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Hippocampal lesions, superior cervical ganglia removal, and behavior in rats
Physiology & Behavior, Vol. 22, pp. 461--466. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Hippocampal Lesions, Superior Cervical Ganglia Removal, and Behavior in Rats I D A N I E L P. K I M B L E , S U S A N A N D E R S O N , R U T H B R E M I L L E R A N D E U G E N E D A N N E N
Department of Psychology, University of Oregon, Eugene, OR 97403 (Received 8 August 1978) KIMBLE, D. P., S. ANDERSON, R. BREMILLER AND E. DANNEN. Hippocampal lesions, superior cervical ganglia removal, and behavior in rats. PHYSIOL. BEHAV. 22(3) 461--466, 1979.--Rats were subjected to either bilateral removal of the superior cervical ganglia (SCG) or to a sham operation of the neck. Subsequently, these rats received either bilateral neopallial lesions, bilateral dorsal hippocampal lesions or no further operation. The SCG removal prevented the anomalous noradrenergic innervation of remaining hippocampal tissue. This anomalous innervation, as reported by Loy and Moore [19], originates from neurons in the SCG. All animals were then evaluated on behaviors previously shown to be sensitive to hippocampal destruction: open field activity, spontaneous alternation and spatial maze learning, in order to determine if any behavioral consequences could be related to the anomalous hippoeampal innervation. No statistically significant differences appeared which could be unambiguously related to the anomalous innervation.
Hippocampus Superiorcervical ganglia Open field activity Spatialmaze learning
Anomalousnoradrenerglc innervation
LOY and Moore [19] have reported the growth of anomalous fibers from the superior cervical ganglia (SCG) into the hippocampal formation following aspiration lesions to the anterior 2-3 mm of the hippocampal formation. This novel pattern of innervation was discovered using fluorescence histochemical procedures [6,18]. This new plexus had the characteristic appearance of peripheral sympathetic fibers. The anomalous innervation extended throughout the remaining portion of the hippocampal formation, including the area dentata and Ammon's horn. Ultrastructural studies currently in progress suggest that these fibers may make synapses, particularly in the hilar zone of the dentate gyrus (R. Y. Moore, R. Loy, personal communication). As indicated in the Loy and Moore report, "such anomalous growth could have significant implications for the sequelae to injury of the central nervous system of both experimental animals and man" [19]. The present experiments were designed to test these functional implications in rats in which this anomalous innervation was present, and in rats in which such anomalous growth was prevented by removal of the source of the fibers, the superior cervical ganglia.
METHOD
Animals The animals were 30 male Sprague-Dawley albino rats (CD, random-bred) obtained from the Charles River Co., Wilmington, MA. They were maintained in individual cages
Spontaneous alternation
on a 12 hr diurnal light cycle. Rats were approximately 75-80 days of age at the time of the first operation (superior cervical ganglia removal). Except for the times specified in the procedure, food and water were available on an ad lib basis.
Apparatus Locomotor activity was measured in a wooden box measuring 78.7x78.7 cm with 11.4 cm high walls. The floor was marked offby white lines into 36 equal size squares and the entire apparatus was painted gray and covered with a sheet of clear Plexiglas. A similar apparatus, painted a slightly darker gray was used in ths maze acquisition phase. This apparatus, together with wooden barriers, a start box and a goal box, constituted a Hebb-Williams maze apparatus [5]. The maze patterns were Nos. 1, 5 and 11 (one "easy", one "moderately difficult", and one "difficult") from the Rabinovitch-Rosvold series [23]. The start box and goal box were the same size, each measuring 30 cm in length, 13.5 cm in width, and 15 cm in height. Both boxes were equipped with fiber-board guillotine-type doors and Plexiglas tops. Spontaneous alternation was evaluated in a wooden T-maze constructed of a 56.3 x 11.4 cm stem and two 31.7x 24.1 cm goal arms, set at right angles to the main stem. Both goal arms were equipped with Plexiglas guillotine-type doors. The walls of the apparatus were 18 cm high.
Procedure Preoperative training. All animals were gentled for sev-
~This project was supported in part by BRSC Grant RR 07080 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/030461-06502.00/0
K I M B L E E l AL.
462 eral days following their arrival. Two weeks after their arrival they were pre-trained in the maze for seven days. The following day animals were placed, one at a time, in the center of the open field and allowed 5 min of free exploration, during which time the number of squares entered was recorded. An entry was scored if a rat placed its two front paws in a square. The following day, each rat was given three trials in the T-maze to determine its preoperative spontaneous alternation rate. Each rat was released from the start box into the main stem of the maze and allowed up to 3 min to enter one of the goal arms. Upon entry into one of the arms, the guillotine door was then gently closed behind the rat, 20 sec allowed to elapse, and the rat replaced in the start box for the next trial. All 30 rats completed three trials, with an overall alternation rate of 93% (100% of first free trial choices). Surgery. Superior cervical ganglia removal: Fifteen animals underwent bilateral superior cervical ganglionectomy. This operation took place five to seven days prior to brain operations. Anesthesia in all cases was induced by sodium pentobarbital (Nembutal), in doses of 50 mg/kg, IP. Atropine sulfate, in doses of approximately 0.04 mg/animal was also administered to reduce mouth and throat secretions. The ganglionectomy procedure was worked out in a series of pilot operations. The superior cervical ganglion in the rat lies in the Y-shaped junction formed by the bifurcation of the common carotid to form the internal and external carotid arteries. To visualize this ganglion, the rat was taped to a board on its back and a mid-line incision made in the skin from just below the mandible to just below the beginning of the chest musculature, a distance of about 3 cm. Upon retraction of the skin, the tissue overlying the SCG was blunt-dissected and laid to the side of the opening. The most superficial structures are the large submaxillary glands and the somewhat smaller and more anterior sublingual glands. These can be freed from the membrane holding them to the underlying musculature quite easily with no bleeding. The thyroid and parathyroid glands may be left undisturbed. Blunt dissection along the edge of the sternohyoideus muscle will expose the carotid which can be identified by its position and obvious pulsing. The carotid is then gently freed from the membranes binding it to its underlying musculature. Upon lifting the carotid at a level approximately 1 mm posterior to the posterior tip of the hyoid process, the SCG becomes visible at the juncture of the internal and external carotids. It is ovoid in shape, grayish-white in color and resembles a very small, uncooked grain of rice. Once visualized, the SCG was grasped with iris forceps and gently pulled away from its connections with the sympathetic chain together with a portion of the sympathetic chain. This operation was performed under low-power (10 to 16X) magnification. The instrument used in this experiment was a Zeiss binocular zoom operating microscope. While this operation can be performed in a few minutes with virtually no bleeding, great care must be taken not to rupture the carotid artery. If broken, bleeding from this artery can be somewhat troublesome to stop, although it can be done. In only two animals was this a problem. Both animals were assigned to the nobrain lesion group. There were no observed behavioral effects of the bleeding. Fifteen animals received sham SCG operations in which the ganglia were visualized but left inact. Brain lesion. Five to seven days following ganglionectomy eight animals underwent bilateral lesions of the neopallium overlying the hippocampus, including cortical tissue and part of the underlying white matter, primarily cor-
pus callosum. Another group of 12 rats underwent bilateral lesions of the dorsal and anterior aspects of the hippocampal formation. All operations were performed in one stage by aspiration, as described previously [12]. Ten additional animals underwent no operations and served as unoperated controls. Half of this group had, however, undergone previous bilateral ganglionectomy. Thus, six experimental groups were formed: (1) Unoperated, sham SCG operation (UNSHAM), (2) Unoperated, superior cervical ganglia removed (UN-SCG), (3) Neopallial-lesioned, sham SCG operation (NL-SHAM), (4) Neopallial-lesioned, superior cervical ganglia removed (NL-SCG), (5) Hippocampal-lesioned, sham SCG operation (HP-SHAM), and (6) Hippocampallesioned, superior cervical ganglia removed (HP-SCG). One animal in Group 1 developed a respiratory infection and was discarded prior to testing; all other rats survived all surgical procedures and completed all phases of the experiment. Postoperative testing. Postoperative testing was done in two stages, immediate and longer-term. The immediate phase lasted from Postoperative Day 2 to Day 18. The longer-term phase lasted from Postoperative Days 52-55. During the early postoperative period, the animals were repeatedly examined on locomotor activity in the open field and for spontaneous alternation, two behaviors which have previously been shown to be quite sensitive to hippocampal damage [3,13] and (in our laboratory) to show rather substantial returns to preoperative levels during the first 3 weeks postoperatively [14]. In the later phase, the animals were tested on the acquisition of spatial maze habits, a behavior which is also sensitive to hippocampal damage but appears to be a "persistent" or permanent sequelae of such lesions [15, 16, 221. Open field locomotor behavior was examined on Postoperative Days 2, 4, 8, 12, and 17. The procedure was identical with that used preoperatively. Spontaneous alternation was measured on Postoperative Days 3, 5, 9, 13 and 18. Procedures were identical with those used preoperatively. Both food and water were available on an ad lib basis. Spatial maze learning capacity was tested using three different maze problems selected from the RabinovitchRosvoid series. The set included maze problems 1, 5 and 11. Each rat was run on each of these problems in that order, on consecutive days. Each rat was run seven trials on each problem. All rats were placed on a 24 hr water deprivation schedule (water available for 10 min) on Postoperative Day 40 and kept on this schedule throughout testing. A water reward was available in the goal box. The rat's path through the maze was recorded on a data sheet containing the floor plan of that maze problem. An error was recorded if two or more paws crossed an error line. HISTOLOGY
Following the completion of the experiment, the brainlesioned animals were sacrificed by decapitation and the unperfused brains removed and frozen in a cryostat at -30°C. Irises were removed from a number of animals with and without ganglionectomy to further check on superior cervical ganglia removal. Irises were removed from the eyes in a procedure described by R. Y. Moore (personal communication). The eyeball was removed, cut along the anteriorposterior midline and the lens removed and discarded. The anterior half of the eye was placed downward and the iris grasped with iris forceps and laid flat (in pieces) on a glass slide. The slides were placed in a dessicator containing
HIPPOCAMPECTOMY AND GANGLIONECTOMY
463
FIG. 1. Frontal sections through hippocampal formation showing anomalous noradrenergic fibers in rat with anterior hippocampal lesion (on left) and absence of such anomalous fibers in rat with hippocampal lesion plus prior bilateral removal of superior cervical ganglionectomy (on fight). Fluorescence pattern in hippocampus on fight is due primarily to locus coeruleus fibers [19].
phosphorous pentoxide and kept there overnight. The slides were then transferred to a closed jar containing paraformaldehyde which had been previously equilibrated in an atmosphere of 70% relative humidity. After incubating for an hour at 80°C, the slides were removed, coverslipped with mineral oil and examined under a Zeiss fluorescent microscope. The brains were sectioned at either 16 or 32 microns. Alternate sections were then subjected to a modified glyoxylic acid technique to examine for catecholamine content [2]. These sections were also examined under the fluorescent microscope. The alternate sections were stained with thionin according to BreMiller's procedure [1].
Results Following the operation to remove the superior cervical ganglia a ptosis of the rat's eyes was observed, due to cutting the superior cervical trunk with a resultant retraction of the eyeball [17]. No behavioral consequences of this ptosis were observed. Examination of the irises from either normal or shamoperated animals revealed an extensive network o f noradrenergic fibers under the fluorescent microscope. Irises from animals with superior cervical ganglia removed showed no such fluorescence. The brain-sections through the hippocampal-lesioned animals which were stained and examined for catecholamine fluorescence showed the extensive pattern of anomalous sympathetic innervation in the intact hippocampal tissue as described by Loy and Moore [19]. Figure 1 shows relevant brain sections through hippocampal tissue from rats with and without SCG removal. The growth was found throughout the hippocampal tissue spared by the lesion, but not elsewhere. This anomalous growth revealed a new plexus of very fluorescent fibers, much thicker and coarser than ever found in normal brains [19] or in our rats with SCG removal. There is also a normal fluorescence observed in the hippocampal formation which is due primarily to fibers from locus coeruleus neurons [19], which can also be seen in Fig. 1. Only the normal growth was observed in those brains from rats with SCG removal. The anomalous growth was observed in all hippocampal-lesioned animals not subjected to SCG removal-.
TABLE 1 MEAN NUMBER OF SQUARES ENTERED IN 5 MIN IN OPEN FIELD, PRE- AND POSTOPERATIVELY Group
Pre-Op 2
Postoperative Day 4 8 12
17
UN-SHAM UN-SCG Combined UN
144 181 164
118 128 124
139 141 140
118 137 129
132 137 134
135 135 135
NL-SHAM NL-SCG Combined NL
186 171 179
111 86 99
98 112 105
138 105 122
139 96 118
132 102 117
HP-SHAM HP-SCG Combined HP
168 173 170
69§ 156t 117
179 179 179¢
148 148 148
117 110 114
32* 90~ 63
*p=0.006, two-tailed, compared with combined NL, p=>0.001 compared with combined UN. tp=0.082, two-tailed; 0.041, one-tailed, compared with HPSHAM. :~p=0.05, two-tailed, compared with either NL or UN. §p=0.008, two-tailed, compared with UN, p=0.114 compared with NL. The thionin-stained sections revealed a pattern of hippocampal damage very similar to that reported several times previously [ 12, 13, 14]. There was substantial ablation of the dorsal and dorsolateral hippocampus and dentate g y m s , with sparing of the more ventral and posterior aspects. Some entorhinal tissue was invaded in some animals, and the subiculum was partially invaded in virtually all animals. In the neopallial-lesioned rats there was extensive removal of both white and gray matter overlying the hippocampus, but the hippocampal formation itself was not invaded. RESULTS
Open Field Locomotion Overall, the results in the open field were similar to those reported earlier [14]. On Postoperative Days 2 and 4 there
K1MBLE E l AL.
464 TABLE 2 MEAN FIRST TRIAL SPONTANEOUS ALTERNATION (TOTAL % ALTERNATION)
Group
Pre-Op 3
5
Postoperative Day 9
13
18
UN-SHAM UN-SCG Combined UN
100% (100) 100% (90) 100% (95)
75% (62) 40% (60) 56% (61)
100%(87) 100%(90) 10(~o(88)
75% (75) 100%(90) 89% (83)
75% (75) 10(1%(100) 89% (88)
75% (87) 10t1%(90) 89% (83)
NL-SHAM NL-SCG Combined NL
100% (87) 100% (87) 100% (87)
50% (50) 67% (50) 57% (50)
10(~v(87) 75% (75) 75% (81)
100%(62) 50% (37) 75% (50)
5ffYb(62) 75% (62) 62% (62)
75% (87) 75% (75) 75% (81)
HP-SHAM HP-SCG Combined HP
100% (92) 100% (100) 100% (96)
0% (33) 50% (33) 39% (33)
60% (60) 50% (36) 55% (48)
33% (30) 83% (75) 58% (52)
17% (33) 50% (50) 33% (42)
60% (50) 50% (42) 55% (46)
was a significant hypoactivity displayed in the open field by the hippocampal-lesioned rats, which was replaced on Postoperative Day 8 by a significant hyperactivity. Table 1 indicates the probability levels associated with these differences, as evaluated with Mann-Whitney U tests [24]. The hypoactivity on r, ays 2 and 4 was attenuated in the group that received ganglionectomies as well as hippocampal lesions (p=0.041, one-tailed, 0.082, two-tailed). No significant differences were seen between the HP-SCG rats and any of the control groups: they displayed no significant open field hypoactivity, although two rats in this group did display some motoric lethargy, as described below. By Postoperative Day 12 there were no further activity differences noted. Overall, the rats in this experiment were about 25% less active in the open field than was true in an earlier experiment [14] using similar procedures. One possible explanation is that the rats in the present experiment were from a SpragueDawley strain obtained from Charles River, Inc. while those used previously were bred at Oregon from a Carworth Farms strain. Although the hypoactivity phase was attenuated in those rats receiving ganglionectomies, there is currently no evidence for or against any anomalous growth within the first four postoperative days. However, this has not been tested adequately. There is definite evidence for some anomalous growth at 8 days postoperatively. According to R. Loy (personal communication) it is not very likely that there is any substantial anomalous growth by Postoperative Day 4. Thus, it is not possible now to relate the attenuation of hypoactivity seem in the SCG rats with hippocampal-lesions to the absence of anomalous innervation, although this remains a theoretical possibility. Other than the overall lowered activity level in the open field, the present results are in good accord with those published previously concerning the effects of hippocampal and neopallial lesions. These effects can be summarized as little or no effect of the neopalliallesions and a transitory hypoactivity followed by a transitory hyperactivity with a return to near-normal levels of activity by about two weeks postoperatively in the hippocampallesioned animals [14]. The extreme "motoric lethargy" observed previously in some hippocampal lesioned rats in the open-field was seen in 5/12 (three from Group 5, two from Group 6) hippocampallesioned rats in the present experiment. In all cases this was present only on Postoperative Days 2 and 4 and disappeared
by Day 8. This phenomenon was not seen in neopalliallesioned rats or unoperated controls. As described previously, those rats displaying this behavior "tended to retain a given posture for long periods of time, and when they did move, they did so in a 'slow-motion', slinking fashion. Also, the exactness with which these animals would retain a particular posture was remarkable; they often held a pose unchanged for several minutes at a time [14]." With the present rats, as with previous rats displaying this behavior, attempts to have these animals retain unusual or abnormal postures (on their backs, facing down against a cage wall, etc.) were not successful. They would assume normal postures which were then often held for several minutes. Thus, the term "waxy flexibility" does not seem appropriate, although there is a considerable inertia of movement. It was not possible to discern any other differences, either in other behaviors, or histological material, from those rats which did and those which did not display this phenomenon.
Spontaneous Alternation There was no discernable effect of gangtionectomy on levels of spontaneous alternation in any of the three brain lesion experimental groups. Both first choice and total percentage alternation were examined, and no significant differences were seen in these measures (Table 2). There was a statistically significant effect of the hippocampal lesion, however, which reduced the level of spontaneous alternation compared with the unoperated controls (U= 1 I, p =0.02) or the neopallial-lesioned rats (U=19, p=0.05). Although the level of spontaneous alternation was somewhat lower in the rats with neopallial lesions, the difference between this rate and unoperated control levels did not reach the 0.05 level of statistical significance (U--18).
Spatial Maze Learning As has been reported many times previously, spatial maze learning is quite sensitive to brain damage. A 2-way ANOVA revealed a significant lesion effect, F(2,28)=77.39, p <0.001, and a significant maze ×lesion interaction, F(4,28) =3.68, p<0.01, but no maze effect, F(2,28)= 1.26, ns. Individual group comparisons revealed highly significant differences in total number of errors on the three mazes between the hippocampal-lesioned animals and the unoperated controls ( U - 0 , p=0.002), and between the hippocampal-
HIPPOCAMPECTOMY AND GANGLIONECTOMY
465
TABLE 3 MEAN (RANGE)ERRORS ON SPATIALMAZES Group
Maze No. 1
No.5
No. 11
Total
UN-SHAM UN-SCG Combined UN
16 (9-25) 11 (6--17) 13 (6-25)
22 (18-32) 27 (H A~) 25 (14--44)
17 (13-26) 25 (8-45) 21 (8--45)
56 (41-83) 63 (28-99) 60 (28--99)
NL-SHAM NL-SCG Combined NL
26 (18-38) 22 (19-25) 24 (18--38)
37 (2%55) 28 (16--48) 33 (16-55)
34 (27--46) 24 (14-37) 29 (14-46)
97 (72-111) 75 (56-106) 86 (56-111)
HP-SHAM HP-SCG Combined HP
74 (39-108) 70 (20-144) 72 (20-144)
59 (44-85) 53 (31-74) 56 (31-85)
88 (42-127) 84 (57-111) 86 (42-127)
221 (194-272) 206 (162-268) 214 (162-272)
lesioned animals and the neopallial-lesioned animals ( U = 0 , p =0.002). No overlap in total errors appeared between the hippocampal-lesioned group and any of the other animals. The " b e s t " hippocampal-lesioned rat made 162 total errors, while the " w o r s t " neopallial-lesioned animal made 111 total errors. The neopallial-lesioned rats were somewhat impaired, making an average of 86 total errors to 60 for the unoperated controls ( U = 14, p =0.05). The mean number of errors made by the hippocampal-lesioned rats was 214. Most of the difference between the neopallial-lesioned animals and the unoperated controls occurred on the RabinovitchRosvold pattern 1, (U=7.5, p=0.02) which we have repeatedly found to be extremely sensitive to brain damage effects. The differences between the neopallial-lesioned animals and the unoperated group were not statistically significant for either of the other two maze patterns used. On the other hand, the hippocampal-lesioned rats made significantly more errors on each of the other two maze patterns than did either of the other two groups (Table 3). Individual group tests did not reveal any significant differences in those animals which received brain lesions in addition to SCG removal as compared with those animals which received brain lesions in addition to only a sham SCG operation. DISCUSSION Our data do not reveal any behavioral results which can
be unambiguously related to the observed anomalous sympathetic innervation of the remaining hippocampal tissue following lesions to the anterior and dorsal hippocampus. We were able to demonstrate both the anomalous sympathetic innervation discovered by Loy and Moore [19] and its elimination by superior cervical ganglionectomy. The expected brain lesion effects on open field locomotion, spontaneous alternation and spatial maze acquisition were observed. It is, of course, possible that other behavioral measures would reveal differences between animals with and without anomalous sympathetic innervation, although spatial maze learning has repeatedly proven to be highly sensitive to hippocampal damage [4, 7, 8, 9, 10, 11, 12, 16, 20, 21, 22, 25]. It is also possible that less extensive hippocampal lesions, thei'eby increasing the amount of remaining hippocampal tissue available for innervation might reveal behavioral differences between animals with and without prior superior cervical ganglionectomy. On the other hand, it does not appear on the basis o f our results, that well-established effects o f hippocampal lesions (e.g., spatial maze learning difficulties) can be ascribed to the anomalous CNS sympathetic innervation of remaining hip pocampal tissue. Nevertheless, the possibility that the abnormal innervation does contribute in some way to the behavioral sequelae of central nervous system injury remains an important if as yet undocumented possibility, warranting further research.
REFERENCES
1. BreMiller, R. A. A rapid technique of preparing frozen sections of small brains. Physiol. Behav. 6: 463-464, 1971. 2. de la Torre, J. C. and J. W. Surgeon. A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The SPG method. Histochemistry 49: 81-93, 1976. 3. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. 4. Gross, C. G., S. L. Chorover and S. M. Cohen. Caudate, conical, hippocampal and dorsal thalamic lesions in rats: Alternation and Hebb-Williams Maze performance. Neuropsychologia 3: 53--68, 1965. 5. Hebb, D. O. and K. Williams. A method of ratinganimal intelligence. J. genet. Psychol. 34: 59-65, 1946.
6. Hokfelt, T. and A. Ljungdahl. Modification of the Falck-Hillarp formaldehyde fluorescence method using the vibratome: simple, rapid sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue. Histochemie 29: 324-339, 1972. 7. Hostetter, G. and G. J. Thomas. Evaluation of enhanced thigmotaxis as a condition of impaired maze learning by rats with hippocampal lesions. J. comp. physiol. Psychol. 63: 105--110, 1967. 8. Isaacson, R. L. Experimental brain lesions and memory. In: Neural Mechanisms of Learning and Memory, edited by M. R. Rosenzweig and E. L. Bennett. Cambridge: MIT Press, 1976, pp. 521-543.
466 9. Jackson, W. J. and P. N. Strong. Differential effects of hippocampal lesions upon sequential tasks and maze learning in the rat. J. comp. physiol. Psychol. 68: 442-450, 1969. 10. Jarrard, L. E. and T. C. Lewis. Effects of hippocampal ablation and intertrial interval on complex maze acquisition. Am. J. Psychol. 80: 66--72, 1967. 11. Kaada, B. R., E. W. Rasmussen and O. Kviem. Effects of hippocampal lesions on maze learning and retention in rats. Expl Neurol. 3: 333-355, 1961. 12. Kimble, D. P. The effects of bilateral hippocampal lesions in rats. J. comp. physiol. Psychol. 56: 273-283, 1963. 13. Kimble, D. P. Choice behavior in rats with hippocampai lesions. In: The Hippocampus, A Comprehensive Treatise, edited by R. L. Isaacson and K. H. Pribram. New York: Plenum Press, 1975, pp. 309-326. 14. Kimble, D. P. Changes in behavior of hippocampai-lesioned rats across a 6-week postoperative period. Physiol. Psychol. 4: 28%293, 1976. 15. Kimble, D. P. Effects of combined entorhinal cortexhippocampal lesions on locomotor behavior, spontaneous alternation and spatial maze learning in the rat. Physiol. Behav. 21: 177-187, 1978. 16. Kimble, D. P. and E. Dannen. Persistent spatial maze-learning deficits in hippocampal-lesioned rats across a 7-week postoperative period. Physiol. Psychol. 5: 409-413, 1977.
KIMBLE El' AL. 17. Koizumi, K. and C. McC. Brooks. The autonomic nervous system and its role in controlling visceral activities. In: Medical Physiology, Vol. 1, edited by V. B. Mountcastle. St. Louis: C. V. Mosby, 1974, p. 783. 18. Lindvall, O. and A. Bjorklund. The glyoxylic acid fluorescence histochemical method: A detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistrv 39: 97-127, 1974. 19. Loy, R. and R. Y. Moore. Anomalous innervation of the hippocampal formation by peripheral sympathetic axons following mechanical injury. Expl Neurol. 57: 645-650, 1977. 20. Myhrer, T. Maze performance in rats with hippocampal perforant path lesions: Some aspects of functional recovery. Physiol. Behav. 15: 433--437, 1975. 21. Niki, H. Response perservation following hippocampal ablation in the rat..lap, psychol. Res. g: 1-9, 1966. 22. Olton, D. S. Spatial memory. Scient. Am. 236: 82-98, 1977. 23. Rabinovitch, M. S. and H. E. Rosvold. A closed-field intelligence test for rats. Can. J. Psychol. 5: 122-128, 1951. 24. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 25. Stein, D. G. and D. P. Kimble. The effects of hippocampal lesions and posttrial strychnine administration on maze behavior in the rat. J. comp. physiol. Psychol. 62: 243-249, 1966.
Hippocampal Lesions, Superior Cervical Ganglia Removal, and Behavior in Rats I D A N I E L P. K I M B L E , S U S A N A N D E R S O N , R U T H B R E M I L L E R A N D E U G E N E D A N N E N
Department of Psychology, University of Oregon, Eugene, OR 97403 (Received 8 August 1978) KIMBLE, D. P., S. ANDERSON, R. BREMILLER AND E. DANNEN. Hippocampal lesions, superior cervical ganglia removal, and behavior in rats. PHYSIOL. BEHAV. 22(3) 461--466, 1979.--Rats were subjected to either bilateral removal of the superior cervical ganglia (SCG) or to a sham operation of the neck. Subsequently, these rats received either bilateral neopallial lesions, bilateral dorsal hippocampal lesions or no further operation. The SCG removal prevented the anomalous noradrenergic innervation of remaining hippocampal tissue. This anomalous innervation, as reported by Loy and Moore [19], originates from neurons in the SCG. All animals were then evaluated on behaviors previously shown to be sensitive to hippocampal destruction: open field activity, spontaneous alternation and spatial maze learning, in order to determine if any behavioral consequences could be related to the anomalous hippoeampal innervation. No statistically significant differences appeared which could be unambiguously related to the anomalous innervation.
Hippocampus Superiorcervical ganglia Open field activity Spatialmaze learning
Anomalousnoradrenerglc innervation
LOY and Moore [19] have reported the growth of anomalous fibers from the superior cervical ganglia (SCG) into the hippocampal formation following aspiration lesions to the anterior 2-3 mm of the hippocampal formation. This novel pattern of innervation was discovered using fluorescence histochemical procedures [6,18]. This new plexus had the characteristic appearance of peripheral sympathetic fibers. The anomalous innervation extended throughout the remaining portion of the hippocampal formation, including the area dentata and Ammon's horn. Ultrastructural studies currently in progress suggest that these fibers may make synapses, particularly in the hilar zone of the dentate gyrus (R. Y. Moore, R. Loy, personal communication). As indicated in the Loy and Moore report, "such anomalous growth could have significant implications for the sequelae to injury of the central nervous system of both experimental animals and man" [19]. The present experiments were designed to test these functional implications in rats in which this anomalous innervation was present, and in rats in which such anomalous growth was prevented by removal of the source of the fibers, the superior cervical ganglia.
METHOD
Animals The animals were 30 male Sprague-Dawley albino rats (CD, random-bred) obtained from the Charles River Co., Wilmington, MA. They were maintained in individual cages
Spontaneous alternation
on a 12 hr diurnal light cycle. Rats were approximately 75-80 days of age at the time of the first operation (superior cervical ganglia removal). Except for the times specified in the procedure, food and water were available on an ad lib basis.
Apparatus Locomotor activity was measured in a wooden box measuring 78.7x78.7 cm with 11.4 cm high walls. The floor was marked offby white lines into 36 equal size squares and the entire apparatus was painted gray and covered with a sheet of clear Plexiglas. A similar apparatus, painted a slightly darker gray was used in ths maze acquisition phase. This apparatus, together with wooden barriers, a start box and a goal box, constituted a Hebb-Williams maze apparatus [5]. The maze patterns were Nos. 1, 5 and 11 (one "easy", one "moderately difficult", and one "difficult") from the Rabinovitch-Rosvold series [23]. The start box and goal box were the same size, each measuring 30 cm in length, 13.5 cm in width, and 15 cm in height. Both boxes were equipped with fiber-board guillotine-type doors and Plexiglas tops. Spontaneous alternation was evaluated in a wooden T-maze constructed of a 56.3 x 11.4 cm stem and two 31.7x 24.1 cm goal arms, set at right angles to the main stem. Both goal arms were equipped with Plexiglas guillotine-type doors. The walls of the apparatus were 18 cm high.
Procedure Preoperative training. All animals were gentled for sev-
~This project was supported in part by BRSC Grant RR 07080 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/030461-06502.00/0
K I M B L E E l AL.
462 eral days following their arrival. Two weeks after their arrival they were pre-trained in the maze for seven days. The following day animals were placed, one at a time, in the center of the open field and allowed 5 min of free exploration, during which time the number of squares entered was recorded. An entry was scored if a rat placed its two front paws in a square. The following day, each rat was given three trials in the T-maze to determine its preoperative spontaneous alternation rate. Each rat was released from the start box into the main stem of the maze and allowed up to 3 min to enter one of the goal arms. Upon entry into one of the arms, the guillotine door was then gently closed behind the rat, 20 sec allowed to elapse, and the rat replaced in the start box for the next trial. All 30 rats completed three trials, with an overall alternation rate of 93% (100% of first free trial choices). Surgery. Superior cervical ganglia removal: Fifteen animals underwent bilateral superior cervical ganglionectomy. This operation took place five to seven days prior to brain operations. Anesthesia in all cases was induced by sodium pentobarbital (Nembutal), in doses of 50 mg/kg, IP. Atropine sulfate, in doses of approximately 0.04 mg/animal was also administered to reduce mouth and throat secretions. The ganglionectomy procedure was worked out in a series of pilot operations. The superior cervical ganglion in the rat lies in the Y-shaped junction formed by the bifurcation of the common carotid to form the internal and external carotid arteries. To visualize this ganglion, the rat was taped to a board on its back and a mid-line incision made in the skin from just below the mandible to just below the beginning of the chest musculature, a distance of about 3 cm. Upon retraction of the skin, the tissue overlying the SCG was blunt-dissected and laid to the side of the opening. The most superficial structures are the large submaxillary glands and the somewhat smaller and more anterior sublingual glands. These can be freed from the membrane holding them to the underlying musculature quite easily with no bleeding. The thyroid and parathyroid glands may be left undisturbed. Blunt dissection along the edge of the sternohyoideus muscle will expose the carotid which can be identified by its position and obvious pulsing. The carotid is then gently freed from the membranes binding it to its underlying musculature. Upon lifting the carotid at a level approximately 1 mm posterior to the posterior tip of the hyoid process, the SCG becomes visible at the juncture of the internal and external carotids. It is ovoid in shape, grayish-white in color and resembles a very small, uncooked grain of rice. Once visualized, the SCG was grasped with iris forceps and gently pulled away from its connections with the sympathetic chain together with a portion of the sympathetic chain. This operation was performed under low-power (10 to 16X) magnification. The instrument used in this experiment was a Zeiss binocular zoom operating microscope. While this operation can be performed in a few minutes with virtually no bleeding, great care must be taken not to rupture the carotid artery. If broken, bleeding from this artery can be somewhat troublesome to stop, although it can be done. In only two animals was this a problem. Both animals were assigned to the nobrain lesion group. There were no observed behavioral effects of the bleeding. Fifteen animals received sham SCG operations in which the ganglia were visualized but left inact. Brain lesion. Five to seven days following ganglionectomy eight animals underwent bilateral lesions of the neopallium overlying the hippocampus, including cortical tissue and part of the underlying white matter, primarily cor-
pus callosum. Another group of 12 rats underwent bilateral lesions of the dorsal and anterior aspects of the hippocampal formation. All operations were performed in one stage by aspiration, as described previously [12]. Ten additional animals underwent no operations and served as unoperated controls. Half of this group had, however, undergone previous bilateral ganglionectomy. Thus, six experimental groups were formed: (1) Unoperated, sham SCG operation (UNSHAM), (2) Unoperated, superior cervical ganglia removed (UN-SCG), (3) Neopallial-lesioned, sham SCG operation (NL-SHAM), (4) Neopallial-lesioned, superior cervical ganglia removed (NL-SCG), (5) Hippocampal-lesioned, sham SCG operation (HP-SHAM), and (6) Hippocampallesioned, superior cervical ganglia removed (HP-SCG). One animal in Group 1 developed a respiratory infection and was discarded prior to testing; all other rats survived all surgical procedures and completed all phases of the experiment. Postoperative testing. Postoperative testing was done in two stages, immediate and longer-term. The immediate phase lasted from Postoperative Day 2 to Day 18. The longer-term phase lasted from Postoperative Days 52-55. During the early postoperative period, the animals were repeatedly examined on locomotor activity in the open field and for spontaneous alternation, two behaviors which have previously been shown to be quite sensitive to hippocampal damage [3,13] and (in our laboratory) to show rather substantial returns to preoperative levels during the first 3 weeks postoperatively [14]. In the later phase, the animals were tested on the acquisition of spatial maze habits, a behavior which is also sensitive to hippocampal damage but appears to be a "persistent" or permanent sequelae of such lesions [15, 16, 221. Open field locomotor behavior was examined on Postoperative Days 2, 4, 8, 12, and 17. The procedure was identical with that used preoperatively. Spontaneous alternation was measured on Postoperative Days 3, 5, 9, 13 and 18. Procedures were identical with those used preoperatively. Both food and water were available on an ad lib basis. Spatial maze learning capacity was tested using three different maze problems selected from the RabinovitchRosvoid series. The set included maze problems 1, 5 and 11. Each rat was run on each of these problems in that order, on consecutive days. Each rat was run seven trials on each problem. All rats were placed on a 24 hr water deprivation schedule (water available for 10 min) on Postoperative Day 40 and kept on this schedule throughout testing. A water reward was available in the goal box. The rat's path through the maze was recorded on a data sheet containing the floor plan of that maze problem. An error was recorded if two or more paws crossed an error line. HISTOLOGY
Following the completion of the experiment, the brainlesioned animals were sacrificed by decapitation and the unperfused brains removed and frozen in a cryostat at -30°C. Irises were removed from a number of animals with and without ganglionectomy to further check on superior cervical ganglia removal. Irises were removed from the eyes in a procedure described by R. Y. Moore (personal communication). The eyeball was removed, cut along the anteriorposterior midline and the lens removed and discarded. The anterior half of the eye was placed downward and the iris grasped with iris forceps and laid flat (in pieces) on a glass slide. The slides were placed in a dessicator containing
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463
FIG. 1. Frontal sections through hippocampal formation showing anomalous noradrenergic fibers in rat with anterior hippocampal lesion (on left) and absence of such anomalous fibers in rat with hippocampal lesion plus prior bilateral removal of superior cervical ganglionectomy (on fight). Fluorescence pattern in hippocampus on fight is due primarily to locus coeruleus fibers [19].
phosphorous pentoxide and kept there overnight. The slides were then transferred to a closed jar containing paraformaldehyde which had been previously equilibrated in an atmosphere of 70% relative humidity. After incubating for an hour at 80°C, the slides were removed, coverslipped with mineral oil and examined under a Zeiss fluorescent microscope. The brains were sectioned at either 16 or 32 microns. Alternate sections were then subjected to a modified glyoxylic acid technique to examine for catecholamine content [2]. These sections were also examined under the fluorescent microscope. The alternate sections were stained with thionin according to BreMiller's procedure [1].
Results Following the operation to remove the superior cervical ganglia a ptosis of the rat's eyes was observed, due to cutting the superior cervical trunk with a resultant retraction of the eyeball [17]. No behavioral consequences of this ptosis were observed. Examination of the irises from either normal or shamoperated animals revealed an extensive network o f noradrenergic fibers under the fluorescent microscope. Irises from animals with superior cervical ganglia removed showed no such fluorescence. The brain-sections through the hippocampal-lesioned animals which were stained and examined for catecholamine fluorescence showed the extensive pattern of anomalous sympathetic innervation in the intact hippocampal tissue as described by Loy and Moore [19]. Figure 1 shows relevant brain sections through hippocampal tissue from rats with and without SCG removal. The growth was found throughout the hippocampal tissue spared by the lesion, but not elsewhere. This anomalous growth revealed a new plexus of very fluorescent fibers, much thicker and coarser than ever found in normal brains [19] or in our rats with SCG removal. There is also a normal fluorescence observed in the hippocampal formation which is due primarily to fibers from locus coeruleus neurons [19], which can also be seen in Fig. 1. Only the normal growth was observed in those brains from rats with SCG removal. The anomalous growth was observed in all hippocampal-lesioned animals not subjected to SCG removal-.
TABLE 1 MEAN NUMBER OF SQUARES ENTERED IN 5 MIN IN OPEN FIELD, PRE- AND POSTOPERATIVELY Group
Pre-Op 2
Postoperative Day 4 8 12
17
UN-SHAM UN-SCG Combined UN
144 181 164
118 128 124
139 141 140
118 137 129
132 137 134
135 135 135
NL-SHAM NL-SCG Combined NL
186 171 179
111 86 99
98 112 105
138 105 122
139 96 118
132 102 117
HP-SHAM HP-SCG Combined HP
168 173 170
69§ 156t 117
179 179 179¢
148 148 148
117 110 114
32* 90~ 63
*p=0.006, two-tailed, compared with combined NL, p=>0.001 compared with combined UN. tp=0.082, two-tailed; 0.041, one-tailed, compared with HPSHAM. :~p=0.05, two-tailed, compared with either NL or UN. §p=0.008, two-tailed, compared with UN, p=0.114 compared with NL. The thionin-stained sections revealed a pattern of hippocampal damage very similar to that reported several times previously [ 12, 13, 14]. There was substantial ablation of the dorsal and dorsolateral hippocampus and dentate g y m s , with sparing of the more ventral and posterior aspects. Some entorhinal tissue was invaded in some animals, and the subiculum was partially invaded in virtually all animals. In the neopallial-lesioned rats there was extensive removal of both white and gray matter overlying the hippocampus, but the hippocampal formation itself was not invaded. RESULTS
Open Field Locomotion Overall, the results in the open field were similar to those reported earlier [14]. On Postoperative Days 2 and 4 there
K1MBLE E l AL.
464 TABLE 2 MEAN FIRST TRIAL SPONTANEOUS ALTERNATION (TOTAL % ALTERNATION)
Group
Pre-Op 3
5
Postoperative Day 9
13
18
UN-SHAM UN-SCG Combined UN
100% (100) 100% (90) 100% (95)
75% (62) 40% (60) 56% (61)
100%(87) 100%(90) 10(~o(88)
75% (75) 100%(90) 89% (83)
75% (75) 10(1%(100) 89% (88)
75% (87) 10t1%(90) 89% (83)
NL-SHAM NL-SCG Combined NL
100% (87) 100% (87) 100% (87)
50% (50) 67% (50) 57% (50)
10(~v(87) 75% (75) 75% (81)
100%(62) 50% (37) 75% (50)
5ffYb(62) 75% (62) 62% (62)
75% (87) 75% (75) 75% (81)
HP-SHAM HP-SCG Combined HP
100% (92) 100% (100) 100% (96)
0% (33) 50% (33) 39% (33)
60% (60) 50% (36) 55% (48)
33% (30) 83% (75) 58% (52)
17% (33) 50% (50) 33% (42)
60% (50) 50% (42) 55% (46)
was a significant hypoactivity displayed in the open field by the hippocampal-lesioned rats, which was replaced on Postoperative Day 8 by a significant hyperactivity. Table 1 indicates the probability levels associated with these differences, as evaluated with Mann-Whitney U tests [24]. The hypoactivity on r, ays 2 and 4 was attenuated in the group that received ganglionectomies as well as hippocampal lesions (p=0.041, one-tailed, 0.082, two-tailed). No significant differences were seen between the HP-SCG rats and any of the control groups: they displayed no significant open field hypoactivity, although two rats in this group did display some motoric lethargy, as described below. By Postoperative Day 12 there were no further activity differences noted. Overall, the rats in this experiment were about 25% less active in the open field than was true in an earlier experiment [14] using similar procedures. One possible explanation is that the rats in the present experiment were from a SpragueDawley strain obtained from Charles River, Inc. while those used previously were bred at Oregon from a Carworth Farms strain. Although the hypoactivity phase was attenuated in those rats receiving ganglionectomies, there is currently no evidence for or against any anomalous growth within the first four postoperative days. However, this has not been tested adequately. There is definite evidence for some anomalous growth at 8 days postoperatively. According to R. Loy (personal communication) it is not very likely that there is any substantial anomalous growth by Postoperative Day 4. Thus, it is not possible now to relate the attenuation of hypoactivity seem in the SCG rats with hippocampal-lesions to the absence of anomalous innervation, although this remains a theoretical possibility. Other than the overall lowered activity level in the open field, the present results are in good accord with those published previously concerning the effects of hippocampal and neopallial lesions. These effects can be summarized as little or no effect of the neopalliallesions and a transitory hypoactivity followed by a transitory hyperactivity with a return to near-normal levels of activity by about two weeks postoperatively in the hippocampallesioned animals [14]. The extreme "motoric lethargy" observed previously in some hippocampal lesioned rats in the open-field was seen in 5/12 (three from Group 5, two from Group 6) hippocampallesioned rats in the present experiment. In all cases this was present only on Postoperative Days 2 and 4 and disappeared
by Day 8. This phenomenon was not seen in neopalliallesioned rats or unoperated controls. As described previously, those rats displaying this behavior "tended to retain a given posture for long periods of time, and when they did move, they did so in a 'slow-motion', slinking fashion. Also, the exactness with which these animals would retain a particular posture was remarkable; they often held a pose unchanged for several minutes at a time [14]." With the present rats, as with previous rats displaying this behavior, attempts to have these animals retain unusual or abnormal postures (on their backs, facing down against a cage wall, etc.) were not successful. They would assume normal postures which were then often held for several minutes. Thus, the term "waxy flexibility" does not seem appropriate, although there is a considerable inertia of movement. It was not possible to discern any other differences, either in other behaviors, or histological material, from those rats which did and those which did not display this phenomenon.
Spontaneous Alternation There was no discernable effect of gangtionectomy on levels of spontaneous alternation in any of the three brain lesion experimental groups. Both first choice and total percentage alternation were examined, and no significant differences were seen in these measures (Table 2). There was a statistically significant effect of the hippocampal lesion, however, which reduced the level of spontaneous alternation compared with the unoperated controls (U= 1 I, p =0.02) or the neopallial-lesioned rats (U=19, p=0.05). Although the level of spontaneous alternation was somewhat lower in the rats with neopallial lesions, the difference between this rate and unoperated control levels did not reach the 0.05 level of statistical significance (U--18).
Spatial Maze Learning As has been reported many times previously, spatial maze learning is quite sensitive to brain damage. A 2-way ANOVA revealed a significant lesion effect, F(2,28)=77.39, p <0.001, and a significant maze ×lesion interaction, F(4,28) =3.68, p<0.01, but no maze effect, F(2,28)= 1.26, ns. Individual group comparisons revealed highly significant differences in total number of errors on the three mazes between the hippocampal-lesioned animals and the unoperated controls ( U - 0 , p=0.002), and between the hippocampal-
HIPPOCAMPECTOMY AND GANGLIONECTOMY
465
TABLE 3 MEAN (RANGE)ERRORS ON SPATIALMAZES Group
Maze No. 1
No.5
No. 11
Total
UN-SHAM UN-SCG Combined UN
16 (9-25) 11 (6--17) 13 (6-25)
22 (18-32) 27 (H A~) 25 (14--44)
17 (13-26) 25 (8-45) 21 (8--45)
56 (41-83) 63 (28-99) 60 (28--99)
NL-SHAM NL-SCG Combined NL
26 (18-38) 22 (19-25) 24 (18--38)
37 (2%55) 28 (16--48) 33 (16-55)
34 (27--46) 24 (14-37) 29 (14-46)
97 (72-111) 75 (56-106) 86 (56-111)
HP-SHAM HP-SCG Combined HP
74 (39-108) 70 (20-144) 72 (20-144)
59 (44-85) 53 (31-74) 56 (31-85)
88 (42-127) 84 (57-111) 86 (42-127)
221 (194-272) 206 (162-268) 214 (162-272)
lesioned animals and the neopallial-lesioned animals ( U = 0 , p =0.002). No overlap in total errors appeared between the hippocampal-lesioned group and any of the other animals. The " b e s t " hippocampal-lesioned rat made 162 total errors, while the " w o r s t " neopallial-lesioned animal made 111 total errors. The neopallial-lesioned rats were somewhat impaired, making an average of 86 total errors to 60 for the unoperated controls ( U = 14, p =0.05). The mean number of errors made by the hippocampal-lesioned rats was 214. Most of the difference between the neopallial-lesioned animals and the unoperated controls occurred on the RabinovitchRosvold pattern 1, (U=7.5, p=0.02) which we have repeatedly found to be extremely sensitive to brain damage effects. The differences between the neopallial-lesioned animals and the unoperated group were not statistically significant for either of the other two maze patterns used. On the other hand, the hippocampal-lesioned rats made significantly more errors on each of the other two maze patterns than did either of the other two groups (Table 3). Individual group tests did not reveal any significant differences in those animals which received brain lesions in addition to SCG removal as compared with those animals which received brain lesions in addition to only a sham SCG operation. DISCUSSION Our data do not reveal any behavioral results which can
be unambiguously related to the observed anomalous sympathetic innervation of the remaining hippocampal tissue following lesions to the anterior and dorsal hippocampus. We were able to demonstrate both the anomalous sympathetic innervation discovered by Loy and Moore [19] and its elimination by superior cervical ganglionectomy. The expected brain lesion effects on open field locomotion, spontaneous alternation and spatial maze acquisition were observed. It is, of course, possible that other behavioral measures would reveal differences between animals with and without anomalous sympathetic innervation, although spatial maze learning has repeatedly proven to be highly sensitive to hippocampal damage [4, 7, 8, 9, 10, 11, 12, 16, 20, 21, 22, 25]. It is also possible that less extensive hippocampal lesions, thei'eby increasing the amount of remaining hippocampal tissue available for innervation might reveal behavioral differences between animals with and without prior superior cervical ganglionectomy. On the other hand, it does not appear on the basis o f our results, that well-established effects o f hippocampal lesions (e.g., spatial maze learning difficulties) can be ascribed to the anomalous CNS sympathetic innervation of remaining hip pocampal tissue. Nevertheless, the possibility that the abnormal innervation does contribute in some way to the behavioral sequelae of central nervous system injury remains an important if as yet undocumented possibility, warranting further research.
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