BRAIN RESEARCH

E L S E V I E R Brain Research 725 (1996) 257-262

Short communica t ion

Chlordiazepoxide improves the performance of septal lesioned but not hippocampal lesioned animals in a Morris maze

H. Terry Farber *

Department of Biology, Bryn Mawr College, Bryn Mawr, PA 19010, USA

Accepted 19 March 1996

Abstract

The effects of hippocampal and lateral septum lesions were compared in rats tested in a water maze spatial memory task, and the effect of chlordiazepoxide (CDP) was examined. There was a significant interaction for lesion and CDP in the septal lesioned subjects, with the lesioned animals performing worse than control animals, and CDP improving the performance of lesioned animals. CDP had no effect on impaired performance in hippocampal lesioned animals.

Keywords: Hippocampus; Lateral septum; Spatial memory; Chlordiazepoxide; Lesion; Anxiety

It has been hypothesized by Gray et al. [6] that the septohippocampal system (SHS) acts as a comparator of sensory inputs from the thalamus with what is predicted about the environment. The hippocampus probably plays a major role in the formation and/or retrieval of memory in the intact animal [12]. Morris et al. [15] discovered that lesions of the hippocampus impaired rats' ability to find the location of a hidden plexiglass platform in a water maze paradigm. In addition to the requirement of retrieval of memory, the model of the hippocampus by Amsel [1] requires positive and negative weights of each decision that is made. Therefore, one might expect that a balance of excitatory and inhibitory inputs into the hippocampus may be required for the optimal functioning of the hippocampus in its decision making capacity.

There is evidence that parts of the SHS provide the excitatory and inhibitory inputs necessary for the optimal functioning of the hippocampus itself. Jarrard and Becker [8] believe that the medial septum, with its connections to the CA3 region of the hippocampus, is responsible in activation or tonic readiness of the animal, though indirect inhibitory influences are also possible. The lateral septum may also provide indirect inhibitory inputs to the hip- pocampus, which are necessary to balance the excitatory inputs from the medial septum [14,18,20]. It therefore appears that both inhibitory and excitatory inputs are re-

* Corresponding author. Fax: (1) (610) 896-7938.

quired for optimal functioning of the hippocampus; the lateral septum and medial septum, may provide these inputs.

However, an alternative explanation for the effects of the lateral septum upon the functioning of the hippocam- pus may be postulated, since it is now known that the lateral septum does not have direct input to the hippocam- pus [9]. The lateral septum lesions as described by Yadin and Thomas [20] may produce a proconflict effect in the rat. The anxious state of the animal may via some mecha- nism interfere with the functioning of the hippocampus, as described by M'Harzi and Jarrard [14]. Evidence exists for the concept of a balance between excitatory and inhibitory inputs possibly correlating with anxigenic and anxiolytic influences for the maximal functioning of the hippocampus [7,11,13,17].

The present study was therefore undertaken in order to explore the relationship between the balance of inhibitory (or anxiolytic) and excitatory (or anxiogenic) influences and a spatial memory task as tested in a water maze paradigm. We also tested the effects of a low dose of chlordiazepoxide on lesioned and nonlesioned animals.

The subjects consisted of 72 experimentally naive male albino Sprague-Dawley rats. The testing apparatus for the experiment consisted of a swimming pool water maze similar to that described by Morris et al. [15]. A white plastic pool (diameter of 2.25 m and height of 0.4 m) provided homogeneous sides and bottom in order to pre- vent any intramaze cues. The pool was filled to a depth of

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258 H.T. Farber / Brain Research 725 (1996) 257-262

21 cm with water at a temperature of 26 + I°C. The maze was designated to have directions North, South, East and West. A cylindrical Plexiglas platform which was l0 cm in diameter and 20 cm high was placed approximately 0.33 m from the side of the pool in the NW quadrant. The testing room in which the pool was placed was well lit and had numerous visual cues present.

Forty-six of the animals were subjected to surgeries involving the lateral septum. The rats were anesthetized using sodium pentobarbital (40 m g / k g i.p.). Twenty-three of the animals underwent bilateral electrolytic septal le- sions. Lesions were performed using a single 0.18 mm stainless steel wire insulated to the tip, with a 2 mA DC constant current delivered via a Grass stimulator through the electrode for 20 s. Twenty-three animals underwent sham lesions in which the electrode was lowered 4 mm below the surface of the skull, but no current was passed through the electrode.

Seventeen animals underwent bilateral hippocampal ab- lations via suction. These animals were anesthetized in a similar manner as the lateral septum lesioned animals, and then 3 mm diameter holes were drilled through the skull. The dura was incised and reflected, and the neocortex and corpus callosum was aspirated through a fine glass pipette attached to a suction device. Aspiration was then contin- ued, removing as much of the hippocampus as possible without damaging any adjacent structures. Nine animals served as sham controls, in which the neocortex and corpus callosum were removed, but the hippocampus was left intact.

After 5 days of rest, the lateral septum lesioned and sham control animals were then randomized into two groups. One group (12 lesioned and 11 sham control subjects) received chlordiazepoxide (CDP) at a dose of 2 m g / k g diluted in saline and injected intraperitoneally 15 min before each test session in the water maze. The second group of animals (11 lesioned and 12 sham control sub- jects) received the saline vehicle at a dose of 1 m l / k g 15 rain before each test session. This randomization resulted in four test groups: sham/sal ine (12 subjects), s h a m / C D P (11 subjects), lesion/saline (11 subjects), and les ion /CDP (12 subjects). A similar randomization was accomplished for the hippocampal lesioned and sham control animals resulting in 4 sham/saline, 5 sham/CDP, 9 lesion/saline and 8 l e s ion /CDP animals.

Each trial in the water maze consisted of randomly placing the subject at one of the four starting positions (North, South, East or West) with its nose pointing toward the center of the pool. The animal was permitted to swim freely for 120 s or until it located the hidden platform on its own. After 120 s (or if the subject located the platform in a shorter time) the animal was placed upon the platform and allowed to remain there for 30 s; it was then removed from the pool. The animals received five trials per day with an ITI of 10 rain, for a total of 3 days of testing sessions. The experimenter was blind to the condition of

( ) Bregma + 0.2

O i

Bregma + 0.7

Bregma + 1.2

Fig. 1. Schematic distribution of typical lateral septum lesion.

the animal, and stood at the same marked spot at the Southeast quadrant of the maze for all trials.

At the end of testing, all animals were sacrificed. The brains were removed and fixed in formalin, after which 40 ~xm sections of the lateral septum (Fig. 1) or hippocampal regions (Fig. 2) were prepared and examined microscopi- cally in order to determine the adequacy of the lesions. Analyses were performed using three way analysis of variance (ANOVA) for the relationships among trial, le- sion and drug conditions; for each trial, relationships be- tween CDP and saline conditions for lesioned and non-le- sioned animals was analyzed using one-way analysis of variance (ANOVA).

The hippocampal lesions had a significant effect on the performance of the animals in the water maze spatial navigation task ( F ( 1 , 2 5 ) = 33.75, P < 0.001) similar to that described by Morris et al. [15]. However, as seen in Fig. 3A, chlordiazepoxide had no significant effect on either lesioned or sham control subjects ( F ( 1 , 2 5 ) = 1.08, P > 0.30). No significant differences were seen across each of the three trials for for lesioned ( F ( I , 1 6 ) = 0.01,

H.T Farber / Brain Research 725 (1996) 257-262 259

P > 0.10) and non-lesioned (F(1,8) = 0.05, P > 0.10) ani- mals when CDP and saline control groups were compared. The interaction between the lesion and CDP approached significance (F(1,25) = 3.70, P = 0.06), probably largely due to the overwhelming effect of the lesions.

As seen in Fig. 3B, the animals with lateral septal lesions with and without CDP performed in a different manner. As compared with the hippocampal lesions, there was a significant interaction between lateral septal lesions and chlordiazepoxide (F(1,45) = 9.93, P < 0.005). No significance for CDP alone was seen, nor for lesion alone (due to the interaction of the two effects). When directly compared across trials, CDP and saline groups with (F(1,22) = 1.0l, P > 0.10) or without (F(1,22) = 0.95, P > 0.1) lesions performed in a similar manner.

When directly compared, hippocampal lesions caused significantly worse performance in the water maze than did lateral septum lesions (F(1 ,39)= 37.37, P < 0.001, Fig. 4A). As seen in Fig. 4B, there is also a significant

difference between hippocampal lesioned animals given CDP, and subjects with lateral septum lesions given CDP (F(1,39) = 10.96, P = 0.001), and there is a significant interaction between lesion and drug effects (F(1 ,39)= 5.06, P < 0.05).

Qualitatively, the performance of the animals in the water maze paralleled the quantitative results. The rats with hippocampal lesions given saline (Fig. 5) or CDP (Fig. 6) circled the periphery of the pool (demonstrating a tendency toward thigmotaxis) without seeming to be able to locate the platform, in contrast with animals who had both types of sham lesions (Figs. 7 and 8) or septal lesions given CDP (Fig. 9), which were able to take a more direct route to the platform. Septal lesions given saline (Fig. 10) seemed to spend time circling in incorrect quadrants of the pool and therefore did not find the platform as readily as septal sham animals or those lesioned animals given CDP, but did not exhibit thigmotaxis nor were they as impaired as hippocampal lesioned rats.

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Fig. 2. A: schematic distribution of sample hippocampal lesion. B: schematic distribution of sample hippocampal lesion.

260 H.T. Farber / Brain Research 725 (1996) 257-262

One of the major findings of this study is the impair- ment in performance of the hippocampal and lateral sep- tum lesioned subjects in a water maze trial. The effect on performance in the hippocampus lesioned animals was similar to that noted by Morris et al. [15]; however, in contrast to Morris' study, the subjects in this study ap-

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Fig. 3. A: average escape latencies (s) of each session of water maze testing for animals given hippocampal lesions or sham lesions and saline or CDP. Note decrease in latencies over session for sham animals compared with hippocampal animals, and lack of improvement with CDP. B: average latencies (s) over sessions for animals given lateral septum or sham lesions and either saline or CDP. Note lack of decrease in latencies for lesioned animals compared with shams (similar to the hippocampal animals), but improvement in performance with CDP com- pared to the lack of effect of CDP on hippocampal lesioned subjects.

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Fig. 4. A: direct comparison between hippocampal and lateral septum lesioned animals, both given saline (average escape latencies in seconds). B: direct comparison between hippocampal and lateral septum lesioned animals with both groups given CDP (average escape latencies in sec- onds).

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Fig. 5. Swim path example of animal with hippocampal lesion given saline from Session 2. Note thigmotaxis of the subject in the pool.

H.T. Farber / Brain Research 725 (1996) 257-262 261

peared to be impaired during all three days of testing, leading to no significant learning effect. Yet, non-lesioned animals rapidly learned to locate the hidden platform. The lateral septum lesioned animals also had impairment in their ability to locate the platform, but unlike the hip- pocampal lesioned animals, the impairment was not as severe, and learning did occur over the three days. Thus, the present study confirms the fact that the lateral septum plays a different role in the functioning of the hippocam- pus from that of the hippocampus itself.

The most important result of this study was the effect of a low dose of chlordiazepoxide on the performance of hippocampus and lateral septum lesioned animals. A sig- nificant improvement in performance by CDP was noted for the animals which had undergone lateral septum le- sions; no such effect was noted for those subjects with hippocampus ablations.

The effect of CDP on lateral septum lesions may be

Fig. 6. Swim path from hippocampal lesioned animal given CDP 15 min before the trial (Session 2). Note persistence of thigmotaxis and impaired navigation despite CDP.

Fig. 7. Swim path from animal with hippocampal sham lesion given saline (Session 2). Note lack of thigmotaxis and increased amount of swimming in the correct quadrant prior to locating the platform.

t Fig. 8. Swim path from lateral septum sham lesion given CDP (Session 2). Note the direct route taken to the platform.

Fig. 9. Swim path of animal with lateral septum lesion given CDP (Session 2). Note the direct route taken to the platform and increased amount of swimming in the correct quadrant.

Fig. 10. Swim path of an animal with lateral septum lesion given saline (Session 2). Note the lack of thigmotaxis compared with hippocampal lesioned animals, but significant amount of time spent in the incorrect quadrant and apparent inability to locate the platform.

explainable by its activation of GABA receptors in the hippocampus, medial septum, or in other parts of the mammalian brain. It is well known that the effect of benzodiazepines, mediated via the GABA receptor, is to provide inhibitory influences in the brain. It is possible that the hippocampus is dependent on both excitatory inputs (mediated via serotonin and catecholamines) and inhibitory inputs (mediated via GABA) for the effective functioning of its comparator system [5]. Any disruption of either excitatory or inhibitory inputs might then impair the func- tions of the hippocampus, and in turn, impair the perfor- mance of the animal. The lateral septal lesions would then reduce the inhibitory input, allowing a predominance of excitatory inputs in the hippocampus, with resultant im- pairment in the decision making process. The role of CDP would be to restore the inhibitory influence on the hip- pocampus by either indirect affects mediated through the medial septum, or through a gating mechanism on thalamic sensory information [10] to allow for maximal functioning.

Several studies support the need for a balance of inputs for maximal hippocampal function. Venault et al. [19] found that mice exposed to low doses of methyl [3-carbo- line-3-carboxylate (a benzodiazepine inverse agonist) al- lowed for enhanced learning in mice tested in an habitua- tion of a new environment model, while higher doses of the drug actually impaired the learning process. Similarly, an optimal dose of o-amphetamine sulfate or epinephrine bitartrate was required to increase LTP frequency in hip- pocampal neurons of rats; high or low doses failed to

262 H.T. Farber / Brain Research 725 (1996) 257-262

increase L T P [4]. As a correlate to the present study,

Brioni et al. [2] found that l inopirdine (which enhances the

release o f ache tychol ine upon st imulation) amel iora ted the

impa i rment in pe r fo rmance on two-p la t form water maze

trials o f media l septal les ioned rats. However , larger doses

fai led to improve the pe r fo rmance o f the rats in the spatial

task. It wou ld therefore appear that an opt imal level o f

arousal (exci ta tory input) is required for the facil i tat ion o f

pe r fo rmance in any learning task [16]. In the present study,

it appears that the lesions o f the lateral sep tum disrupted

that opt imal level by e l iminat ing inhibi tory inf luences and

therefore a l lowing for an unbalanced excess o f exci ta tory

input; the low dose C D P prov ided the miss ing inhibi tory

inf luence which is needed. H ippocampa l ablat ion e l imi-

nates the site o f the decis ion mak ing process itself, and

could therefore not be improved through the use o f CDP.

Another explanat ion for the effects seen in this study is

that o f the effect o f anxiety on learning. Fi le and Pe l low

[3] conc luded that an inverse -U relat ionship exists be tween

anxie ty and pe r fo rmance in a study invo lv ing two 13-

carbol ines tested in a rat pass ive avoidance parad igm and

holeboard explora t ion technique. It is therefore possible

that the lateral septum lesions caused the animals in this

study to b e c o m e ' anx ious ' s imilar to the effect seen by

Thomas [18] and Yadin and Thomas [20], which then

interfered with their pe r fo rmance in the s w i m m i n g pool

navigat ion task. Since C D P is an anxiolyt ic agent, it would

then a l low the animals to per form in a manner s imilar to

the controls. The C D P would have had no effect on

h ippocampal les ioned animals since the basic mechan i sm

for m e m o r y and pe r fo rmance in the water maze was

ablated. However , d i f ferences in sizes o f the h ippocampus

and lateral sep tum lesions may also expla in the di f ferences

in pe r fo rmance and response to C D P which was seen.

Fur ther research using other tests o f anxie ty are warranted.

Acknowledgements

The author wishes to thank Professor Earl Thomas for

the use o f laboratory facilit ies, Hol ly Grishkat and Susan

Choi for technical assistance, and Jack M c G a r v e y for

graphic presentat ion.

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