Brain Research Bulletin, Vol. 3, pp. 461-474.
printed in the U.S.A.
Olfactory Pathway Evoked Potentials in Response to Hypothalamic Stimulation R. GUEVARA-AGUILAR Departamento
AND H. U. AGUILAR-BATURONI
de Fisiologia, Facultad de Medicina, Universidad National Autonoma de Mexico Apartado Postal 70250, Mexico, 20. D.F. (Received
10 January
1978)
GUEVARA-AGUILAR,
R. AND H. U. AGUILAR-BATURONI. Olfactory pathway evoked potentinls in response to BRAIN RES. BULL. 3(S) 467-474, 1978.-Ipsilateral and contralateral stimulation of lateral, ventromedial and posterior hypothalamic nuclei produced evoked responses in the olfactory bulb and in the prepyriform cortex. No differences in the latencies were found by stimulation of each nucleus in the homo and contralateral olfactory structures. The high amplitude of the fast component (Nl) was obtained with stimuli applied to the ventral zones and the slow components (N2,N3) were obtained with more dorsal stimulation. An ipsilatetal pathway is indicated at the supramammillary and posterior commissure level, since severing these structures abo!ishes the evoked responses. A bilateral projection is proposed for the olfactory bulb.
hypofhukunic
stimulation.
Evoked potentials
Olfactory system
Centrifugal fibers
EFFERENT fibers to the olfactory system have been extensively investigated in recent years. Such fibers originate
from within [2, 10, 11, 14,23,24,25,28] as well as outside [3, 9, 211 the rhinencephalon. Following lesions of the olfactory pathways degenerating fibers have been traced to the homolateral olfactory bulb. Lesions damaging the anterior commissure result in degeneration that is found in both bulbs [26]. The distribution of commissural fibers are believed to connect both anterior olfactory nuclei with each other but do not extend to the olfactory bulb [ 19,20,3 11. The existence of ascending fibers in the medial forebrain bundle has been demonstrated [ 13,221. The distribution of this bundle in the rabbit has been shown to project to the rostra1 aspect of the olfactory tubercle [33]. Some of these fibers run through the precommissural and posterior region of the anterior olfactory nuclei. The crossing over of fibers of the dorsomedial part of the medial forebrain bundle to the opposite dorsal longitudinal fasciculus and medial forebrain bundle has also been described [5]. Histochemical fluorescence studies [ 11,301 of the ascending catecholaminergic fibers in the dorsolateral aspect of the medial forebrain bundle reveal that some fibers of this system cross the midline in the commissura supraoptic dorsalis and a large number of fibers run to the olfactory tubercle through the lateral olfactory tract and to the diagonal band of Broca and the homolateral septal nuclei. The response evoked bilaterally in olfactory structures by stimulation of the posterior hypothalamic nucleus and medial forebrain bundle [l] indicate functional direct connections between these structures. The purpose of the present study is to determine possible projections of the ventromedial and lateral areas of the hypothalamus to the olfactory bulb and prepyriform cortex including the decussations of the pathways involved in this system.
Copyright 0 1978 ANKHO International
Olfactory bulb
Prepyriform
cortex
METHOD
Experiments were conducted on 35 adult cats of both sexes, weighing 2.5 to 3.5 kg. Anesthesia was ether and chloralose, 70 mg/kg. Electrical activity of homolateral and contralateral olfactory bulbs and prepyriform cortex (respectively, HOB, COB, HPPC, CPPC) was recorded through stainless steel monopolar and bipolar electrodes implanted stereotactically, following the atlas by Snider and Niemer [29]. Stimulation stereotaxic coordinates were F:7.5 to 13.5, L:O.5 to 4.0 and H:O.O to -7.0. In each preparation, one to three recording electrodes were placed at different locations in order to map the field of evoked potentials in both bulbs and prepyriform cortices. Stimulation consisted of l-5 mA, 0.1-0.5 msec single shocks from a Grass S-88 stimulator through a stimulus isolation unit. Thresholds were measured in each preparation and for each recorded response. In a group of 20 cats, electrolytic lesions (2-10 mA DC current for 30 set) or knife cuts (using a stereotactically calibrated knife) were placed in corpus callosum, anterior commissure (F: 14.5, L:O.O, H: + 1 to -2.O), supramammillar commissure (F8.0, L:O.O, H: -3 to -5.0) and medial forebrain bundle (F:13.5 to 12.0, L:3.0 to 4.5, H: -2.0 to -5.0). Electrical activity was recorded from the olfactory bulbs and prepyriform cortex during hypothalamic stimulation before and after lesions were made. The position of the electrode tips used for stimulation and recording was marked by passing a direct 50 PA current for 10 sec. At the end of the experiment, animals were perfused with 10% Formalin containing potassium ferrocyanide. The location of electrode tips and extent of the lesions were determined by examining 20 p thick frozen sections or projecting unstained sections, mounted on slides, on photographic paper.
Inc.-0361-9230/78/050467-08$01.30/O
GUEVARA-AGUILAR
468
AND AGUILAR-BATURONI
Fr =;
Fr =8!
A
E
8
F
C
G
D
H
5
Fr =I00
FIG. 1. Drawings of three different frontal planes from where potentials where evoked in the OB homo and contralateral. The amplitude of each bar represents the amplitude of the first component (Nl) recorded to different depths. The largest response was taken as 100% for each preparation. Bottom left shows the evoked potentials in homolateral olfactory bulb by posterior hypothalamic nuclei stimulation.
RESULTS Potentials
Recorded
from the Olfactory
Pathway
Previously it was demonstrated [I] that single shocks in the posterior hypothalamic nuclei elicited bilaterally in the olfactory bulbs and prepyriform cortex, biphasic potentials with negative components with latencies: 2.1 * 0.3 msec (Nl), 12.0 ? 2.0 msec (N2) and 54.8 2 6.1 msec (N3) for the homolateral response, and 2.7 + 0.05 msec (Nl), 11.0 + 1.3 msec (N2), and 60.2 t 6.1 msec (N3) for the contralateral bulb response. In order to find out if stimulation of other nuclei could evoke these responses, stimulation electrodes were placed in various ventromedial and lateral hypothalamic areas as follows: F:7.5 to 13.5, L:O.S to 4.0 and H:O.O to -7.1. Figure 1 shows the sites of stimulation which evoked the first Nl component in the homo and contralateral olfactory bulbs, in three different frontal planes. The amplitude of the first component has been represented by the
FIG. 2. Evoked potentials from the HOB. recorded at various depths in steps of 500/*. elicited by VMH stimulation. Superposition of three sweeps. Coordinates F:9.0, L:1.5, H: A -2.0, B: -2.5, C: -3.0, D: -3.5, E: -4.0, F: -4.5, G: -5.0, H: -5.5. Observe that the first component began to appear at -3.0 of depth (C). Calibrations: 100 fiV; 10 msec.
height of each bar. The largest evoked response was taken as 100% for each preparation. Only responses which were obtained 80% of the time were retained. The largest amplitude obtained with stimulation of the lateral hypothalamic region (LH) was below the medial forebrain bundle (MFB), posterior hypothalamic nuclei, and the region of the medial and lateral mammillary bodies. Figure 2 shows the responses elicited at the homolateral olfactory bulb when the ventromedial hypothalamic nuclei (VMH) is stimulated. The largest amplitude corresponds to ventral regions of the VMH in the case of the short latency component (Nl) and to the dorsal regions in the case of the longer latency (N2) or (N3) components. No significant other differences were found in latencies between the different components in the olfactory bulbs when posterior, ventromedial, or lateral hypothalamic nuclei were stimulated. Figure 3 shows the response evoked in both olfactory bulbs by stimulation of the posterior hypothalamic nucleus. As the stimulation electrode was driven more ventrally, while still recording, the amphtude of the first component was larger for the basal regions (Fig. 3
EVOKED
POTENTIALS
IN OLFACTORY
469
SYSTEM
FIG. 3. Evoked potentials, from HOB upper traces and COB lower traces, recorded at various depths in steps of 1000 p elicited by PH stimulation. Superposition of three sweeps. Coordinates F: 8.5, L: 1.0, H: A -1.0, B: -2.0, C: -3.0, D: -4.0, E: -5.0, F: -6.0. Calibrations: 100 pV; 10 msec.
C-F), either at homolateral or contralateral olfactory bulb, while in the case of the slow component (N3) the largest amplitude was obtained from the dorsal regions (Fig. 3A). Intravenous administration of Nembutal (10 mgikg) attenuated the slow components (N2) and (N3); whereas, it did not effect the fast component (Nl). At higher doses, all components were attenuated for both ventromedial, lateral or posterior hypothalamic nuclei stimulation. Pathways
to Contrulaterul
Olfactory Structures
In order to examine the fiber systems which project to the contralateral olfactory bulb and prepyriform cortex, partial sections of different structures were performed in some animals. Figure 4 shows potentials evoked at homo and contralateral olfactory bulbs by stimulation of posterior (A,B,C) and lateral (D,E,F) hypothalamic nuclei. Tracings A and D represent responses prior to any sections while stimulating the posterior and lateral hypothalamus, respectively. In tracing B and E, the posterior commissure and the part of the corpus callosum situated above it were cut in an anteroposterior plane. Both the duration and amplitude of the slow component changed (mainly at the contralateral bulb), and the fast component (both homo and contralateral) decreased in amplitude. In tracing C and F, the supramammillary
commissure and the corresponding part of corpus cahosum were cut: the first component increased in amplitude (mainly the PHN one), and the amplitude of all slow components decreased. Figure 5 depicts the effects observed at the PPC and OB following stimulation of the posterior hypothalamus. Tracing A represents a control recording of potentials elicited at homo (upper tracing) and contralateral (lower tracing) olfactory bulbs. Tracing B represents a control recording of potentials elicited at the PPC. Tracing C is a transcortical recording obtained by stimulating one side of the PPC and recording at the other. In tracing D and E, the posterior commissure and supramammillar commissure, together with the neighboring regions of the corpus callosum, were severed. Tracing D, where the response to stimulation was recorded in the olfactory bulbs, shows increased amplitude fast components, and smaller slow N3 components, both homo and contralaterally. Tracing E, where the response to stimulation was recorded in the prepyriform cortex, illustrates that the homolateral N3 lasts longer but the contralatera1 N3 has disappeared. The flat tracing at F confirms that the transcortical fibers were cut, stimulating one prepytiform cortex area had no effect in the other. In another group of experiments only the corpus callosum was cut. Under these conditions, there were no significant changes of the amplitude or duration of homo and contralat-
470
GUEVARA-AGUlLAR
AND AGUILAR-BATURON~
FIG. 4. Evoked potentials, from HOB upper traces and COB lower traces, in response to stimulation of PH (A,B,C) anf LH (D,E,F). A and D show control responses B and E are responses after the posterior commissure and part of the corpus callosum were cut (F: 4.0 to 6.0, L:O.O, H: +2.0). C and F after the supramammillar~ commissure was cut (F5.0 to X.5, L:O.O. H: -6.0). Superposition of 3 sweeps. Calibrations: 100 /*V; IO msec.
era1 evoked potentials in the olfactory bulb or prepyriform cortex produced by hypothalamic stimulation. When the anterior commissure was severed in addition to the posterior and supramammillary commissure, no further change occurred in the shape or amplitude of potentials evoked at the olfactory bulb and prepy~form cortex. When the anterior commissure was severed first, the amplitude of the first component increased just as it did after section of the supramammiUary commissure, but the slow component did not change. In another five animals the medial forebrain bundle was severed by either knife cuts [27] or by electrolytic lesions. Figure 6 shows the changes induced in the components evoked in the homoiateral olfactory bulb following stimulation of the posterior hypothalamic nucleus of a cat in which the homoIater~ medial forebrain bundle has been severed by a knife cut. Lesion is indicated in the upper right part of the figure. A is a control tracing; B was recorded immediately after the section; both fast and slow components are smaller. At C, 1 hr later, Nl has increased, but N3 tends to flatten out after the fourth successive stimuli. No such effect was observed at A. The lower part of Fig. 7 presents the modification of Nl immediately after the section, using a faster sweep. When electrolytic lesions (Z-10 mA DC for 30 see) were placed rostraily in the medial forebrain bundle, as illustrated in the three slices from the same cat by arrows in left part of Fig. 7, the slow and fast
components
in homolateral
and
contralateral olfactory bulbs became smaller. These effects are illustrated in part A and B of Fig. 7. In part A the normal response is depicted. In part B the smaller response following the electrolytic lesion of the medial forebrain bundle is illustrated. However, evoked potentiais did disappear at the homoiateral prepyriform cortex. These effects are also iliustrated in parts C and D of Fig. 7. In part C the normal PPC response is illustrated and in part B no homolateral response is observed while no change was observed at the contralatera1 prepyriform cortex. DISCUSSION
These results confirm the existence of centrifugal fibers to the olfactory system from the posterior, ventromedial, and lateral hypothalamic regions. StimuIation of any of these nuclei failed to show any differences in latency of the different components in the evoked potentials. Apparently, the fibers from each hypothalamic nucleus have separate and inindependent projections to the olfactory system. However, such an interpretation might be misleading due to the limitations of the present technique, in which different size fibers were stimulated simultaneously and small difference in latencies are difficult to evaluate. We are presently conducting single cell recordings to delineate this problem. When the stimulating electrode was placed more ventrally in the hypothalamus amplitude changes in the different components of the evoked potentials were observed. Large
EVOKED
POTENTIALS
IN OLFACTORY
SYSTEM
f--i FIG. 5. Evoked potentials in OB and PPC (A and B, respectively), homolateral upper traces and contralateral lower traces, by stimulation of PH. C is a transcortical record, D and E are responses after the posterior commissure and supramammillary commissure were cut, F shows that transcortical fibers were cut. Supposition of four sweeps. Calibrations: 100 pV, 10 msec.
amplitude stimulation
long latency in the dorsal
components
were
obtained
with
regions. Shorter latencies were obtained with more ventral stimulation. This observation is compatible with a double projection system to the olfactory bulb. The aminergic ascending fibers are arranged in both dorsal and ventral bundles which terminate in the diencephalon [30]. These two systems might remain distinct until they reach more rostra1 structures; for example, the olfactory tubercle where different fibers from the bulboreticular regions to the olfactory bulb also terminate 1121. Procaine administered to more lateral zones of the tubercle modifies the slow component of the potential evoked by stimulation of the posterior hypothalamic nucleus. Only when more medial regions are anesthetized by procaine can a change in the short latency component be seen [I] which supports a dual system interpretation. Worseradish peroxidase accumulates in the lateral hypothalamus following its local application in the olfactory bulb which indicates the existence of centrifugal fibers from the hypothalamus to the olfactory bulb. Peroxidase was found in the contralateral locus coeruleus and in the raphe nuclei but it was not found in the contralateral hypothalamus 161.
From these results, it can be concluded that the hypothalamic projections to the prepyriform cortex differs from the one to the olfactory bulb; one is only ipsilateral and the other is bilateral. Since severing the supramammillary and posterior commissures abolishes the evoked potential at the contralateral prepyriform cortex, the decussation of this bilateral projection might be found at this level. Olfactory bulb fibers could decussate more posteriorly; for instance in the pons, locus coeruleus, or raphe nuclei [61 and stimulation in these areas also might result in evoked responses in olfactory structures. A great deal of experimental data [4,15,16,17,18, 321 suggest the presence of ascending fibers in the medial forebrain bundle. These fibers originate in the tectum, raphe nuclei, and locus coeruleus and ascend to the hypoth~amus with some reaching the olfactory tubercle. Lesions were replaced in the locus coeruleus on one side of the brain and tyrosine hydroxylase activity was measured in the contralateral nuclei. Changes in the activity were found mainly on the fourth day after the lesion [7] which might indicate a functional interaction between the two nuclei. Since the present study indicates that lesions which transect the medial forebrain bundle modify evoked potentials at ipsilateral pre-
472
GUEVARA-AGUILAR
AND AGUILAR-BATURONI
FIG. 6. Evoked potentials from HOB elicited by stimulation of PH. A shows the response before MFB has been severed. B was recorded immediately after and C 1 hr later. D represents the response of only the short latency component, using a faster sweep before the MFB damage. E immediately after. Upper left shows the position of the stimulating electrode (F:8.5, L:l.O, H: -3.5). Upper right shows the damage in the MFB (F: 13.5, L:3.0, H: -3.5). Superposition of four sweeps. Calibrations: 100 pV, 20 msec (A,B,C) and 5 msec (D and E).
pyriform cortex and olfactory bulb, the medial forebrain bundle as well as some fibers which cross over to the contrilateral nuclei are probably part of the projecting pathway to the olfactory system. This would explain the changes observed at the contralateral olfactory bulb.
ACKNOWLEDGEMENTS
The authors are grateful to Professor M. J. Wayner, F. C. Barone and C. C. Loullis for their help and suggestions in preparing this report. The technical assistance of Mr. Carlos de 10s Santos is acknowledged.
473
EVOKED POTENTIALS IN OLFACTORY SYSTEM
FIG. 7. Evoked potentials from the OB and PPC (A and C are responses from OB and PPC, respectively). Homolateral responses upper traces and contralateral lower traces by stimulation of PH before electrolytic lesions (5 PA DC for 30 set) to the MFB. B and D after lesion. Left shows the extent of the lesion in the MFB. Coordinates F:14.5, L:3.5, and H: 4.5. Calibrations: 100 pV; 10 msec.
REFERENCES 1. Aguilar-Baturoni, H. U., R. Guevara-Aguilar, H. Arechiga and C. Alcocer-Cuaron. Hypothalamic influences on the electrical activity of the olfactory pathway. Bruin Res. Bull. 1: 263-272, 1976. 2. Allison, A. A. The structure of the olfactory bulb and its relationship to the olfactory pathway in the rabbit and the rat. J. camp. Neural. 98: 309-353, 1953. 3. Arduini, M. and M. Moruzzi. Sensory and thalamic synchronization in the olfactory bulb. Electroenceph. din. Neumphysiol. 5: 235-242,
Ban, T. tonomic 5. Bodian, 11. The
1953.
Fiber connections in the hypothalamus and some aufunctions. Pharmuc. Biochem. Behuv. 3: 3-13, 1975. D. Studies on the diencephalon of the Virginia opossum fiber connections in normal and experimental material. J. camp. Neurcjl. 72: 207-298, 1940. 6. Broadwell, R. D. and D. M. Jacobowitz. Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retinobulbar formation. J. camp. Neural. 170: 321-346, 1976. 7. Buda, M., B. Roussel, B. Renaud and J. F. Pujol. Increase in tyrosine hydroxylase activity in the locus coeruleus of the rat brain after contralateral lesioning. Bruin Res. 93: 564-569, 1975.
4.
8. Carreras, M., D. Mancia and M. Mancia. Centrifugal control of the olfactory bulb as revealed by induced DC potential changes. Bruin Res. 6: 548-560,
1%7.
Cowan, W. M., R. W. Guillery and T. P. S. Powell. The origin of mammillary peduncle and other hypothalamic connections from the midbrain. J. Amt. 98: 345-363, 1964. 10. Cragg, B. G. Centrifugal fibers to the retina and olfactory bulb and composition of the supra-optic commissure in the rabbit. Expl Neural. 5: 406-427, 1962. 11. Dahlstrom, A., D. Fuxe, L. Olson and U. Ungerstedt. Ascending system of catecholamine neurons from the lower brain system. Actu physiol. scund. 62: 485-486, 1964. 12. Guevara-Aguilar. R., H. U. Aguilar-Baturoni, H. Arechiga and C. Alcocer-Cuaron. Efferent evoked response in the olfactory pathway of the cat. Electroenceph. c/in. Neuroph.vsiol. 34z 9.
23-32, 13. 14.
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Guillery, R. W. Degeneration in the hypothalamic connections of the albino rat. J. Ancrt. 91: 91-115, 1957. Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system of the rat. An experimental anatomical study. J. Amt. 103: 413-439, 1968.
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15. Kataoka, K., M. Sorimachi, S. Okuno and M. Mizuno. Enzymatic evidence for a mesolimbic dopaminergic innervation in the olfactory tubercle of the rabbit. Bruin RPS. 88: 513-517, 1975. 16. Kataoka, K., M. Sorimachi and M. Mizuno. Innervation of hypothalamic and limbic areas by the cholinergic, Gaba-ergic and catecholaminergic nerve fibers, a quantitative analysis. Pharmac. Biochem.Behav. 3 Suppl. 1: 61-73, 1975. 17. Levin. B. E.. C. H. Sadowskv and J. M. Stolk. Axoplasmic transport of norepinephrine in the rat brain after locus coeruleus lesions. Brain Res. 106: 198-203, 1976. 18. Lindvall, 0. and A. Bjorkland. The organization of the ascending catecholamine neuron system in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta physiol. stand. Suppl. 412: l-48, 1974. 19. Lohman, A. H. M. and H. J. Lammers. On the connections of the olfactory bulb and the anterior olfactory nucleus in some mammals. Bruin Res. 3: 149-162, 1%3. 20. Lohman, A. H. M. and G. M. Mentink. The lateral olfactory tracts, the anterior commissure and the cells of the olfactory bulb. Brain Res. 12: 396413, 1%9. 21. Mancia, M. Specific and unspecific influences upon the olfactory bulb. Electroenceph. din. Neurophysiol. 14: 42U25, 1%2. 22. Nauta, W. J. H. Hypothalamic regulation of sleep in rats. An experimental study. J. Neurophysiol. 9: 285-316, 1963. 23. Powell, T. P. S. and W. M. Cowan. Centrifugal fibers in the lateral olfactory tract. Nature 199: 12961297, 1963.
GUEVARA-AGUILAR
AND AGUILAR-BATURONI
24. Powell, T. P. S., W. M. Cowan and G. Raisman. The central olfactory connections. J. Anat. 99: 791-813, 1965. 25. Price, J. L. and T. P. S. Powell. An exnerimental studv of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat. 1. Anat. 107: 215-237, 1970. 26. Ramon y Cajal, S. Textura del sistema nervioso central del hombre y 10s vertebrados. Madrid, Ed. Movtr. 1904. 27. Sclafani, A. and S. P. Grossman. Hyperphagia produced by knife cuts between the medial and lateral hypothalamus in the rat. Physiol. Behav. 4: 533-537, 1969. 28. Shepherd, G. M. Synaptic organization of the mammalian olfactory bulb. Physiol. Rev. 52: 864-916, 1972. 29. Snider, R. S. and W. T. Niemer. A Stereotuxic Atltrs oJ’the Cclt Brtrin. Chicago, IL: The University of Chicago Press, 1961. 30. Ungerstedt, U. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta ph.vsio/. .vccmd. Suppl. 367: 148. 1971. 31. Valverde, F. Sfudir.\ 011the Pw(fiwm Lo/w. Cambridge. MA: Harvard University Press, 1965. 32. Worth, W. S., J. Collins, D. Kett and J. H. Austin. Serial changes in norepinephrine and dopamine in rat brain after locus coeruleus lesions. Brain Rcs. 106: 198-203, 1976. 33. Zyo, K., T. Oki and T. Ban. Experimental studies on the medial forebrain bundle, medial longitudinal fasciculus and supraoptic decussations in the rabbit. Med. J. U.wXa 13: 197-239. 1963.
printed in the U.S.A.
Olfactory Pathway Evoked Potentials in Response to Hypothalamic Stimulation R. GUEVARA-AGUILAR Departamento
AND H. U. AGUILAR-BATURONI
de Fisiologia, Facultad de Medicina, Universidad National Autonoma de Mexico Apartado Postal 70250, Mexico, 20. D.F. (Received
10 January
1978)
GUEVARA-AGUILAR,
R. AND H. U. AGUILAR-BATURONI. Olfactory pathway evoked potentinls in response to BRAIN RES. BULL. 3(S) 467-474, 1978.-Ipsilateral and contralateral stimulation of lateral, ventromedial and posterior hypothalamic nuclei produced evoked responses in the olfactory bulb and in the prepyriform cortex. No differences in the latencies were found by stimulation of each nucleus in the homo and contralateral olfactory structures. The high amplitude of the fast component (Nl) was obtained with stimuli applied to the ventral zones and the slow components (N2,N3) were obtained with more dorsal stimulation. An ipsilatetal pathway is indicated at the supramammillary and posterior commissure level, since severing these structures abo!ishes the evoked responses. A bilateral projection is proposed for the olfactory bulb.
hypofhukunic
stimulation.
Evoked potentials
Olfactory system
Centrifugal fibers
EFFERENT fibers to the olfactory system have been extensively investigated in recent years. Such fibers originate
from within [2, 10, 11, 14,23,24,25,28] as well as outside [3, 9, 211 the rhinencephalon. Following lesions of the olfactory pathways degenerating fibers have been traced to the homolateral olfactory bulb. Lesions damaging the anterior commissure result in degeneration that is found in both bulbs [26]. The distribution of commissural fibers are believed to connect both anterior olfactory nuclei with each other but do not extend to the olfactory bulb [ 19,20,3 11. The existence of ascending fibers in the medial forebrain bundle has been demonstrated [ 13,221. The distribution of this bundle in the rabbit has been shown to project to the rostra1 aspect of the olfactory tubercle [33]. Some of these fibers run through the precommissural and posterior region of the anterior olfactory nuclei. The crossing over of fibers of the dorsomedial part of the medial forebrain bundle to the opposite dorsal longitudinal fasciculus and medial forebrain bundle has also been described [5]. Histochemical fluorescence studies [ 11,301 of the ascending catecholaminergic fibers in the dorsolateral aspect of the medial forebrain bundle reveal that some fibers of this system cross the midline in the commissura supraoptic dorsalis and a large number of fibers run to the olfactory tubercle through the lateral olfactory tract and to the diagonal band of Broca and the homolateral septal nuclei. The response evoked bilaterally in olfactory structures by stimulation of the posterior hypothalamic nucleus and medial forebrain bundle [l] indicate functional direct connections between these structures. The purpose of the present study is to determine possible projections of the ventromedial and lateral areas of the hypothalamus to the olfactory bulb and prepyriform cortex including the decussations of the pathways involved in this system.
Copyright 0 1978 ANKHO International
Olfactory bulb
Prepyriform
cortex
METHOD
Experiments were conducted on 35 adult cats of both sexes, weighing 2.5 to 3.5 kg. Anesthesia was ether and chloralose, 70 mg/kg. Electrical activity of homolateral and contralateral olfactory bulbs and prepyriform cortex (respectively, HOB, COB, HPPC, CPPC) was recorded through stainless steel monopolar and bipolar electrodes implanted stereotactically, following the atlas by Snider and Niemer [29]. Stimulation stereotaxic coordinates were F:7.5 to 13.5, L:O.5 to 4.0 and H:O.O to -7.0. In each preparation, one to three recording electrodes were placed at different locations in order to map the field of evoked potentials in both bulbs and prepyriform cortices. Stimulation consisted of l-5 mA, 0.1-0.5 msec single shocks from a Grass S-88 stimulator through a stimulus isolation unit. Thresholds were measured in each preparation and for each recorded response. In a group of 20 cats, electrolytic lesions (2-10 mA DC current for 30 set) or knife cuts (using a stereotactically calibrated knife) were placed in corpus callosum, anterior commissure (F: 14.5, L:O.O, H: + 1 to -2.O), supramammillar commissure (F8.0, L:O.O, H: -3 to -5.0) and medial forebrain bundle (F:13.5 to 12.0, L:3.0 to 4.5, H: -2.0 to -5.0). Electrical activity was recorded from the olfactory bulbs and prepyriform cortex during hypothalamic stimulation before and after lesions were made. The position of the electrode tips used for stimulation and recording was marked by passing a direct 50 PA current for 10 sec. At the end of the experiment, animals were perfused with 10% Formalin containing potassium ferrocyanide. The location of electrode tips and extent of the lesions were determined by examining 20 p thick frozen sections or projecting unstained sections, mounted on slides, on photographic paper.
Inc.-0361-9230/78/050467-08$01.30/O
GUEVARA-AGUILAR
468
AND AGUILAR-BATURONI
Fr =;
Fr =8!
A
E
8
F
C
G
D
H
5
Fr =I00
FIG. 1. Drawings of three different frontal planes from where potentials where evoked in the OB homo and contralateral. The amplitude of each bar represents the amplitude of the first component (Nl) recorded to different depths. The largest response was taken as 100% for each preparation. Bottom left shows the evoked potentials in homolateral olfactory bulb by posterior hypothalamic nuclei stimulation.
RESULTS Potentials
Recorded
from the Olfactory
Pathway
Previously it was demonstrated [I] that single shocks in the posterior hypothalamic nuclei elicited bilaterally in the olfactory bulbs and prepyriform cortex, biphasic potentials with negative components with latencies: 2.1 * 0.3 msec (Nl), 12.0 ? 2.0 msec (N2) and 54.8 2 6.1 msec (N3) for the homolateral response, and 2.7 + 0.05 msec (Nl), 11.0 + 1.3 msec (N2), and 60.2 t 6.1 msec (N3) for the contralateral bulb response. In order to find out if stimulation of other nuclei could evoke these responses, stimulation electrodes were placed in various ventromedial and lateral hypothalamic areas as follows: F:7.5 to 13.5, L:O.S to 4.0 and H:O.O to -7.1. Figure 1 shows the sites of stimulation which evoked the first Nl component in the homo and contralateral olfactory bulbs, in three different frontal planes. The amplitude of the first component has been represented by the
FIG. 2. Evoked potentials from the HOB. recorded at various depths in steps of 500/*. elicited by VMH stimulation. Superposition of three sweeps. Coordinates F:9.0, L:1.5, H: A -2.0, B: -2.5, C: -3.0, D: -3.5, E: -4.0, F: -4.5, G: -5.0, H: -5.5. Observe that the first component began to appear at -3.0 of depth (C). Calibrations: 100 fiV; 10 msec.
height of each bar. The largest evoked response was taken as 100% for each preparation. Only responses which were obtained 80% of the time were retained. The largest amplitude obtained with stimulation of the lateral hypothalamic region (LH) was below the medial forebrain bundle (MFB), posterior hypothalamic nuclei, and the region of the medial and lateral mammillary bodies. Figure 2 shows the responses elicited at the homolateral olfactory bulb when the ventromedial hypothalamic nuclei (VMH) is stimulated. The largest amplitude corresponds to ventral regions of the VMH in the case of the short latency component (Nl) and to the dorsal regions in the case of the longer latency (N2) or (N3) components. No significant other differences were found in latencies between the different components in the olfactory bulbs when posterior, ventromedial, or lateral hypothalamic nuclei were stimulated. Figure 3 shows the response evoked in both olfactory bulbs by stimulation of the posterior hypothalamic nucleus. As the stimulation electrode was driven more ventrally, while still recording, the amphtude of the first component was larger for the basal regions (Fig. 3
EVOKED
POTENTIALS
IN OLFACTORY
469
SYSTEM
FIG. 3. Evoked potentials, from HOB upper traces and COB lower traces, recorded at various depths in steps of 1000 p elicited by PH stimulation. Superposition of three sweeps. Coordinates F: 8.5, L: 1.0, H: A -1.0, B: -2.0, C: -3.0, D: -4.0, E: -5.0, F: -6.0. Calibrations: 100 pV; 10 msec.
C-F), either at homolateral or contralateral olfactory bulb, while in the case of the slow component (N3) the largest amplitude was obtained from the dorsal regions (Fig. 3A). Intravenous administration of Nembutal (10 mgikg) attenuated the slow components (N2) and (N3); whereas, it did not effect the fast component (Nl). At higher doses, all components were attenuated for both ventromedial, lateral or posterior hypothalamic nuclei stimulation. Pathways
to Contrulaterul
Olfactory Structures
In order to examine the fiber systems which project to the contralateral olfactory bulb and prepyriform cortex, partial sections of different structures were performed in some animals. Figure 4 shows potentials evoked at homo and contralateral olfactory bulbs by stimulation of posterior (A,B,C) and lateral (D,E,F) hypothalamic nuclei. Tracings A and D represent responses prior to any sections while stimulating the posterior and lateral hypothalamus, respectively. In tracing B and E, the posterior commissure and the part of the corpus callosum situated above it were cut in an anteroposterior plane. Both the duration and amplitude of the slow component changed (mainly at the contralateral bulb), and the fast component (both homo and contralateral) decreased in amplitude. In tracing C and F, the supramammillary
commissure and the corresponding part of corpus cahosum were cut: the first component increased in amplitude (mainly the PHN one), and the amplitude of all slow components decreased. Figure 5 depicts the effects observed at the PPC and OB following stimulation of the posterior hypothalamus. Tracing A represents a control recording of potentials elicited at homo (upper tracing) and contralateral (lower tracing) olfactory bulbs. Tracing B represents a control recording of potentials elicited at the PPC. Tracing C is a transcortical recording obtained by stimulating one side of the PPC and recording at the other. In tracing D and E, the posterior commissure and supramammillar commissure, together with the neighboring regions of the corpus callosum, were severed. Tracing D, where the response to stimulation was recorded in the olfactory bulbs, shows increased amplitude fast components, and smaller slow N3 components, both homo and contralaterally. Tracing E, where the response to stimulation was recorded in the prepyriform cortex, illustrates that the homolateral N3 lasts longer but the contralatera1 N3 has disappeared. The flat tracing at F confirms that the transcortical fibers were cut, stimulating one prepytiform cortex area had no effect in the other. In another group of experiments only the corpus callosum was cut. Under these conditions, there were no significant changes of the amplitude or duration of homo and contralat-
470
GUEVARA-AGUlLAR
AND AGUILAR-BATURON~
FIG. 4. Evoked potentials, from HOB upper traces and COB lower traces, in response to stimulation of PH (A,B,C) anf LH (D,E,F). A and D show control responses B and E are responses after the posterior commissure and part of the corpus callosum were cut (F: 4.0 to 6.0, L:O.O, H: +2.0). C and F after the supramammillar~ commissure was cut (F5.0 to X.5, L:O.O. H: -6.0). Superposition of 3 sweeps. Calibrations: 100 /*V; IO msec.
era1 evoked potentials in the olfactory bulb or prepyriform cortex produced by hypothalamic stimulation. When the anterior commissure was severed in addition to the posterior and supramammillary commissure, no further change occurred in the shape or amplitude of potentials evoked at the olfactory bulb and prepy~form cortex. When the anterior commissure was severed first, the amplitude of the first component increased just as it did after section of the supramammiUary commissure, but the slow component did not change. In another five animals the medial forebrain bundle was severed by either knife cuts [27] or by electrolytic lesions. Figure 6 shows the changes induced in the components evoked in the homoiateral olfactory bulb following stimulation of the posterior hypothalamic nucleus of a cat in which the homoIater~ medial forebrain bundle has been severed by a knife cut. Lesion is indicated in the upper right part of the figure. A is a control tracing; B was recorded immediately after the section; both fast and slow components are smaller. At C, 1 hr later, Nl has increased, but N3 tends to flatten out after the fourth successive stimuli. No such effect was observed at A. The lower part of Fig. 7 presents the modification of Nl immediately after the section, using a faster sweep. When electrolytic lesions (Z-10 mA DC for 30 see) were placed rostraily in the medial forebrain bundle, as illustrated in the three slices from the same cat by arrows in left part of Fig. 7, the slow and fast
components
in homolateral
and
contralateral olfactory bulbs became smaller. These effects are illustrated in part A and B of Fig. 7. In part A the normal response is depicted. In part B the smaller response following the electrolytic lesion of the medial forebrain bundle is illustrated. However, evoked potentiais did disappear at the homoiateral prepyriform cortex. These effects are also iliustrated in parts C and D of Fig. 7. In part C the normal PPC response is illustrated and in part B no homolateral response is observed while no change was observed at the contralatera1 prepyriform cortex. DISCUSSION
These results confirm the existence of centrifugal fibers to the olfactory system from the posterior, ventromedial, and lateral hypothalamic regions. StimuIation of any of these nuclei failed to show any differences in latency of the different components in the evoked potentials. Apparently, the fibers from each hypothalamic nucleus have separate and inindependent projections to the olfactory system. However, such an interpretation might be misleading due to the limitations of the present technique, in which different size fibers were stimulated simultaneously and small difference in latencies are difficult to evaluate. We are presently conducting single cell recordings to delineate this problem. When the stimulating electrode was placed more ventrally in the hypothalamus amplitude changes in the different components of the evoked potentials were observed. Large
EVOKED
POTENTIALS
IN OLFACTORY
SYSTEM
f--i FIG. 5. Evoked potentials in OB and PPC (A and B, respectively), homolateral upper traces and contralateral lower traces, by stimulation of PH. C is a transcortical record, D and E are responses after the posterior commissure and supramammillary commissure were cut, F shows that transcortical fibers were cut. Supposition of four sweeps. Calibrations: 100 pV, 10 msec.
amplitude stimulation
long latency in the dorsal
components
were
obtained
with
regions. Shorter latencies were obtained with more ventral stimulation. This observation is compatible with a double projection system to the olfactory bulb. The aminergic ascending fibers are arranged in both dorsal and ventral bundles which terminate in the diencephalon [30]. These two systems might remain distinct until they reach more rostra1 structures; for example, the olfactory tubercle where different fibers from the bulboreticular regions to the olfactory bulb also terminate 1121. Procaine administered to more lateral zones of the tubercle modifies the slow component of the potential evoked by stimulation of the posterior hypothalamic nucleus. Only when more medial regions are anesthetized by procaine can a change in the short latency component be seen [I] which supports a dual system interpretation. Worseradish peroxidase accumulates in the lateral hypothalamus following its local application in the olfactory bulb which indicates the existence of centrifugal fibers from the hypothalamus to the olfactory bulb. Peroxidase was found in the contralateral locus coeruleus and in the raphe nuclei but it was not found in the contralateral hypothalamus 161.
From these results, it can be concluded that the hypothalamic projections to the prepyriform cortex differs from the one to the olfactory bulb; one is only ipsilateral and the other is bilateral. Since severing the supramammillary and posterior commissures abolishes the evoked potential at the contralateral prepyriform cortex, the decussation of this bilateral projection might be found at this level. Olfactory bulb fibers could decussate more posteriorly; for instance in the pons, locus coeruleus, or raphe nuclei [61 and stimulation in these areas also might result in evoked responses in olfactory structures. A great deal of experimental data [4,15,16,17,18, 321 suggest the presence of ascending fibers in the medial forebrain bundle. These fibers originate in the tectum, raphe nuclei, and locus coeruleus and ascend to the hypoth~amus with some reaching the olfactory tubercle. Lesions were replaced in the locus coeruleus on one side of the brain and tyrosine hydroxylase activity was measured in the contralateral nuclei. Changes in the activity were found mainly on the fourth day after the lesion [7] which might indicate a functional interaction between the two nuclei. Since the present study indicates that lesions which transect the medial forebrain bundle modify evoked potentials at ipsilateral pre-
472
GUEVARA-AGUILAR
AND AGUILAR-BATURONI
FIG. 6. Evoked potentials from HOB elicited by stimulation of PH. A shows the response before MFB has been severed. B was recorded immediately after and C 1 hr later. D represents the response of only the short latency component, using a faster sweep before the MFB damage. E immediately after. Upper left shows the position of the stimulating electrode (F:8.5, L:l.O, H: -3.5). Upper right shows the damage in the MFB (F: 13.5, L:3.0, H: -3.5). Superposition of four sweeps. Calibrations: 100 pV, 20 msec (A,B,C) and 5 msec (D and E).
pyriform cortex and olfactory bulb, the medial forebrain bundle as well as some fibers which cross over to the contrilateral nuclei are probably part of the projecting pathway to the olfactory system. This would explain the changes observed at the contralateral olfactory bulb.
ACKNOWLEDGEMENTS
The authors are grateful to Professor M. J. Wayner, F. C. Barone and C. C. Loullis for their help and suggestions in preparing this report. The technical assistance of Mr. Carlos de 10s Santos is acknowledged.
473
EVOKED POTENTIALS IN OLFACTORY SYSTEM
FIG. 7. Evoked potentials from the OB and PPC (A and C are responses from OB and PPC, respectively). Homolateral responses upper traces and contralateral lower traces by stimulation of PH before electrolytic lesions (5 PA DC for 30 set) to the MFB. B and D after lesion. Left shows the extent of the lesion in the MFB. Coordinates F:14.5, L:3.5, and H: 4.5. Calibrations: 100 pV; 10 msec.
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