Brain Research, 584 (1992) 117-122
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00 BRES 17890
Hippocampal laminar glucose utilization and theta rhythm following unilateral fimbria-fornix lesions in rats G.A. Zimmerman Departments of
S.E. Fox b, L.A. Freed
and D.L. Dow-Edwards
Neurosurgery, b Physiology and c Anatomy, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York (USA)
(Accepted 11 February 1992)
Key words: Theta rhythm; Hippocampus; Septohippocampal pathway; Fimbria-fornix lesion; Glucose metabolism; 2-Deoxyglucose
Laminar profiles of glucose utilization were related to the presence or absence of movement-related hippocampal theta rhythm in CA1 and dentate gyrus of rats after aspirative unilateral combined lesions of the fimbria-fornix and cingulum. Three groups were studied: (1) sham-operated rats, (2a) lesioned rats with an ipsilaterai loss of theta activity at 4 weeks post-lesion that persisted at 12 weeks post-lesion, and (2b) lesioned rats with a loss of theta activity at 4 weeks post-lesion, but a recovery of theta rhythm at 12 weeks post-lesion. Fimbriafornix/cingulum lesions served both to abolish ipsilateral theta rhythm and to decrease ipsilateral glucose metabolism in all cell layers of CA1 and the dentate gyrus, when normalized to the contralateral hemisphere. Although glucose metabolism in lesioned animals with a recovery of theta rhythm was not as high as control levels, in several laminae it was significantly higher than that of lesioned animals with persistent loss of theta rhythm. These laminae included the dentate hilus and strata oriens, pyramidale and lacunosum-moleculare of CA1. The increased glucose metabolism associated with the return of theta rhythm suggests a functional reinnervation of these layers of the hippocampus in such animals.
INTRODUCTION Two types of h i p p o c a m p a l theta rhythm, or rhythmic slow-wave activity, have b e e n described in rats: m o v e m e n t - r e l a t e d theta r h y t h m occurs primarily with translational m o v e m e n t s of the rat such as walking; 'immobility' theta r h y t h m occurs during certain motionless behaviors, and u n d e r u r e t h a n e or ether anesthesia 1°. G e n e r a t o r s of theta rhythm have been identified in the stratum oriens of CA1, molecular layer of the dentate gyrus ~, cingulate cortex 8, and entorhinal cortex 15. T h e p r o d u c t i o n of theta rhythm is d e p e n d e n t on rhythmic activity of ' p a c e m a k e r ' cells in the medial septal nucleus and the nucleus of the diagonal b a n d 2°. Projections f r o m these nuclei 24 are the major source of cholinergic innervation of the h i p p o c a m p u s 11. Acetylcholinesterase (ACHE) staining corresponds to the laminar distribution o f septal projections to the hippo-
c a m p u s 12 and is indicative of an intact septohippocampal pathway. H i p p o c a m p a l deafferentation with timbria-fornix lesions has b e e n shown both to diminish cholinergic input to the h i p p o c a m p u s by 90% 6 and to disrupt theta activity 7. Thus, the loss of septal input to the hippocampus, indicated by a reduction in A C h E staining, causes a loss o f the theta rhythm. H i p p o c a m pal 'function' as m e a s u r e d by p e r f o r m a n c e of behavioral tasks involving spatial m e m o r y is also seriously disrupted by loss of s e p t o h i p p o c a m p a l afferents 16,26. Local cerebral glucose utilization ( L C G U ) m e a s u r e d using the 2-deoxyglucose ( 2 D G ) m e t h o d of Sokoloff is an index of the level of n e u r o n a l activity in brain structures 22. This study was designed to indicate w h e t h e r or not there was a relationship between movement-related theta rhythm and hippocampal L C G U , utilizing unilateral aspirative c o m b i n e d fimbria-fornix and cingulum lesions, and a laminar analysis of L C G U in the hippocampus. T h e laminar analysis was per-
D.D.E. and L.A.F. are currently associated with the Department of Pharmacology, SUNY Health Science Center at Brooklyn, G.A.Z. is currently associated with New York Hospital, Cornell Medical Center, New York, NY. Correspondence: D. Dow-Edwards, Department of Pharmacology, Box 29, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.
118 formed in CA1 and the dentate gyrus because of their well-established involvement in the generation of the hippocampal theta rhythm25. MATERIALS AND METHODS Twelve female L o n g - E v a n s rats (225-275 g) were used in this study. Hippocampal recording electrodes were implanted stereotaxically. Animals were then randomly divided into 2 groups: (1) shamlesion and (2) unilateral fimbria-fornix/cingulum aspirative lesion. On the basis of the results of electrophysiological recordings taken at 12 weeks after surgery, the lesioned animals were further divided into 2 groups: (2a) lesioned rats with persistent loss of theta activity and (2b) lesioned rats with return of theta activity.
Surgery Animals were anesthetized with sodium pentobarbital (50 m g / k g , i.p.), secured in a stereotaxic apparatus (Kopf) and prepared for burr-hole craniotomies using sterile technique. The hippocampal recording electrode was implanted near the hippocampal fissure, 4.3 m m caudal to bregma, 2.5 m m lateral to midline, and 3.2 m m deep to dura mater, according to established procedures 4. After one week recovery, baseline theta recordings (see below) were obtained. O n the following day all rats were re-anesthetized with sodium pentobarbital (50 m g / k g , i.p.), secured in a the stereotaxic apparatus and prepared for sterile surgery. A craniotomy in the parietal bone overlying the right neocortex was performed by drilling. Aspirative lesions were m a d e to the depth of the lateral ventricle at a distance of 1.3 m m caudal to bregma, extending from the midline to 2.0 m m lateral to the midline. These lesions included portions of the neocortex, corpus caliosum, and fimbria-fornix. Sham-operated rats received a burr hole craniotomy in the same location but no aspiration was performed. After surgery, rats were housed under standard laboratory conditions with a 12-hour l i g h t / d a r k cycle and food and water ad libitum.
Theta recording O n e week after electrode implantation, and at bi-weekly intervals thereafter, E E G from hippoeampal electrodes was amplified and recorded on a polygraph (Grass Instruments) and on magnetic tape using an FM recording adapter (Vetter). Recordings were carried out in a partially enclosed motor-driven treadmill while the rat walked at a constant speed for 2 min. Enforced treadmill walking served to control the behavior of the rat and to provide long periods of continuous theta rhythm. Theta recordings were scored by a computer, using a previously described autocorrelation method z3. Briefly, filtered E E G recordings (high-pass corner frequency = 0.3 Hz, - 6 dB/octave, low-pass corner frequency = 100 Hz, - 2 4 d B / o c t a v e ) were digitized at 120 Hz and digitally filtered to eliminate non-theta frequencies (flat bandpass between 4.7 and 8.4 Hz). Autocorrelation functions were computed for these filtered data using a Fourier method and normalized by the total power in the unfiltered data. The root-mean-square value of the normalized autocorrelation function over the time lags from zero to 1.07 s represented the 'theta score'. T h e value of this score for a perfect sine wave at the theta rhythm frequency is 1.0. A theta score of 0.37 or above was used as the criterion for theta rhythmicity.
Preanesthetic body temperature was maintained throughout the procedure with a heat lamp. Just prior to isotope infusion, 0.2 ml of whole blood was withdrawn from the arterial catheter in a heparinized syringe for blood gas and pH determination, corrected for body temperature (Radiometer model ABL-2 Acid-Base Laboratory, Copenhagen). Packed red cell volume was determined using a Monoject capillary tube centrifuged for 1 min in a Beckman model B Microfuge. Mean arterial blood pressure was determined using a hand-blown mercury m a n o m e t e r with saline interface. Baseline plasma glucose levels were determined from a 50 ~zl arterial blood sample collected in a lithium-fluoride-heparin tube (Beckman Instruments) and analyzed in a glucose analyzer (Yellow Springs Instrument Company, model 23A). The animals were placed on a treadmill for 10 min of forced walking. They were then quickly removed from the apparatus and infused intravenously with 1 2 5 / x C i / k g [14C]2-deoxyglucose (American Radiolabeled Chemicals, Inc.) over 30 s. Timed arterial blood samples were taken for the determination of plasma glucose and 2 D G concentrations using the YSI glucose analyzer and liquid scintillation spectroscopy (Beckman LS 1800) respectively. The rats were required to walk on the treadmill for two-minute periods prior to the 5, 10, and 15 min post-infusion time points and for five-minute periods prior to the 25, 35 and 45 min time points. At all other times, the rats were freely moving while the samples were collected. Fortyfive min after the administration of 2DG, the animals were overdosed with pentobarbital (40 rag), the time of death was recorded and the brains were rapidly removed and frozen in isopentane chilled to - 3 5 ° C with dry ice. The brains were later sectioned (20 /~m coronal sections) in a cryostat (AO Reichert). Some sections were taken for A C h E histochemistry and Nissl (thionin) staining and others were thaw-mounted on coverslips, heat fixed and placed against Kodak O M film with methylmethacrylate standards in lead cassettes in the dark for 12 days.
Image analysis After development, autoradiographs were analyzed using a computerized imaging system ( A m e r s h a m / L o a t s ) . Brain sections were digitized with the spatial resolution of 14 tzm by 14 /xm per pixel. Boundaries of cell layers in the CA1 region and the dentate gyrus were determined by superimposing 2 D G images on adjacent Nissl sections. Layers were delineated according to criteria put forth by Bayer 2. In order to assess the possible influence of other brain structures on hippocampal function, glucose utilization was also computed for several brain regions known to innervate the hippocampus. Those included were the medial and lateral septal nuclei and the entorhinal cortex. For each animal, six consecutive autoradiographic images were analyzed. The L C G U value for a particular cell layer in each animal was calculated by averaging final L C G U values for all six 2DG images examined. Sections used for hippocampal analysis were taken from an area 4.2 m m to 4.7 m m anterior to the interaural plane according to the Paxinos and Watson atlas 19.
Data analysis Raw laminar L C G U values for the hippocampus (/zmol glucose/100 g / m i n ) were normalized. Normalized L C G U values (x) for a given cell layer were calculated as follows: U x = ~- x 100%
2DG method Twelve weeks after the second surgery the 2DG method of Sokoloff 2z was carried out. Following an overnight fast, the rats were anesthetized with a mixture of 2 - 5 % halothane in nitrous oxide and oxygen (70:30). Arterial and venous femoral catheters (PE 50) were inserted and tunneled subcutaneously to the level of the cervical spine. The animals were allowed to recover from anesthesia (minimum 2 h) and remained freely moving in Plexiglas rat cages.
where U is the L C G U value of the layer to be normalized in ~zmol glucose/100 g / m i n and S is the sum of L C G U values (/~mol gluc o s e / 1 0 0 g / m i n ) for the seven layers analyzed in the control hemisphere of the respective rat. Statistical differences between respective cell layers of different animals and different hemispheres were tested by three-way Analysis of Variance ( A N O V A ) and appropriate post-hoc comparisons (one-way ANOVA).
119 RESULTS Theta activity in sham-operated rats (group 1, n = 4) was consistently present in all recording sessions. Unilateral combined lesions of the fimbria-fornix and cingulum abolished the hippocampal theta rhythm ipsilateral to the lesion. A coronal section through a typical lesion is depicted in Fig. 1. At 4 weeks post-lesioning, theta activity was absent both in fimbria-fornix/cingulum lesioned rats with a persistent loss of theta activity (group 2a, n = 4) and fimbria-fornix/cingulum lesioned rats that showed an eventual return of theta activity (group 2b, n = 4). At 12 weeks post-lesion, group 2a rats exhibited no change in theta activity from previous recordings, whereas group 2b rats showed a spontaneous return of theta rhythm. Typical recordings are illustrated in Fig. 2 for each of the 3 groups prior to the second surgery (baseline), and both four and twelve weeks after the second surgery (4 and 12 weeks post-operatively, respectively). Neither the size nor the location of the lesion was correlated with the recovery of theta rhythm that occurred in the group 2b fimbria-fornix/cingulum lesioned rats. At the time of the 2DG experiment, there were no statistically significant differences between the 3 groups in measured values of physiological variables including mean arterial blood pressure, blood gases and pH, hematocrit, baseline glucose levels or body temperature (data not shown). The presence of AChE staining in the hippocampus of some lesioned animals showing recovery of the theta rhythm suggested that some reinnervation had occurred. Due to the variability of this staining method, no quantitative correlations could be made.
Fig. 1. Camera-lucida drawing of a representative fimbriafornix/cingulum lesion at its greatest extent, taken from a Nisslstained coronal section.
4 WEEKS POST-OP
12 WEEKS POST-OP
e , 0.41
Fig. 2. Representative hippocampal theta rhythm tracings, before (BASELINE) and after surgery (POST-OP). The effect, over time, of sham and fimbria-fornix/cingulum lesions on hippocampal formation theta rhythmicity during type 1 theta behavior is shown. Note that the division of fimbria-fornix/cingulum lesioned animals, group 2, into 2a and 2b was based on the presence or absence of theta recovery at 12 weeks post-lesion. Group 1 is sham-lesioned animals. Values below electroencephalographic traces are their respective theta scores (0), having a possible range from zero to one, 0.37 being chosen as the criterion for rhythmicity in these experiments.
Glucose metabolism in hippocampi from all 3 groups showed patterns specific to the presence or absence of theta activity at 12 weeks after the fimbriafornix/cingulum lesion. Sham-operated rats, with no disruptions of theta rhythm, displayed no apparent differences between hemispheres. The persistent loss of theta rhythm in group 2a fimbria-fornix/cingulum lesioned rats was accompanied by a characteristic decrease in glucose utilization. Those fimbriafornix/cingulum lesioned rats that exhibited a spontaneous return of theta rhythm, group 2b, also demonstrated a greater hippocampal glucose metabolism than the depressed levels seen in lesioned rats with a sustained loss of theta rhythm, group 2a. The average raw LCGU values for the control hemispheres in lesioned rats were not significantly different from those of sham-operated r a t s [F2, 9 = 0.23, P = 0.79 (ANOVA)]. The mean _+ S.E.M. for the sham-operated group was 58.4_ 5.6; for the lesioned group with a sustained loss was 64.9 + 7.6 and for the lesioned group with a return of theta rhythm was 61.2 _+ 7.1. Because there was variation in the raw LCGU values across rats that was unrelated to the control and experimental groups, the data for each rat were normalized with respect to the control hemisphere according to the equation given above. The results for each lamina were
LAMINAR LCGU VALUES 2O 18
Group 1 control Group 1 lesion
Group 2a control
Group 2a lesion
Group 2b control
Group 2b lesion
LAMINAE Fig. 3. Graph of normalized laminar local cerebral glucose utilization (LCGU) values. Mean normalized LCGU in seven hippocampal laminae of control, contralateral and sham or fimbria-fornix/cingulum lesioned hemispheres is plotted for all groups. Both hemispheres of fimbriafornix/cingulum lesioned rats demonstrate similar LCGU patterns. Lesioned hemispheres of group 2a and 2b underwent significant reductions in LCGU compared to control hemispheres. Group 2b, however, which experienced a recovery of theta rhythm, shows higher LCGU values than group 2a, which did not. On the Y-axis, the mean of the 'normalized' LCGU is expressed as a percentage of the sum of the LCGU values from the seven hippocampal laminae of the control hemisphere. Abbreviations of laminae are: OR, oriens; PYR, CA1 stratum pyramidale; RAD, CA1 stratum radiatum; L-M, CA1 stratum lacunosum-moleculare; MOL, dentate molecular layer; GR, dentate stratum granulosum; DH, dentate hilus.
then expressed as a percent of the total measured LCGU in the control hemisphere. Figure 3 shows mean normalized LCGU values in seven hippocampal laminae, including control and lesioned hemispheres. Sham-operated rats demonstrated similar patterns of hippocampal glucose metabolism in both hemispheres, the lacunosum-molecular layer showing the highest rates. Group 2a rats, with a persistent loss of theta rhythm, showed significant LCGU reductions (P < 0.05, ANOVA) in all seven laminae
LCGU values for other limbic sites Limbic region
Control (sham lesion) group 1
Lesioned (no theta) group 2a
70.7 " (3.9) b
Lateral septum (lesion side)
Lateral septum (control side)
Entorhinal cortex (lesion side)
Entorhinal cortex (control side)
a Mean of three observations. b Standard error of the mean.
Lesioned (theta return) group 2b
compared to the control hemisphere with the largest changes occurring in strata oriens and pyramidale of CA1. Hippocampi from group 2b animals, with a return of theta rhythm, also underwent significant (P < 0.05, ANOVA) reductions in laminar metabolic activity compared to the control hemisphere. However, concomitant with the return of theta rhythm in group 2b rats, LCGU values in the hippocampi ipsilateral to the lesions were higher than the values for group 2a ipsilateral hippocampi. These were significantly higher (P < 0.05, ANOVA) in strata oriens, pyramidale and lacunosum-moleculare of CA1 and in the dentate hilus. Rates of glucose utilization in the septum and entorhinal cortex are shown in Table I. Since these structures lie outside the hippocampus, their rates of glucose utilization were not normalized to the rates within the hippocampal profile. None of the variations in glucose utilization across groups were statistically significant (ANOVA). DISCUSSION
Unilateral combined lesions of the fimbria-fornix and cingulum produce a substantial decline in the local cerebral glucose utilization (LCGU) values in all ipsilateral hippocampal laminae. The decrease in LCGU values is presumably caused by the loss of afferents from the brain stem, diencephalon a n d / o r the medial
121 septal region of the basal forebrain, since these are the main fiber tracts interrupted by the lesion. The reduction in LCGU values is probably not due to loss of adrenergic or serotonergic brain stem afferents, since specific lesions of these afferent populations have been shown to have little effect on hippocampal L C G U 3'21. Loss of hippocampal commissural afferents is also without major effect on LCGU values. Both sides of lesioned animals sustain a loss of those afferents, yet the raw LCGU values in control hemispheres of lesioned animals are not different from those of shamoperated animals. Also, since there were no differences in glucose utilization in the medial and lateral septum or in entorhinal cortex across any of the groups of animals, changes in the activity of these regions do not explain the recovery of theta rhythm in the hippocampus. The decrease in hippocampal LCGU after fimbria-fornix/cingulum lesions is linearly related to acetylcholinesterase staining 9, suggesting that it is the loss of septal cholinergic innervation and consequent reduction in muscarinic activation of hippocampal neurons that is responsible for the decreased metabolic activity. At 4 weeks post-lesion, unilateral fimbriafornix/cingulum lesions produce an ipsilateral loss of the hippocampal theta rhythm. The ipsilateral loss of theta rhythm undoubtedly results from the loss of the innervation of the hippocampus arising from the medial septal region, the presumed 'pacemaker' for theta rhythm2°'23. By 12 weeks after the lesion, a movementrelated rhythm returns in some animals. The most likely explanation for this recovery is compensatory collateral sprouting of septal afferents arriving at the hippocampus via the ventral amygdaloid pathway 6'14. It is not clear why the theta rhythm recovered after 12 weeks in some lesioned animals, while it did not recover in others. Prior to analysis, lesioned animals were selected for the completeness and uniformity of their lesions. We could find no measures of the lesion that correlated with recovery of theta rhythm. Perhaps, given sufficient recovery time, the theta rhythm eventually would have returned in all of the fimbriafornix/cingulum lesioned rats. The animals with theta rhythm recovery at 12 weeks post-lesion also have significantly higher ipsilateral "LCGU values in several laminae compared to animals without recovery. Higher LCGU values in strata oriens and lacunosum-moleculare of CA1 and the dentate hilus probably result directly from septal reinnervation, since these are normally the most prominent targets of septal afferents TM. Higher LCGU values in stratum pyramidale probably result from increased activity of interneurons during theta rhythm5, since IPSPs, as well
as EPSPs, are effective in increasing LCGU values 1 The interneurons are probably driven directly by cholinergic septal afferents 13. Though LCGU values on the lesioned side are higher in animals with recovery of the theta rhythm compared to those without recovery, in no case are they as great as those of sham-lesioned animals, or contralateral control values. The residual reduction in LCGU values on the lesion side as compared to controls is presumably due to the incomplete nature of the reinnervation of the hippocampal formation. The observed pattern of higher LCGU values, however, does occur in relation to changes that are sufficient to enable the production of a theta rhythm. The largest differences in LCGU values of animals with recovery of theta rhythm compared to animals with no recovery occur in the strata oriens and pyramidale. This is in substantial agreement with the results of Monmaur et al. 17, who compared LCGU values for rats in a running wheel to LCGU values for rats engaged primarily in non-theta mode behaviors. It is remarkable that two studies with such different methodologies arrive at the same conclusion; in the presence of the hippocampal theta rhythm, higher LCGU values occur in the stratum oriens of CA1 than in the absence of theta rhythm. Moreover, in the results presented here, the difference was observed even though both groups of rats were engaged in the same behavior during the 2DG exposure. The advantage of the 2DG method is that it can provide information on the average level of activity of a whole population of afferents to a lamina 22. However, it does not indicate changes in the pattern of activity (e.g., non-rhythmic to rhythmic). There are undoubtedly changes in the patterns of firing of several sets of afferents associated with the presence of theta rhythm that are not accompanied by changes in mean rate of firing, and are therefore undetected by this method. On the other hand, these data, taken together with those of Monmaur et al. 17 indicate that an increase in rate of firing of septal afferents accompanies the hippocampal theta rhythm and may be sufficient to enable its production, since the septal afferents themselves are known to fire rhythmically2°,23. Acknowledgements. This work was supported in part by NIH grants NS 22766 to D.D-E. and NS 17095 to S.E.F. The contributions of Drs. Roy Vingan, Richard Johnson and John Kubie to the early stages of this project are greatly appreciated.
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