as illustrated in the upper photograph. The traces at right begin within a few 100 msec after cessation of stimulation of the posterior hypothalamus at the locus indicated by the tip of the bar shown in the diagram at center (A, 10.0; L, 1.0; H, -4.5). Note bilateral mydriasis induced by stimulation (lower photograph). With the type of stimuli applied to hypothalamus (0.5 mA, 1.0 msec, 40 HZ for 5 set) only the ipsilateral OB shows the typical bursting response, without changes in respiratory or heart rates. Cal, ROB 100 ILV, LOB 500 CCV, FrfZ lflflIIV FKC All uV Time: 1 sec.
HYPOTHALAMIC
INFLUENCES
ON OLFACTORY
response. When the intensity of stimulation was gradually increased, the following responses were successively detected: (a) spread of bursting response to the contralateral bulb, (b) generalization to the cortex, (c) at the highest intensities (3.5 mA), the various behavioral components of the defense reaction. The effects shown in Fig. 1 for the olfactory bulb were also found in other areas of the olfactory pathway (i.e. LOT, PPC and OT) and it was possible to evoke them by stimulation of the MFB. Figure 2 shows the effect of left MFB stimulation on various olfactory structures. In this experiment, the rate and duration of stimuli, and the length of the stimulus train were the same as in Fig. 1, but the intensity was greater (1.5 mA) in order to illustrate the propagation of activity to both sides. As can be seen, both the prepyriform cortices and lateral olfactory tracts change their pattern of activity. The spindle bursts in PPC and LOT, however, differ in frequency (30-39 Hz) from those in OB. In this experiment, the neocortex also displays bursting activity after stimulation. No behavioral changes were observed aside from those described above as attentive behavior. An analysis of the temporal distribution of the spindles revealed that those in OB usually preceded by 100-200 msec those in PPC. Another primary olfactory structure where a similar change in activity can be induced by hypothalamic stimulation is the olfactory tubercle (OT). The lack of respiratory change following stimuli which modify the electrical activity of the olfactory pathway strongly suggests that these effects are central in origin, the influence of peripheral changes was further explored, however, in the tracheotomized animal, thus eliminating the effects of ventilation. The trachea was sectioned, the oral stump was tied and cannula was inserted into the bronchial end. Figure 3A illustrates the characteristic response to stimulation of the left posterior hypothalamus. At 24 hr after tracheotomy (Fig. 3B) no changes can be evoked, and it is only after 72 hr (Fig. 3C) that spindles can be induced. At 120 hr, the full pattern is elicitable, with characteristics entirely similar to those of controls (Fig. 3D). These experiments point to the existence of direct neural influences from hypothalamus to the olfactory structures, and the rest of this paper deals with possible channels for these influences. The Pathway from factory Structures
the Posterior
Hypothalamus
265
PATHWAY
to Ol-
In 58 acute preparations electrodes were placed in PH and the following structures: (a) prepyrifo~ cortices; (b) olfactory tubercles; (c) lateral olfactory tracts; (d) olfactory bulbs. Stimulation of PH with single shocks evoked potentials bilaterally at the 4 levels of the olfactory pathways. Several deflections could be detected in the evoked response at each olfactory station, and consistent differences in the waveform of the evoked potential were recognized. The waveform at any given station was nevertheless consistent from one preparation to another, and specific for that particular position. The notation followed in Table 1 to describe the latencies to the peak of the various deflections takes into account only the order to appearance, although as shown below, their origins can be quite different, e.g., N, in the response in contralateral PPC is not due to a direct hypothetic projection, and therefore its latency is comparable to that of N, at other
LPPC LLOT
RPPC
RLOT
A
ECG
ENG
PnG
LPPC
LLOT
RPPC
RLOT
B ECQ
EKG
PttG
FIG. 2. Bursting response induced in various olfactory structures by stimulation of left medial forebrain bundle. (A) control, (B) after stimulation. RPPC, Right Prepyriform Cortex, LPPC, Left Prepyriform Cortex, RLOT, Right Lateral Olfactory Tract, LLOT, Left Lateral Olfactory Tract, ECG, Electrocorticogram, EKG, Electrocardiogram, Pn G, Pneumogram. The more intense stimulation (1.5 mA, 40 Hz, 5 set) results in a bilateral bursting response. Calibration: 100 pV, 1 sec.
olfactory stations. The number of components listed in the table is the maximum recorded, although at some levels one or more components could be missing. No attempt is made in this paper to analyze the source of each component. The potentials evoked in OB by stimulation of PH showed 3 components (see Fig. 4): an initial negative wave with a latency of about 2.1 msec (N, ); a positive wave at 4.9 ms (P, ), second negative wave (N, ) at 12.0 msec and a slow negative deflection with a latency of 54.8 msec (N,). The potentials evoked in PPC were very similar, showing the same 3 components and only 2 in OT (see Table 1). The olfactory tubercle as a relay station for efferent fibers to the olfactory bulb. Since OT is a major relay
station for the fibers running from the bulbar reticular fo~ation to the olfactory pathway, the effects of blocking the activity of OT by topical application of procaine were studied. In the experiment illustrated in Fig. 4, 4S-~1 of 10% procaine, divided in 3 injections of 15 ~1 were applied to OT (at A = 18.5, L = 7.0, 5.0 and 3.0, H = -2.0, - 3.0 and -4.0). The slow components of the evoked potentials disappear, while N, remains. Yet, when procaine is applied at more medial coordinates (L = 1.0-2.0) even the fast components could be abolished. The potentials at PPC are
266
AGUILAR-BATURONI
A08
Figure 5 summarizes the connections which we surmise must exist between PH and the olfactory structures. The organization of this pathway is presently under investigation.
LOB A ROE
Sympathetic Path way
LOB
ROE LOB
B ROB LOB
ROB
C
LOB ROB LOB
ROE LO8
D ROB
LOB
6‘T A 1..
I
FIG. 3. Effect of tracheotomy on bursting responses in right (ROB) and left olfactory bulb (LOB) to hypothalamic stimulation. From A to D, upper 2 traces show activity before hypothalamic stimulation, and lower 2 traces are recordings taken immediately after stimulation with parameters specified in Fig. 1. (A) Control, (B) lack of response 24 hr after tracheotomy. (C) 72 hr after operation; only a very slight response is evoked. At 120 hr (D) the response is recovered on left. Calibration: 100 nV, 1 sec.
modified by procaine in OT regardless of the site of application, while N, persists, and the slow components are abolished (see Fig. 4). The efferent fibers to the olfactory bulb run in the lateral olfactory tract. Section of LOT results in a total
disappearance of the potential evoked in OB by hypothalamic stimulation, while those in PPC, OT and the proximal stump of LOT remain unchanged. Section of AC, on the other hand, does not affect any of the components in the homolateral bulb, but the response in the contralateral bulb is partially suppressed. The conduction velocity of efferent fibers in LOT was determined in a manner similar to that used to measure that for fibers from the bulbar reticular formation @RF). A stimulating electrode was placed at the usual coordinates in PH, and a recording electrode was positioned at various distances along LOT; the velocity, as calculated from the measure latencies, is 5-10 m/set, i.e. the same range as found for fibers from BRF [ 161.
Influences
on the Activity of the OlfhctorJ
In chronic preparations electrodes were implanted unilaterally on the cervical sympathetic trunk, and in various olfactory structures. In quiet, unrestrained animals 5 set trains of stimuli (1.0 mA, 1 msec, 15 Hz pulses) were applied to the CS. These parameters of stimulation induce bilateral mydriasis, without any other vegetative or behavioral changes being apparent, e.g., the cats frequently showed electrophysiological and behavioral evidence of sleep during this stimulation. In Fig. 6 the changes in the pattern of electrical activity in the olfactory bulb are shown. The typical response was a prolonged discharge (3-4 min) of spindles with characteristics entirely similar to those described above for hypothalamic stimulation. The electrocortical activity, however, is consistently desynchronized by the stimulation. This result suggests a possible sympathetic channel in the integration of the hypothalamic influence on the olfactory pathway. A series of experiments was consequently carried out in order to define what role the CS might play in this modulatory function. In 6 cats, electrodes were placed in the usual hypothalamic and olfactory areas, and the effects of hypothalamic stimulation were assessed. Both CS’s were then severed, and a 1 cm segment of the distal stump removed. In order to be certain that no regeneration had taken place in CS during the period of observation, the separation between the stumps was verified at necropsy. The animals were left to recover for 24 hr and the tests for hypothalamic effects on the activity of olfactory structures were resumed. The characteristics of the response are significantly affected (Fig. 7). The latency to the onset of the bursting response, the duration of response, and its intensity, measured as the rate of bursting (number of spindles/min) reveal a lessened responsiveness. It was consistently found that the latency to appearance of bursting was not greatly affected on the first day, but was greatly prolonged by the second and third days (Fig. 7). Why the prolongation of latency does not appear immediately is not understood. Even after 21 days the responsiveness has not yet attained full recovery (Fig. 7). The depression of the intensity of the bursting response and its total duration is even deeper and more prolonged. No changes in the response to hypothalamic stimulation were observed after sham operations, involving manipulations of the sympathetic trunk. Since no information is available about the projections of cervical sympathetic fibers into the olfactory bulb, some experiments were performed recording at various depths of OB while stimulating CS with single shocks. At the same level at which evoked potentials can be recorded after stimulation of PH, monophasic, bimodal potentials are evoked by CS stimulation with a latency of 6.1 + 0.2 ms. These potentials follow the stimulation rate one-to-one only up to 20 Hz. Higher rates result in loss of the response, yet such high rates resulted in post-tetanic potentiation of the evoked response in OB. In order to find whether an interaction of this pathway exists with the direct pathway from PH to the olfactory
HYPOTHALAMIC
267
INFLUENCESONOLFACTORYPATHWAY
I
2
3
FIG. 4.Potentials evoked in olfactory bulb (l), olfactory tubercle (2) and prepyriform cortex (3), by stimulation of homolateral (left) posterior hypothalamus. (A) before, (B) after local repetitive injection of 5 ~1 at different lateral deep to the total of 45 ~1 of 10% procaine in olfactory tubercle. Section at center shows area of placement of cannulae (A, 18.5; L, 7.0, 5.0 and 3.0; H, -2.0, -3.0 and -4.0). The graphs shows the effect upon the different components of the evoked potentials in the OB by administration of procaine. Note that the N, component is depressed only when the procaine is applied more medially (L 1.0). Calibration: 100 MV, 10 msec. Superimposition of 5 traces.
TABLE 1 LATENCIES (AVERAGE
AND S.E.) FOR VARIOUS
THALAMIC
NUCLEI TO DIFFERENT
COMPONENTS OF EVOKED RESPONSES LEVELS OF OLFACTORY PATHWAY
FROM HYPO-
=I 32.9*0.4
n.221
ECG
B
LOB
ECG
C
LOB LOB ECG
FIG. 5. Diagram of pathways postulated from posterior hypothalamus to olfactory structures (shaded areas), ventral view of brain. OB, olfactory bulb, OT, olfactory tubercle, PPC prepyriform cortex, PH posterior hypothalamus. Connections indicated by arrows.
bulb, different types of experiments were carried out. Repetitive stimulation of HP (1.5 mA, 0.1 ms and 50 Hz) was maintained during 1 min, while single test shocks were applied to CS (Fig. 8). The amplitudes of evoked potentials in OB are greatly reduced as compared with controls; the effect lasting 5 min. In another series of experiments, the recovery cycle of OB responses to CS stimuli was determined by application of pairs of shocks at varying intervals. When PH is stimulated, an occlusion is apparent between the two pathways, thus suggesting they share a common postsynaptic neuronal pool. Finally, another possible path for hypothalamic influences was explored. Since peripheral effects for sympathetic action on olfactory receptors have also been described [4,6], a cannula was inserted in the nasal cavity, in animals with electrodes chronically implanted in OB. Epinephrine was then topically applied to the nasal mucosa. This evoked trains of spindles (induced by nasal instillation of 0.5 ml of 10e6 g/l) in OB (Fig. 9). Again the characteristics of the bursting response were entirely similar to those resulting from PH or CS stimulation. Similar instillations of Ringer solution, or procaine failed to elicit spindles. DISCUSSION
Three influence
pathways olfactory
by which the hypothalamus might structures can be discerned from the
FIG. 6. Bursting response induced in right (ROB) and left olfactory bulb (LOB), by electrical stimulation of left cervical sympathetic trunk. Upper trace of each pair, from superficial electrode, lower trace, from electrode 6 mm deep in OB. (A) control, (B) response to sympathetic stimulation (horizontal bar 1.0 mA, 1.0 msec, 15 Hz, 5 set). (C) record continuous with that of B. ECG, electrocorticogram from marginal gyrus. Respiration (not shown) did not change. Calibration: ROB, 1 mV, LOB, 0.5 mV, ECG, 100 @V, I sec. above: (1) a direct path from posterior hypothalamus to both the olfactory cortex and tubercle, (2) a direct projection from the hypothalamus to the olfactory bulb, (3) by the sympathetic system via CS. We are presently endeavoring to determine if the hypothalamic projections are bilateral and, if so where decussation occurs. In a previous paper we described the projections of the bulbar reticular formation to the olfactory structures [ 161. The posterior hypothalamus receives fibers from the brain stem [ 101, and recent evidence suggests that the hypothalamo-olfactory pathway is in series with a reticulohypothalamic projection (Guevara-Aguilar, in preparation). Therefore, it is not surprising that the projections from the posterior hypothalamus to the olfactory areas show the same distribution as those from the brain-stem, and that the latencies, albeit shorter, bear the same relationships, and as does the conduction velocity of efferent axons in the lateral olfactory tract for the two systems. It seems likely, given the effects of MFB stimulation, that other hypothalamic areas are also connected to the olfactory pathway [21 I. The hypothalamus thus seems to be an integrative center for influences acting on the olfactory pathway: other regions connected with olfactory structures, such as the septum and amygdala, also send projections to the hypothalamus [3, 26, 301. The concept that the hypothalamus influences the olfactory system is not new. It was common in the past, on anatomical grounds, to consider the
HYPOTHALAMIC
269
INFLUENCES ONOLFACTORYPATHWAY
200
I50
f 5
2
x
200
100
8
8 8
f
50
DLIYS FIG. 7.Sympathectomy depresses bursting response to PH stimulation; mean and standard error for 6 cats. At left, latency for bursting response in olfactory bulb to ipsilateral (left) PH stimulation. Heavy arrows indicate time of sympathectomy. Latencies (average and S.E.) for successive days in samples from the 1st to the 21st. At right, duration (in set on left ordinates in light columns), and number of spindles (right ordinate, shaded columns) in responses to PH stimulation as modified by sympathetic transection. Abscissa, days after CS section.
FIG. 8. Influence of hypothalamic stimulation upon potentials evoked in OB by stimulation of cut right CS. Hypothalamic stimulation, 50 Hz, 1.5 mA, 0.1 msec pulses, applied for 1 min between 2 and 3 min on graph. Stimulation ofCS, 5 Hz: 1, control (at min); 2, 4 min (2 min after cessation of PH stimulation at arrow); 3, 5 min;4, 8 min. Calibration: 100 GV, 2 msec. Time course of the depression is shown on inset, taking amplitude of response before PH stimulation as 100%. Abscissa, time, min.
210
A LOB
ROB
LOB
FIG. 9. Effect on electrical activit of left (LOB) and right olfactory bulb (ROB) of topical application of 0.5 ml epinephrine hydrochloride (1 X lo- B gr/l) to olfactory mucosa in the alert cat. (A) activity before, (B) after instillation of epinephrine (signaled by arrow B) in the left nostril. Although the cat often sneezed or sniffed immediately upon application of the solution, the electrical effects endured long after such respiratory effects were apparent, and it can be seen that the electrical activity is initially altered only on the side homolateral to the site of application. Also note the decease in ROB during the initial, protracted burst in LOB. Calibration: 100 PV, 1 sec.
hypothalamus as an integral part of the olfactory pathway [23]. Although our results are restricted to the olfactory projections of the posterior hypothalamus and MFB, a more detailed analysis, exploring different hypothalamic regions, has shown that the olfactory areas recieve projections from at least two more hypothalamic areas in the lateral and anterior hypothalamus (Guevara-Aguilar, in preparation). The dual effect of hypothalamic stimulation on the spontaneous activity of the olfactory structures is well in line with the role commonly assigned to its modulatory action. On the one hand, in chronic preparations, the usual effect was the triggering of a hypersynchronous bursting pattern of activity, entirely similar to the arousal spindles described by Lavin et al. [22]. Unfortunately there is no solid ground to discuss the cellular basis of the various patterns of rhythmic activity recorded from the olfactory areas with gross electrodes [32], and therefore it would be premature to speculate about the role of this hypothalamic pathway on the transfer of information along the olfactory pathway. Clearly more detailed studies on this subject are necessary. The bilateral effects of unilateral stimulation of the CS in the intact animal require comment. It seems likely that the commissural connections between the olfactory bulbs could account for a bilateral effect, one bulb altering the activity of the other when it itself is excited via the CS. The bilateral mydriasis, however, must be explained as stimulation either of unknown afferents in the CS or more likely, recurrent collateral effects from antidromic stimulation. The possibility of the mydriasis being attributable to inadvertent stimulation of nonCS afferents seems to be excluded by the lack of any behavioral arousal consequent to such stimulation, and the fact that mydriasis was strictly unilateral following proximal section of CS. In other
respects stimulation of CS was comparable to that in other components of the autonomic nervous system in that the optimum rates of stimulation, although somewhat higher than the average, are within the range described for the fibers to various autonomic effecters [31]. Other evidence of sympathetic influence on olfaction is the fact that norepinephrine when applied iontophoretically [ 5,7] ; has been found to influence the activity of a number of neurons in the olfactory bulb. Monoaminergic nerve terminals have been identified in the olfactory bulb of the rabbit [ 121. Norepinephrine has also been demonstrated to be taken up at the level of periglomerular cells of the olfactory bulb of the mouse [ 24 ] and to be released in the olfactory bulb of rabbit [8 I. However, not all noradrenergic fibers in the olfactory bulb can be taken as sympathetic in origin. The central pathways from the hypothalamus to OB also contain noradrenergic elements. The medial forebrain bundle is a well known noradrenergic system [ 1 1,271, which from our results, sends projections to the olfactory pathway. It will certainly be necessary to have a differential mapping of the noradrenergic fibers ending on the OB in order to define their functional role. The indirect hypohtalamic influence through the sympathetic fibers reaching the nasal blood vessels and olfactory receptors is also known from Beidler’s description of an enhancement of electrical activity in the olfactory nerve fibers after stimulation of the sympathetic bundle in the ethmoidal nerve of the rabbit [6]. This was ascribed to a vascular effect, yet, in isolated preparations of the frog’s olfactory receptors, Ar6chiga and Alcocer [4] found an enhancement in amplitude of the electroolfactogram after topical application of epinephrine and norepinephrine to the olfactory mucosa. They suggested, therefore, a dual facilitatory effect, acting in parallel on olfactory receptors and parareceptor structures. There is an
HYPOTHALAMIC
INFLUENCES
ON OLFACTORY
211
PATHWAY
obvious similarity in this effect on electrical activity of OB described herein, when epineph~e was topically applied to the nasal mucosa, and the effect of hypothalamic or sympathetic stimulation. The fact that the response to hypothalamic stimulation is greatly modified after sympathectomy indicates a role for this peripheral pathway in the overall, normal pattern of hypothalamic influence on olfactory input. The existence of neural connections between posterior hypothalamus and the olfactory structures does not exclude the possibility of influences from other sources. The transient suppression of the bursting response to PH stimulation immediately after tracheotomy confirms the influence of respiration on this pattern of activity, as postulated by Gault and Leaton and by Gault and Coustan [ 14,15 1. On the other hand, the sensitivity of olfactory receptors to epinephrine, the induction of the typical
bursting pattern of bulbar electrical activity by this substance, suggests that a humoral channel could be activated via sympatho-adren~ mechanisms. This, since the early studies of Cannon [ 9 ] and his associates, is know to occur with the defense reaction. The posterior hypothalamus has also been related to the cycles of sleep and wakefulness (see [ 341). Hypothalamic projections to the cerebral cortex have only recently been described [ 331. The possibility has been raised in another context that the hypothalamus influences sensory input [25]. Thus, the centrifugal pathways described in this paper may well play an important role in such modulation of olfactory input. ACKNOWLEDGEMENT
The authors are grateful to Professor Miguel R. Covian and F’mfessor H6ctor BrustCarmona for his suggestions for this paper.
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