Functional dissociation within hippocampus

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BRAIN RESEARCH

F U N C T I O N A L DISSOCIATION WITHIN HIPPOCAMPUS

LESTER D. GRANT AND LEONARD E. JARRARD Psychology Department, Carnegie-Mellon University, Pittsburgh, Pa. (U.S.A.)

(Accepted March 13th, 1968)

INTRODUCTION It has long been known that the hippocampus can be divided into several cytoarchitectonic fields. The fields have been designated C A l, 2, 3, and 4 by Lorente de N614,15. The relative size of the pyramidal cells comprising each field roughly corresponds to the label assigned to the field, i.e., the smallest pyramids are found in field CA1, the next largest in field CA2, and the largest in field CA4. The four cell fields are arranged successively from CAI to CA4 as one moves within the pyramidal layer of the hippocampus toward and around the cornu ammonis and back to within the hilus of the dentate gyrus. It has generally been assumed that the several cell areas are functionally homogeneous. Consequently, few empirical investigations have been concerned with the possibility that neuroanatomical and/or neurophysiological differentiation within the hippocampus may underlie dissociation of functions within the structure. However, as Jarrard s has pointed out, certain recent data on efferent and afferent connections of the hippocampus have provided possible support for such a view. Raisman et al. 19 showed, by means of the Nauta stain, that hippocampal cell fields in rat have differential connections to extrahippocampal structures. Cells of field CA1 in anterodorsal hippocampus project efferents over dorsal and postcommissural fornix to terminate in anterior thalamic nuclei and the mammillary bodies, but CA3 and CA4 ceils project via fimbria and precommissural fornix to septal region nuclei. Efferents from posterior CA1 cells project over both dorsal fornix and fimbria to all extrahippocampal areas mentioned above. Data presented by other investigators on hippocampal afferent connections in rat indicate that fibers which terminate in hippocampus have varying biochemical characteristics. Specifically, the work of Fuxe 4 indicates that hippocampus receives noradrenergic fibers, while data presented by Lewis and Shute 13 suggest that hippocampus also receives cholinergic innervation. The monoaminergic and cholinergic terminals were found throughout both anterodorsal and posteroventral hippocampus. The differences discussed above in regard to the termination points of efferents from hippocampal cell fields and in regard to the chemical characteristics of hippocampal afferents seem to provide excellent neuroanatomical and biochemical bases Brain Research, 10 (1968) 392-401

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for functional dissociation within the hippocampus of the rat. The present study was undertaken to see if, on a behavioral level, functional dissociation within the hippocampus could indeed be demonstrated in the control of certain motivational states and general arousal. More specifically, the present study tested the hypothesis that direct chemical stimulation of the hippocampus would lead to varied effects on eating, drinking, and general activity, depending upon the cell field stimulated and the neurohumoral substance applied. METHOD

Subjects The subjects used in the present study were 28 Sprague-Dawley derived albino male rats of the Charles River strain. The rats were 180 days old and weighed 375475 g when chemical stimulation cannulas were implanted.

Cannulas Each double-walled cannula assembly consisted of an outer guide cannula, which was permanently implanted, and an inner cannula. The inner cannula could be raised and lowered within the guide cannula, thus allowing for repeated chemical stimulation of the same brain site. The guide cannulas consisted of 22 gauge stainless steel hyperflex needle tubing, and the removable inner cannulas were constructed of 30 gauge tubing.

Su~e~ Surgery was carried out while subjects were under sodium pentobarbitol anesthesia. Of the 28 subjects, 18 had cannulas stereotaxically implanted in both anterodorsal and posteroventral hippocampus. The other 10 subjects had one cannula implanted in either cingulum or fornix. Inner cannulas were left in the implanted guide cannulas to keep the latter free of debris.

Experimental procedures After the cannulas were implanted, all the rats were returned to home cages and allowed a 10-day postoperative recovery period. Following the recovery period, the animals were placed in observation cages for 1 h on each of 2 consecutive days in order to habituate them to the experimental testing situation. The observation cages were 15 in. × 9 in. × 7 in. and were constructed of 0.25 in. hardware cloth with a hinged door at the front allowing for the entrance and exit of a subject. On the day following the second habituation session, experimental testing was initiated, with each subject undergoing one of four treatments via one implanted cannula. Subsequently, a single treatment was administered to each rat on every other

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Fig. 1. Representative photomicrographs of hippocampal placements. Brain section A (top) shows a cannula track terminating in anterodorsal CA 1 cells. Brain section B (bottom) shows a posteroventral CA3-CA4 placement.

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day. One and then the other cannula was used in alternating fashion for those animals with two implanted guide cannulas. The treatments were administered in random order and consisted of (a) monoaminergic stimulation with a 1--3 Fg dose of crystalline norepinephrine, (b) cholinergic stimulation with an identical dose of carbachol, (c) control stimulation with NaCI, and (d) sham stimulation with an empty 30 gauge inner cannula. Each treatment was replicated twice over the course of experimental testing, The amounts of food and water consumed were recorded for 1 h after the administration of each treatment. The amount of time alter the application of a treatment until eating or drinking of 1 min duration first occurred was also recorded. In addition, general activity was monitored by means of photoelectric cell units.

Histology Following experimental testing, the subjects were sacrificed and perfused with 10% formalin. The brains were then removed, frozen, sectioned at 30 #, and stained with tbionin. Microscopic inspection of brain sections was performed for 22 animals for which complete data had been obtained for both replications of each treatment. Two judges, without reference to recorded experimental data, classified placements according to where the cannula tracks terminated. Data analysis included only those placements that could be classified by both judges as clearly ending in a particular hippocampal cell field or related fiber tract. Five hippocampal placements were judged as definitely ending in the anterodorsal CA I area, four in the anterior CA3-CA4 area, four in the posteroventral CA I area, and six in the posterior CA3-CA4

CELL

FIELD S T I M U L A T E D : F I E L D C A - 1 = OPEN S Y M B O L S F I E L D C A - 3 & 4 = CLOSED S Y M B O L S

A M O U N T OF WATER C O N S U M E D : LESS T H A N 5 m [ = S Q U A R E S 5-15ml = CIRCLES MORE THAN 15ml = STARS

Fig. 2, Mean amount of water consumed with both replications of the carbachol treatment at hippocampal placements. Left: anterodorsal hippocampal sites stimulated, Right: posteroventral hippocampal sites stimulated. Numbers on brain sections refer to corresponding plates in K~nig and Klippel tz rat brain atlas. Abbreviations: HI, hippocampus; FH, fimbria hippocampi; TO, tractus opticus; IP, nucleus interpeduncularis. Brain Research, 10 (1968) 392-401

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area. In addition to the hippocampal placements, four fiber tract placements clearly ended in the cingulum, dorsal to the anterior hippocampal placements. Another four were in the fornix, anterior to the hippocampus and just medial to the lateral ventricle. Fig. 1 presents representative photomicrographs of placements terminating in anterodorsal and posteroventral hippocampus. Fig. 2 shows specific locations of termination points for all hippocampal placements. RESULTS

The results of the present study are summarized in Table I. The means and standard deviations presented in Table I were calculated on the basis of scores obtained across both replications of each treatment. General behavioral observations on effects of the various treatments at the brain sites tested and the results of statistical analyses are discussed below. During the hour-long habituation sessions prior to experimental testing, most subjects showed exploratory behavior for approximately 15 rain after being placed in the observation cages. The exploratory activity took the form of intermittent sniffing around the cages and occasional brief tasting of food and water. After the initial exploratory behavior subsided, subjects typically moved to the rear of the cages TABLE 1 MEANS AND STANDARD DEVIATIONS (IN PARENq[HESES)OF AMOUNTS OF WATER CONSUMED (IN ml), FOOD CONSUMED (IN g)AND ACTIVITY (NUMBER OF PHOTOCELL BEAM INTERRUPTIONS), FOLLOWING EACH STIMULATION TREATMENT

Abbreviations: C, cholinergic stirnulation; M, monoaminergic stimulation; N, NaCl stimulation; S, sham stimulation. Stimulation sites

Water consumption Treatments C M N S

Food consumption Treatments C M N S

General activity Treatments' C M N

S

12.7 (6.1) 12.6 (7.2)

126 (33) 151 (60)

Anterodorsal hippocampus CAlcells (N 5) CA3 CA4cells (N 4)

1.1 (1.4) 4.1 (1.2)

3.0 (1.5) 3.3 (1.9)

2.8 (1.3) 3.5 (2.3)

2.6 (2.7) 1.8 (1.8)

1.8 (2.2) 2.6 (1.8)

1.4 (1.4) 0.6 (I.2)

0,6 (1.1) 1.6 (1.3)

201 129 154 (106) (68) (60) 174 180 158 (43) (47) (56)

3.4 (2.0) 2.8 (2.5)

3.4 (1.2) 2.8 (1.8)

2.6 (2.4) 3.7 (2.1)

3.9 (1.5) 2.5 (2.1)

1.3 (1.0) 1.5 (I.9)

1.5 (1.8) 2.7 (2.2)

1.4 (1.8) 1.8 (1.2)

0.9 (1.0) 0.8 (I.I)

288 161 138 168 (143) (58) (65) (59) 271 122 162 165 (115) (25) (53) (65)

14.4 (9.2) 14.0 (6.1)

2.3 (3.8) 2.5 (1.8)

2.0 (1.7) 3.1 (I.9)

2.5 (I.6) 3.8 (2.3)

0.8 (l.0) 3.8 (2.7)

1.1 (1.4) 2.1 (2.2)

0.4 (0.7) 2.1 (I.2)

0.6 (0.7) 1.3 (1.8)

91 65 87 103 (98) (94) (35) (61) 171 145 161 184 (83) (51) (62) (27)

Posteroventral hippocampus CAI cells ( N - 4) C A 3 - C A 4 cells (N 6)

Related fiber tracts Fornix (N ~ 4)

Cingulum (N

4)

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and groomed or settled in a relaxed manner. Only occasionally during the remainder of an observation period would any animal move about or eat or drink briefly. Following either NaC1 or sham stimulation, the subjects behaved very much as they had during the habituation sessions. With monoaminergic stimulation, however, subjects often exhibited increased eating of several minutes duration, beginning within I0 min after the application of norepinepherine. An analysis of variance showed that monoaminergic stimulation led to a slight, but statistically significant, increase in overall food consumption over that observed with NaCI or sham stimulation (P < 13.001). Also, 2 animals, one with stimulation of the fornix and the other with stimulation of anterodorsal hippocampal CA1 cells, became excited intermittently and rushed back and forth picking up pieces of laboratory chow and stacking them at the rear of the cages, q-he same behavior occurred after both replications of the norepinepherine treatment at the same placements in the 2 subjects. The food carrying responses may be indicative of neural elements underlying hoarding behaviors having been monoaminergically affected. Aside from increases in food consumption and the two cases of apparent hoarding, monoaminergically stimulated animals behaved in a manner similar to when they received NaC1 or sham stimulation. In contrast to the relatively minor behavioral changes evoked by monoaminergic stimulation, very marked behavioral changes occurred with cholinergic stimulation. Within minutes after the insertion of carbachol, animals stimulated in anterodorsal hippocampal, cingulum, or fornix sites engaged in drinking for periods of several minutes duration. The periods of prolonged drinking, with some intervening pauses~ typically lasted between 20 and 40 min. Carbachol stimulation of posteroventral hippocampal sites did not noticeably facilitate drinking responses. Carbachol stimulation of the posteroventral sites, however, did induce some eating, as did such stimulation at the other brain loci tested. An analysis of variance showed that water consumption was significantly greater with carbachol stimulation than with norepinephrine, NaCI, or sham stimulation at anterodorsal hippocampal CAI and CA3CA4 sites (P < 0.001). Carbachol stimulation at these positive sites (see Fig. 2) was also found to produce greater water intake than similar stimulation at posteroventral hippocampal sites (P -< 0.001). In addition, carbachol stimulation, like norepinephrine stimulation, was found to produce slight, but statistically significant, increases in overall food consumption over control stimulation levels (P < 0.001). However. at least some of the food consumption increases observed at particular brain sites may have been due to non-specific activation effects of the neurohumoral stimulation treatments, since, as indicated in Table 1, NaCI stimulation in some instances raised food consumption over sham stimulation levels. Besides having effects on eating and drinking, carbachol evoked pronounced increases in activity over control stimulation levels when inserted into the hippocampus (P < 0.001). Posteroventral hippocampal stimulation, however, increased activity more than did anterodorsal hippocampal stimulation (P < 0.01). Subjects cholinergically stimulated anywhere in the hippocampus became very alert and excitedly moved about while sniffing the interior of the observation cage and the available food and water. Subjects stimulated in the posteroventral hippocampus remained Brain Research, 10 (1968) 392-401

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hyperactive throughout the hour-long observation period and stopped moving about only to sample food or water briefly. With anterodorsal hippocampal stimulation, the animals similarly were hyperactive for the duration of the observation period, but interrupted their exploratory activity more often in order to drink large quantities of water. Occasionally, as some hippocampal stimulated animals moved about the cages, minor motor seizures occurred, which gradually developed into major convulsions followed by brief periods of catatonic posturing and insensitivity to external stimuli. Responses described above as being elicited with carbachol stimulation have also been reported by MacLean 16 with electrical stimulation of hippocampus. Of particular interest is the finding that hyperactivity and sniffing resulted from low voltage stimulation, while motor seizures and catatonic responses occurred during hippocampal afterdischarges following higher voltage stimulation. DISCUSSION

The results of the present study indicate that functional dissociation within the hippocampus does exist. In regard to the control of thirst, the dissociation seems to be based both on neuroanatomical and biochemical factors. Biochemically, some cholinoceptive hippocampal neurons are apparently involved in the control of thirst or drinking behaviors, but monoaminoceptive neurons are not so involved. Neuroanatomically, dissociation of thirst functions clearly exists at least between anterodorsal and posteroventral hippocampus. Additional evidence strongly suggests that an even more specific localization of function occurs with hippocampal involvement in thirst functions being limited to cholinoceptive anterodorsal CA 1 cells. When subjects were cholinergically stimulated in anterior CA I cells, prolonged drinking of 1 rain or more duration was exhibited within an average time of 4.1 rain, while subjects stimulated in anterior CA3-CA4 cells took a mean time of 6.8 rain to show similar effects (P < 0.01). This difference in latency to drink was probably due to anterior CA3-CA4 drinking effects having actually been caused by carbachol spreading over the short distance (approximately 1 ram) up along the implanted guide cannulas to anterior CA 1 cells. In addition to thirst functions being dissociated within hippocampus, general behavioral arousal control also appears to be dissociated. The dissociation of arousal control is most clearly based on biochemical factors, with cholinergic, but not monoaminergic, mechanisms probably being involved in behavioral arousal. Certain results of the present study, specifically those which showed significantly greater activity with posteroventral carbachol stimulation than with anterodorsal carbachol stimulation, could be taken as indicating that dissociation of behavioral arousal also exists on a neuroanatomical basis. It should be noted, however, that carbachol stimulation of anterodorsal CA I and CA3-CA4 sites did lead to hyperactivity, although the amount of activity was attenuated by drinking effects evoked concurrently by carbachol, i.e., subjects had to stop moving about in order to drink. Routtenberg z° has recently advanced the argument that cholinergic and antiBrain Research, 10 (1968) 392-401

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cholinergic effects on behavior observed with direct chemical stimulation of certain brain areas may actually be due to spread of chemicals into the ventricular system and then to other brain sites. Four lines of evidence mitigate against this as an explanation for the present results. One is the fact that the mean latency for drinking after carbachol stimulation of the fornix (5.3 min) was identical to that for the cingulum and longer than that for anterodorsal hippocampai CA I cells, with the site of fornix stimulation being immediately adjacent to the lateral ventricle and the cingulum being separated from the ventricle by the corpus callosum. A second line of evidence comes from the fact that hyperactivity followed stimulation of hippocampal but not cingulum or fornix sites, even though fornix sites are closest to the ventricle. The third line of evidence is that MacLean 16 observed cholinergic effects reported here (i.e., drinking, behavioral alerting and hyperactivity) with electrical stimulation of the hippocampus of rat. A final, and perhaps the most conclusive argument against ventricular spread as an explanation for the present results, is the fact that Myers and Cicero ts recently found that carbachol injected directly into the rat ventricular system failed to elicit drinking. In contrast to the hippocampal dissociation which appears to exist for thirst and arousal functions, it seems that dissociation of hunger functions within the hippocampus exists neither on a neuroanatomical nor a biochemical basis since stimulation with either norepinephrine or carbachol of any hippocampal cell field increased food consumption over sham stimulation levels. This finding is not consistent with a report by Coury 1 indicating that dorsalmedial hippocampus is positive in regard to norepinephrine-induced eating, while anterior and lateral hippocampal areas are not. Coury's description of cannula locations does not permit his results to be related to specific hippocampal cell fields. Nevertheless, many of our positive anterodorsal CA I and CA3-CA4 sites were clearly in the 'anterior' part of the hippocampus. The inconsistencies between the present findings and those of Coury might be due to the fact that conclusions drawn from the present results have been based on statistically significant effects, while Coury defines positive sites on the basis of an a priori selected amount of food consumption, in any case, it seems safe to conclude that no functional dissociation in control of hunger has yet been clearly shown to exist among various areas of the hippocampus. The present study has potential implications for several areas of brain research. The biochemical data presented here relate to work being done on the tracing of 'chemically coded" CNS circuits underlying motivational and arousal functionsa, 3,5-7,17. They may also bear on the issue of whether neurons that are receptive to a particular neurotransmitter act by a similar or different chemical mode 2,2j, since the present findings indicate that there are probably monoaminoceptive and cholinoceptive neurons within hippocampus, but Fuxe 4 and Lewis and Shute ~3 report that neither monoaminergic nor cholinergic efferents are projected from the hippocampus. The present findings showing neuroanatomically-based dissociation of thirst functions within hippocampus would appear to be of importance to workers involved in the investigation of hippocampal functions on a behavioral level. Conflicting Braht Research, 10 (1968) 392-401

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results can be found in the literature on hippocampal functioning in learning 9,11 and motivation 1,3,10. Although some of the inconsistencies in the literature can perhaps be explained by differences in behavioral testing procedures, the present findings suggest that differences in location of lesions and/or stimulation sites may underlie many of the conflicting results. Since it is now apparent that dissociation of thirst and arousal occur within the hippocampus based on neuroanatomical and/or biochemical factors, further research should be directed to the possibility that other functions are also dissociated within the structure. In fact, perhaps ageneral reevaluation of previous hippocampal research would be useful in light of the results reported here. SUMMARY

Recent data on efferent projections of the hippocampus and biochemical characteristics of hippocampal afferents suggest that functional dissociation may exist within the structure. On the basis of the specific efferent and afferent data reported, the present study was designed to test the hypothesis that chemical stimulation of the hippocampus would lead to varied effects on eating, drinking and behavioral arousal, depending on the hippocampal cell field stimulated and the neurohumoral substance administered. The results of the study indicated that control of drinking was dissociated within hippocampus both on a neuroanatomical and biochemical basis. Behavioral arousal was also found to be dissociated within the structure, but only on a biochemical basis. In regard to the control of eating, the hippocampus was found to be functionally homogeneous. Thus, it appears that not only does neuroanatomical and biochemical differentiation exist within the hippocampus, but functional dissociation also occurs within the structure.

ACKNOWLEDGEMENTS

The present study was supported in part by NSF Grant GB-5519 (to L.E.J.). We thank M. DeMarco for technical assistance. We also thank Merck, Sharp and Dohme Research Lab, Rahway, N.J. for supplying carbachol. L.D.G. was a NASA Predoctoral Trainee at the time of this research, which was done in partial fulfillment of the requirements for the Master of Science Degree at Carnegie-Mellon University.

REFERENCES l COURY,J. N., Neural correlates of food and water intake in the rat, Science, 156 (1967) 1763-1765. 2 FELDBERG,W., ANt) VOGT, M., Acetylcholine synthesis in different regions of the central nervous system, J. Physiol. (Lond.), 107 (1948) 372-381. 3 FISr~ER, A. E., AND COURV, J. N., Cholinergic tracing of a central neural circuit underlying the thirst drive, Science, 138 (1962) 691-693. 4 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol, seand., 64, Suppl. 247 (1965) 39-85.

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5 GROSSMAN, S. P., Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms, Amer. J . Physiol., 202 (1962) 872-882. 6 GROSSMAN, S. P., Behavioral effects of chemical stimulation of the ventral amygdala, J. comp. physiol, Psychol., 57 (1964) 29-36. 7 GROSSMAN, S. P., Effects of chemical stimulation of the septal area on motivation, J. comp. physiol. Psychol., 58 (1964) 194-200. 8 JARRARD, L. E., On the importance of different hippocampal regions for behavioral investigations, Ammon's Horn, Spring (1967) 15-21. 9 KAADA, B. R., RASMUSSEN, E. W,, AND KVEIM, O., Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic, and insular lesions in rats, J. comp. physiol. Psychol., 55 (1962) 661-670. 10 IQMBLE,D. P., AND COOVER, G. D., Effects of hippocampal lesions on food and water consumption in rats, Psychon. Sci., 4 (1966) 91-92. 11 K~MURA,D., Effects of selective hippocampal damage on avoidance behavior in the rat, Canad. J. Psychol., 12 (1958) 213-218. 12 KON1G, J. F. R., AND KLIPPEL, R. A., The Rat Brahl: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 13 LEw~s, P. R., AND SHUTE, C. C. D., The cholinergic limbic system: Projections to hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-540. 14 LORENTE DE N6, R., Studies on the structure of the cerebral cortex. 1. The area entorhinalis, J. Psychol. Neurol. (Lpz.), 45 (1933) 381-438. 15 LORENTEDE N6, R., Studies on the structure of the cerebral cortex. 11. Continuation of the study of the ammonic system, J. Psychol. Neurol, (Lpz.), 46 (1934) 113-177. 16 MACLEAN, P., Chemical and electrical stimulation of the hippocampus in unrestrained animals. II. Behavioral findings, Arch. Neurol. Psyehiat. (Chic.), 78 (1957) 128-142. 17 M~LLER, N., Chemical coding of behavior in the brain, Science, t48 (1965) 328-338. 18 MYERS, R. D., AND CICERO, T. J., Are the cerebral ventricles involved in thirst produced by a cholinergic substance?, Psychon. Sci., 10 (1968) 93-94. 19 R.AISMAN,G., COWAN,W., AND POWELL,Y., An experimental analysis of the efferent projection of the hippocampus, Brain, 89 (1966) 83-108. 20 ROUTTENBERG, A., Drinking induced by carbachol: Thirst circuit or ventricular modification, Science, 157 (1967) 838-839. 21 SHUTE, C. C. D., AND LEWIS, P. R., The ascending cholinergic reticular system: Neocortical, olfactory, and subcortical projections, Brain, 90 (1967) 497-520.

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