Behavior during hippocampal microinfusions. II. Muscarinic locomotor activation

105

Brain Research Reviews, 4 (1982) 105-327 Efsevier Biomedical Press

BEHAVIOR DURING HIPFOCAMPAL MICRO~NFUSIONS. II. MUSCAR~NIC LDCOMOTOR ACTIVATION

CHARLES

FLICKER

and MARK A. GEYER

T-004, Department of Neuroscienees and (M.A.G.) M-003, Department of Psychiatry, School of Medicine, ~nivers~iy of California, San Diego, La Joiia, CA 92093 (U.S.A.] (Accepted October 6th, 1981) Key words: hippocampus carbachol -

- acetylcholine atropine - theta

septum -

dentate gyrus -

.~

-

muscarinic receptors -

_.-_l_

CONTENTS

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1. Introduction.

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2. Methods 3. Results

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4. Discussion 5. Summary

.................................. ...................................

Acknowledgements References

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105

106 108 120 124 124 125

1. INTRODUCTION

The septo-hippocampal pathway is an established central cholinergic pathway. High levels of acetylcholine (ACh), acetylcholinestetase (AChE), and choline acetyltransferase (CAT), are found in the hippocampus and these are depleted by lesions of the medial septum and diagonal bandaeys4~25Je~sa.The distribution of label or degenerating terminals observed in the hippocampus is quite similar after histochemical staining for AChE a73Qa~46, lesions of the medial septumsOJ2, or injection of tritiated amino acids into the medial septum 27~48.Binding studies suggest that the localization of chohnergic receptors in the hippocampus is also comparable to the presumed pattern of the septal innervation ss,dr. Horseradish peroxidase injections into the hippocampus produce retrograde labeling of cells in the medial septal nucleus42. Electrical stimulation of the medial septum increases the release of acetylcholine in the hippocampus, an effect blocked by lesions of the septo-hippocampal pathwayios44; spontaneous release of ACh in the hippocampus is likewise reduced by lesions of the 0165-0l73~82,QOOO-0000/$02.750 Eisevier Biomedical Press

106 medial septumle. Cells in the hippocampus are responsive to ACh iontophoresis and the effects are reversed by cholinergi~ blocking agents2~s,s~~~,s~,In short, the septohippocampal pathway, one of the two major afferents to the hippocampal formation, appears to utilize ACh as a neurotransmitter. Pharmacological studies have shown that cholinomimetics produce desynchronization of the cortical electroencephalogram, the normal correlate of wakefulness and arousal, and that cholinolytics such as atropine can antagonize this effect”“J*. However, even though atropine injection produces a sleep-like synchronization of the cortical EEG, it causes an increase in locomotor activityi7. This ‘dissociation’ between EEC and behavioral arousal seen in response to the systemic administration of cholinergic drug@ might simply be a consequence of effects upon distinct cholinergic systems. Cholinergic neurons in the brain have a diffuse distribution and give rise to a variety of ascending, descending, and local projection pathway@+@. Even without considering the peripheral sites of action of cholinergic agents, it is obvious that more neuroanatomically precise techniques are necessary for the study of the behavioral role of specific central cholinergic pathways. In these experi~~ents the effect of hippocampal cholinergic receptor stimulation upon the spontaneous behavior of the rat was examined. The behavioral analysis system used’” was capable of measuring changes in overall motor activity, locomotion, stimulus responsivity, and the inspection of novel objects; and, in addition, as has been shown with hippocatnpai infusions of norepinephrineii, of detecting subtle changes in the animals’ sequential movement patterns. Unrestrained rats in this computerized holeboard~a~tivity apparatus received continuous, bilateral infusions into the dentate gyrus of the mixed muscarinic/nicotinic agonist carbachol. 2. METHODS

The ex~rimental methods have been described in detail elsewherell~l3. The experimental chamber was a computerized holeboard box 12 in. x 24 in. x 15 in., with 10 holes in the walls and floor, an 8 x 4 perpendicular array of photo~ams, and a 6in. high stainless steel touchplate to detect wall rearings. The animal’s position was localized in time and space with a resolution of 100 ms and 1.5 in. respectively. Male Sprague-Dawley tats were implanted with guide cannulas in the anterodorsal hippocampal formation. After two acclimation sessions at 3-day intervals, the rats were administered bilateral infusions into the dentate gyrus at the rate of 0.025 FIlmin throughout the duration of a 40-&n experimental session. Novel objects were placed at the back of 3 of the 6 side wall holes, In one experiment, strong odor-emitting objects were used. These included an incense cone, a piece of scented soap, and a rolled up laboratory tissue with a drop of oil of rose. The pharma~logi~1 agents infused included the following : carbamylcholine chloride (carbachol), atropine sulfate, nicotine, and D-tubocurarine chloride. Immediately before sacrifice, each animal was infused with Evan’s blue dye (3 mg/ml) for histological veri~cation of the infusion site and spread of the infusate. The dependent variables included the number, duration and mean duration of

107 pokes and rearings, cumulated over either lo-min or full-session 40-min blocks. Holepokes were divided into varied or repeated pokes depending on whether or not there had been an intervening rearing or poke into a different hole between two pokes into the same hole. The number and duration of pokes into the 3 side wall holes into which novel objects had been placed and the number and duration of pokes into the 3 corresponding wail hoies without novel objects were separately totaled. These vatiables were used to assess the animals’ responsiveness to novel stimuli. From the state of the 8 x 4 array of wall beams the animal’s (x,y) position was calculated and used to assign the rat to one of eight Gin. square ‘sectors’ and one of 9 unequally sized ‘regions.’ Duration in and frequency of entries into each sector and region was recorded. ‘Crossovers’ were defined as the total number of sector entries, and used as a measure of Iocomotion. Regions were also used to define five different types of ‘traverses.’ A short wall or long wall traverse was from one corner to another along either a short wall or long wall, respectively. A center traverse was from one short wall to the other via the center. The other two were half traverses from a short wail and back, into either the middle of a long wall or the center. Total number of photobeam interruptions was used as a measure of overall motor activity. Treatment effects were determined by analysis of variance with a two-factor mixed design, the betweensubjects factor being the comparison between experimental and saline-infused control groups and the Titan-subjects repeated measure being the time-block factor. Specific comparisons between saline and experimental groups were tested according to the Newman-Keuls procedure. The group n ranged from 6 to 18. The spatial distribution and sequential patterns of the animals’ locomotor activity were graphically and statistically described in several ways. Based upon the (x,y) position data, a hard-copy plot program produced a continuous ink line trace of the animals’ successive (x,y) positions. A real-time, variable-speed plot program displayed this same information by the movement of a cursor inside a two-dimensional reconstruction of the holeboard on a video terminal. The durations of each animal’s stay in each of the 9 holeboard regions were rank-ordered, pooled, and depicted in a group region duration histogram. Two measures of the repetitiveness or variedness of the animals’ patterns of movement were derived. The coefficient of variation of the 9 region durations was used to assess the randomness of the spatial distribution of each animal’s activity with respect to time. Based upon a 5-region division of the holeboard, the frequency of all possible two-region transitions was caiculated, and the coefficient of variation of these transition frequencies was taken as an index of the variedness of the rat’s sequential pattern of locomotor activity. Animals exhibiting a restricted set of two-region transitions produced high coefficients of variation whereas animals exhibiting a wider and more evenly distributed range of movements produced low coefficients of variation. Together with visual observation, the above methods provided a comprehensive description of the experimental animals’ patterns of movement and permitted the detection of effects upon that pattern per se.

108 3. RESULTS

Bilateral hippocampal infusions of intermediate (0.1 mg/ml) and high (3.0 mg/ml) doses of carbachol produced a powerful motor activation of the rats that was reflected in virtually every measure of their spontaneous activity, including locomotion, holepoking, and rearing. At the 3.0 mg/ml dose there was evidence of druginduced seizure activity. In control animals, during the first minute or two after insertion of the infusion needles, several ‘wet dog’ shakes were typicahy seen -- rapid back and forth body rotations along the longitudinal axis, probably secondary to mechanical damage of h~ppo~ampal tissue and a wave of spreading depression, More of these were observed only in rats infused with high-dose carbachol. All 3 doses of carbachol produced increased locomotion, measured as number of crossovers during the 40-min session, and this effect was significant for the middle and high doses (F(2,33) = 5.06, E’(2,17) = 4.39, P < 0.05, see Fig. 1). In both of these cases there was also an interaction between the drug effect and the repeated time-blocks measure. In neither experiment was carbachol’s effect apparent in the first 10 minutes of the session. Analysis of the simple main effects showed that, at the high dose, the increase in locomotor activity was maximal in the second IO-min block whereas at the 0.1 mg~ml dose the increase was still non-significant in the second IO-mm block and largest in the fourth IO-min block. In short, the onset of carbachols effect upon locomotion appears to have been dose-dependent.

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Fig. 1. Effects of carbachol and atropine in crossovers. The mixed cholinergic agonist carbachol, infused bilaterally into the dentate gyrus in concentrations of 0.1 mg/mi (A) and 3.0 mg/ml (B) increased the locomotor activity of rats. Abscissa is time in min; ordinate is number of crossovers. Carbachol’s effect was maximal during the second IO-mitt block at the high dose and during the fourth IO-min block at the middle dose. The muscarinic receptor blocker atropine sulfate, when co-infused with carbachol in an equal weight/volume concentration, blocked carbachol’s effect upon locomotion at both doses. Newman-Keuls comparisons reveaLed significant differences between carbachol and carbachol + atropine groups and no significant group differences between carbachol + atropine and saline. At the lower dose, however, post-hoc comparisons of simple main effects showed that atropine failed to antagonize carbachol’s effect upon locomotion during the third IO-min block.

109 TABLE! I Center entries

Effect of bilateral infusions of the mixed cholinergic agonist carbachol and the muscarinic antagonist atropine into the dentate gyrus upon the number of entries into the center region during the 40-min session. In both experiments carbachol significantlyincreasedcenter entries and the effect was block-

ed by the co-infusion of atropine. The carbachol + atropine groups differ significantly from the carhachof groups and not from the saline groups. Infusions of atropine alone non-significantlydecreased the frequency of center entries. ---0.1 mgjml 0.3 n&II _...Saline Carbachol Atropine Carbachol + Atropine

56.2 j, 92.7 & 53.1 + 67.1 +

59.4 i 129.5 f 38.3 & 59.7 i

4.0 22.1* &I 10.7

8.6 32.0* 6.7 10.4 -_..X...

* Significantly different from saline, P < 0.05.

Carbachol’s effect upon locomotion was especially apparent in the number of entries into and the amount of time spent in the center region of the experimental chamber, particularly at the high dose (F(2,17) = 4.58,4.37, P < 0.05, see Table I). This finding might suggest that the carbachol-infused animals were less fearful than the saline-infused controls. In contrast, the low dose of carbachol (0.01 mg/ml) significantly increased long wall traverses only (F(f,17) = 7.06, P < 0.05).

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Fig. 2. Effects of carbachol and atropine on rearings and holepokes. A: carbacholO.1 mgjml increased rearing during the second half of the 40-min session relative to saline-infused controls. Abscissa is time in min; ordinate is number of rearings against the wall plate. Atropine sulfate, 0.1 mg/ml, co-infused with carbachol in the same solution, antagonkd carbachol’s effect. B: carbachol infusions (0.1 mg/ml) increased holepoking during the latter part of the 40-min session and the effect was antagonized by co-infusion of the muscarinic receptor blocker atropine sulfate. Abscissa is time in min; ordinate is number of varied hoIepokes. As with the crossover measure, carbachol’s effect was maximal in the fourth block, not fully antagonized by a&opine in the third block, and absent in the first block,

110 TABLE 11 Coefficient of variation of region-to-region transition frequencies Based upon a S-region division of the holeboard, the frequency of all possible two-region transitions was calculated for each animal. From this two-act transition probability matrix, the coefficient of variation of the transition frequencies was calculated (see Methods). Animals exhibiting a limited number of two-region transitions have higher coefficients of variation than animals with a more varied pattern of sequential movement.Bilateral hippocampal infusions of carbachol decreased the coefficient of variation relative to saline controls (F(3,33) ..= 4.78, P < 0.05) and the effect was significant at the high and low doses of carbachol but not at the middle dose _-... .._ _. ._..~~~_._-.. Saline

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0.549 f 0.030*

* Significantly different from saline, P ‘: 0.05.

In the low dose group, rearing was increased non-significantly by over a third. There was, however, a significant group X time-blocks interaction (F(3,51) = 2.86, P < 0.05) attributable to increased rearing during the first half of the session. At the middle dose rearing was increased si~ificantly relative to control, as determined by post-hoc comparison, but especially in the third and fourth blocks, again resulting in a group X time-blocks interaction (F(6,99) = 2.83, P < 0.05, see Fig. 2A). In contrast, the high dose of carbachol decreased rearing duration throughout the session (F(2,17) = 4.62, P < 0.05). TABLE HI Poke duration in tenths of seconds Mean holepoke durations for groups of animals receiving hippocampal infusions of either saline or 0.1 mg/ml carbachol. Before the 40-min experimental session. objects that were either strongly odorous or relatively odor-free were placed in the back of 3 of the 6 wall holes (see Methods). In the odorfree experiment carbachol increased poking into floor holes and the holes without objects but blocked the sahne group’s increased responsivity to the holes with novel objects. In the experiment with odoremitting stimuli the carbachol group still showed increased floor poking and now also exhibited increased responding to object holes, but failed to show the saline group’s increased responsivity to all wall holes __~_ _._.”----.--- --.- --- ~~ Saline _...“. Objects Wall with Wall without Floor

._.. ____.___ .__._ ._-.l__-_---.l 75.2 ri: 6.5** 43.8 :t 4.6 92.2 rir 20.8

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* Significantly different from saline control, P < 0.05. ** Si~ific~tIy

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different from without object control, P -: 0.05.

111 Increased holepoking by carbachol-infused animals was strictly related to the distribution of their increased locomotor activity. The low dose group, which differed from saline on long wall traverses only, made significantly more pokes into the side wall holes but not all holes. The high dose increased poking only slightly, except for a significant increase in floor pokes (F(2,17) = 5.60, P < 0.05). This effect should perhaps be ascribed to the increase in center duration seen with the high dose rather than to any change in the rats’ tendency to poke. The intermediate dose caused an increase in poke number and duration during the latter part of the session. In this group increased poking paralleled the time course of the increase in locomotion and was mostly dependent upon nonrepeated holepokes (see Fig. 2B). It is especially difficult to attribute the increase in holepoking by the intermediate dose group to an increase in stimulus responsivity in the light of their reaction to the holes with novel objects in them. Unlike the saline-infused rats, which spent over 50 %

Fig. 3. Locomotor pattern during high-dosecarbachol infusions. Hard copyx,y plots showing the spatial patterns of locomotor activity of rats receiving bilateral infusions into the dentate gyrus of (A) saline or(B) carbachol3.0 mg/ml at the rate of O.O25,4/min throughout a 40-min experimental session. Note that, compared to saline controls, carbachol animals were more active in the center of the chamber, had a much weaker home corner orientation, exhibited more laterally and diagonally directed movements, and generally covered a wider area of the holeboard floor. These saline animals were among the most active in their group.

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Fig. 4. Locomotor patterns during middle-dose carbach& infusions. Hard copy x,y plots of the sponta~~us activity of rats receiving hipp~pa1 infusions of (A) saline or (B) carbacholO.1 mg/ml during the last 10 min of the session, when carbachol’s effects were maximal. The movement patterns of the ~arbachoi-infused rats were more varied and widespread.

more time investigating the object holes than the object-free holes, the carbacholinfused animals showed no such preference (see Table III). As revealed by viewing of the real-time, variable-speed x,y video plots of the animals’ sequential movements, the pattern of locomotor activity of rats receiving high dose carbachol infusions was most different from controls during the middle of the session, Ten to fifteen minutes after the start of the session these animais often began to exhibit an unusual amount of lateral movements, relative to the long axis of the box, and diagonal traverses through the center region. This highly varied pattern of sequential movement was reflected in a lowered coefficient of variation of their region-toregion transition frequencies (see Table X1and Fig. 3). During the latter part of the session, when they were still more active than saline-infused animals, the high dose carbachol rats commonly circied the chamber, oken in only one direction, or else made back-and-forth trips either through the center region or along one of the long walls. They spent more time at the end of the chamber opposite the home area than saline-infused rats. Some of the rats receiving infusions of the intermediate dose of carbachol

113

Fig. 5. Time-course of locomotor patterns during carbachol infusions. Hard copy x,y plots of successive IO-mm Wocks of the locomotor activity of an animal receiving an intermediate dose carbach infusion. The rat was relatively quiescent 20-30 min into the session (C) but, during the last 10 min of the session (D), was more mobile than during any of the previous IO-min blocks. The time course of the changes in this animal’s pattern of movement is indicative of the abrupt onset of carbachol-induced locomotor activation rather than a deceleration of the gradual process of habituation.

Fig. 6. Effacts of carbachol on region duration histograms. Increased locomotion by rats infused with the middle and high doses ofcarbachol was accompanied by a more widespread spatial d~s~ibution of then activity over time. This effect is highly visible during the tast ten minutes of the infusion, i~~~~ated here. The amount of time spent in each region was r~nk~r~red on an ind~v~d~1 animal basis and cumulated for each group to produce the above region duration histogmms (see Tvfethods). Ascissa is the region number in order of preference for each animal over the entire session. Ordinate is base ten iogarithm of the number of seconds in each region during the session’s fourth IO-min block. Carbachol animals had a home region but they spent less time there than saline controls. The more restricted spatial distribution of the control animals’ time was quanti&d as a significantly higher coefficient of variation of their region durations for the entire session, an index of the increased skew in then region duration histogram (saline 1.65 & 0.06, carbacholO.01 mg/ml 1.70 & 0.19, carbachoi 0.1 mg/ml 1.33 Jo 0.11, carbachol3.0 mg/ml 0.88 f 0.09, F(3,36) = 10.35, P < 0.05). The effect was not s~~ificant for the low dose group.

showed similarly varied patterns of locomotor activity toward the end of the session (see Figs. 4 and 5). This took the form of circling the walls, cutting through the center, and increased rearings, especially in the corners. Like the high dose group, the intermediate dose carbachol animals displayed a weak orientation towards the home corner, sometimes even reversing direction after initiating locomotion towards the home end, a very rare occurrence in saline-infused animals, This tendency to remain out of the home region and distribute their time in a wider variety of loca~ons was s~tisti~lly quantified as a lower coefficient of variation of their region durations (see Fig. 6). The decrease in the coefficient of variation of their regio~~to~region transition frequencies, however, was not signi~c~t at this dose. For at least part of the session the increased spontaneous activity of most i~term~iate dose carbacb~l”infused animals was found to consist of repeated back-and-forth locomotion along one side of the holeboard box. Saline rats often restricted their territorial boundaries so as to exclude one wall of the chamber or the center, but most often they followed a different path and moved more rapidiy during the return haIf of round-trip excursions from the home end. These tendencies were less apparent in ~rb~~~-~~~5ed rats. This pattern of locomotor activity tended to raise the coefficient of variation of their region-toregion transition frequencies closer to that of the saline group (see Table II). In contrast the low dose carbachol group spent approximately the same amount of time as the saline group on their excursions out of the home area, as reflected in tht: absence of a significant change in the coefficient of variation of their re@ion durations

115

(see legend to Fig. 6). The spatial distributions of their patterns of ambulation were more widespread than control, however, The increase in long wall traverses in this group was determined by observation of the x,y video plots to be a consequence of circular movement around the experimental chamber (see Fig. 7). These excursions were not limited to a single direction and were not usually interrupted by rearings in each corner, like the circling sometimes seen with the higher dose infusions. This failure to restrict their locomotion to one side of the holeboard as the session professed, as control animals tended to do, resulted in a decrease in the ~~~cient of variation of their region-to-re~on transition probabilities (see Table II). In summary, the locomotor activation induced by earbachol was both quantitatively (see Fig. 12) and qualitatively dose dependent. As revealed by examination of video reconstructions of the animals’ sequential movement patterns, increasing concentrations of carbachol infused bilateraily into the dentate gyrus changed their most prominent pattern of locomotor activity from full circles to back-and-forth trips on one side of the box to lateral movements through the center and affected numerous dimensions of that pattern in a dose-dependent manner, decreasing home corner orientation, increasing the amount of time spent in the different regions of the experimental chamber, and shortening the spatial displacement between successive changes in the direction of movement. The mus~arinic blocker atropine sulfate, when co-infused in an equal weightvolume concentration, markedly antagonized carbachol’s effects, pa~icularIy its effects upon locomotion. In both experiments, the crossover frequency of the

Fig. 7. Locomotor patterns during low-dose carbachol infusions. The law dose of earbachot produced increased circling of the ex~rimental chamber, as iliustrat~ in this x,y plot af the sequential x,y positions in the holeboard box of a rat receiving a bilateral hipp~mpal infusion of carbachol in a concentration of 0.01 mg/ml throughout the 40-min experimental session, Saline-infused rats usually exhibited a more restricted pattern of locomotion after the first few minutes of the session (see Fig. 3A). The mare diverse pattern of movement of the low dose carbachoi rats was reflected in a decrease in the coefficient of variation of their region-to-re~on transition frequencies. The amount of time they spentin different regions of the holeboard was not increased, however, a~rding to the coe&ient of variation of their region durations.

116

--e-SRCINE -r-CRRB -xc-.CARB

3.0 + ATRO

Fig. 8. Effects of carbachol and atropine on rearing duration. The rearing durations of rats receiving hipp~mpal infusions of 3.0 mg/ml carbachol were si~ifi~ntly decreased. Co-infusion of the muscarinic receptor biocker a&opine antagonized carbachol’s effect only slightly, with the carbachol + atropine group not significantly different from carbachol and still significantly below saline. Abscissa is time in min; ordinate is time in set spent rearing.

carbachol + atropine group was significantly less than that of the carbachol only group and did not significantly differ from control (see Fig. 1). The same was true for number of entries into the center region (see Table I), The carbachol -+- atropineinfused animals were significantly more active than controls, however, during the third IO&n block at the 0.1 mg/ml dose level. This increase was present in the holepoke and rearing data as well, although in both instances carbachol’s overall group effects were blocked (see Fig. 2). In the case of varied pokes, however, the carbachol + atropine group mean was significantly lower than carbachol but higher than saline. At the 3.0 mg/ml dose atropine only shghtly antagonized the carbachol-induced increase in floor pokes and decrease in rearing duration (see Fig. 8). The ineffectiveness of muscarinic blockade against these high dose effects of carbachol suggests that they were a consequence of either nicotinic receptor stimulation or else of non-specific side effects of carbachol.

-e-SRLINE -r-RTRO -x- .ATFiO

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Fig. 9. Effects of atropine on crossovers. The muscarinic antagonist atropine sulfate non-significantly depressed locomotor activity when infused bilaterally into the dentate gyrus in concentrations of 0.1 mg/ml and 3.0 mg/ml. Abscissa is time in min; ordinate is number of crossovers.

117

Atropine alone, in concentrations of 0.1 mg/ml and 3.0 mg/ml, non-significantly decreased locomotion, rearing, and holepoking (see Fig. 9). The high dose of a&opine did, however, alter the animals’ sequential pattern of stimulus responding and movement. Unlike saline-infused rats, which spent significantly more time making varied pokes than repeated pokes (F(1,ll) = 5.13, P < 0.05), the repeated poke durations of atropine-infused rats were actually longer than their varied poke durations. When displayed by the video x,y plot program the locomotor activity of these animals was also found to exhibit certain perseverative characteristics. After leaving their home end atropine animals were less likely than controls to change their direction of movement before reaching the opposite wall of the experimental chamber. Locomotion along the long wails was typicahy rapid. Movement through the center was slower and more hesitant, although this was often the case in saline animals as well, at least when they were moving away from the home area, In traversing the center region, the atropine rats would sometimes drift laterally into closer proximity with the wall or else occasionaily make a wide turn back into the home end. They failed to demonstrate the short back-and-forth trips that normal animals appear to utilize in securing and expanding their home territory 13. The atropine-infused rats did not, however, seem to have a particular preferred direction of movement throughout the session. In order to quantify this apparent decrease in redirected locomotion, the relative frequency of wall-to-wall traverses (short wall traverses, full center traverses, and full long wall traverses) vs the frequency of traverses requiring the spontaneous reversal of the animal’s direction of movement (half-center traverses and half long wall 9

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Fig. 10. Effects of nicotine and curare on activity and rearing. Effects of hippocampal infusions of a nicotinic agonist and antagonist on (A) locomotor activity and (B) rearing. The nicotinic agonist nicotine, infused in a concentration of 0.05 mgfml, slightly increased locomotor activity and rearing throughout the session. The nicotinic receptor blocker o-tubocurare, 0.1 mg/ml, had biphasic effects on activity. Locomotion was nonsignificantly depressed by curare infusions between 10 and 20 min after the start of the session, but si~ifi~ntly increased relative to saline controls during the second half of the session. Curare significantly increased rearing during the second half of the session as well. Abscissa is time in min; ordinates are (A) number of crossovers and (B) number of rearings against the walls of the chamber.

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MINUTES

Fig. 11. Effects of carbacbof and odors on crossovers. In the presence of novel odorous stimuli, bilatera1 intrah;pp~ampal infusions of 0.1 mgjml carbachoI increased locomotor activity relative to saline-infused control rats. Abscissa is time in min; ordinate is number of crossovers. The effect was maximal in the fourth IO-min block and absent in the first.

traverses) was calculated for sahne and atropine rats. The ratios were found to be significantly different as predicted from observation of the x,y plots (saline: 1.75 _ir 0.24, atropine: 0.97 f 0.17, F(1,16) = 4.57, P < 0.05). Nicotine. is approximately 5 times more potent than carbachol as a nicotinic receptor agonist. Infused in a concentration of 0.05 mg/ml, nicotine non-significantly increased locomotion, rearing, and holepoking (see Fig. 10). Curare’s effects on motor activity were biphasic, interacting with the time-blocks factor (Q&93) =: 2.59, P < 0.05). The nicotinic blocker caused a mild depression of locomotion and holepoking during the second 10 min of the session. These effects of nicotine and curare tend to suggest that there is a weak nicotinic contribution to the activating effects of carbachoi. In the second half of the session, however, curare-infused animals exhibited a sharp increase in locomotor activity and rearing (see Fig. IO). In addition, like carbachol, curare decreased responsivity to the holes with novel stimuli (poke duration with/without: saline 2.66 :-k0.64, curare 0.96 3- 0.21, F( I ,20) = 6.35, P < 0.05). These results might be related to the observation in pilot studies that high dose infusions of both carbachol and curare produce wet dog shakes and forced motor activity. These actions were most likely not the consequence of nicotinic receptor antagonism since blockade with near molar concentrations of nicotine failed to evoke such phenomena. The replacement of the novel objects in 3 of the 6 side wall holes with novel odor-emitting stimuli altered the behavioral profile of both control animals and animals receiving bilateral hippocampai infusions of 0.1 mg/mi carbachok As in the odor-free experiment, carbachol increased locomotor activity (F(1,24) :--: 9.43, P c: 0.05) and the effect was absent at the start of the session and maximal at the end (see Fig. 1I). Total holepokes and rearings, on the other hand, were no longer significantly increased. As can be seen in Table III, the novel odors in the 3 wail holes appear to have powerfully attracted the saline-infused animals to all the wall holes, so that their total wall poke duration almost doubled. Unlike the object stimuli, the odor-emitting stimuli appear to have been su~~jently salient to elicit signific~tly increased response

119 TABLE IV Rearing duration in seconds -

carbachol

Effects of hippocarnal infusions of the cholinergic agonist carbachol, 0.1 mg/ml, and of novel, odoremitting stimuli upon the mean rearing duration (& S.E.M.) of rats during a 40-min experimental session. Powerfully odorous or relatively odor-free objects were introduced to three wall holes before infusions. The presence of the odors significantly increased the amount of time spent rearing against the wall by the saline infused animals but not by the carbachol-infused animals.

Objects Odors

Saline

Carbachof

91.3 f 11.9 184.1 & 18.5*

108.2 * 16.3 120.2 & 13.2

* Significantly different from odor-free condition, P < 0.05.

durations from carbachol-infused animals as well as saline-infused animals. In contrast to the saline-infused animals, however, the carbachol animals failed to generalize their increased responding to the other wall holes. As a result, in the experiment with odor-emitting stimuli carbachol increased holepoking into floor holes only. The odors likewise appear to have been responsible for the disappearance of carbachol’s effect upon rearing. As can be seen in Table IV, the addition of odorous stimuli to 3 of the wall holes more than doubled the amount of time saline animals spent rearing against the walls (F(1,34) = 17.75, P < 0.05). The total rearing duration of carbachol animals increased only slightly. The increase in locomotion induced by carbachol in experiments with and without odors does not, therefore, seem to have been dependent upon an increase in stimulus responsivity. Animals receiving infusions of carbachol(O.1 mg/ml) into one dentate gyrus and saline into the other exhibited behavior similar to animals receiving bilateral calbachol infusions. The effects wele milder, though, appearing as interactions with the timeblocks factor for overall motor activity, varied holepokes, and rearing (F(3,48) = 3.28, 5.22, and 4.63, P < 0.05); analysis of simple main effects showed significant increases in varied holepokes and photobeam intenuptions during the first and last 10 min of the session respectively (see Table V). TABLE V Photobeam interruptions Overall motor activity of rats receiving combined infusions of 0.1 mg/ml carbachol into the dentate gyrus on one side of the brain and saline into the other side did not significantly differ from controls although the measure rose significantly during the last 10 min of the session. Bilateral infusions of 0.1 mg/ml carbachol into the dorsal hippocampal alveus that produced heavy leakage of infusate into the overlying lateral ventricle did not affect the motor activity of the rats. Saline Unilateral carbachol I.C.V. saline I.C.V. carbachol

1262.5 1396.2 1420.9 1506.1

+ f + f

61.4 72.3 89.8 72.0

120 The behavior of rats administered bilateral infusions of carbachol 0. I mg/ml into the alveus and overlying ventricle was not markedly different from controls. The manipulation very slightly and nonsignificantly increased overall motor activity (see Table V) and did not affect rearing or holepoking at all. Results from the histological examination of dye-infused brains have been described in detail previously 11. In almost all cases, the tip of the infusion needle was located inside the dentate hilus at approximately A 3200-3400 of Konig and KlippeP. In coronal sections the dye, at its maximal extent, usually covered the dentate gyrus of the anterodorsal hippocafllpai formation except for its dorsolateral and ventrolateral extremes. Heaviest dye accumulation was seen along the dorsal and ventral granule cell layers and inside the dentate hilus. This dye distribution matched closely the fluorescence distribution observed in Falck-Hillarp treated tissue after infusions of ~,L-norepinephrinel~. 4. DISCUSSION

Taken together, the results indicate that the dose-dependent increase in locomotor activity, holepoking, and rearing produced by the bilateral intrahippocanlpai infusion of carbachol in the rat was a consequence of the stimulation of muscarinic choiinergic receptors in the dentate gyrus. The strongest evidence for muscarin~c mediation of carbachol’s effects was the near complete antagonism by the muscarinic blocker atropine (see Fig. 12). Alone, atropine produced non-significant effects in the opposite direction. Nicotine, a far more potent nicotinic agonist than carbachol, only faintly mimicked the actions of the mixed cholinergic agonist, perhaps suggestive of a slight nicotinic component to the carbachol effect. The nieotinic biocker curare likewise produced only a slight, transient depression of activity during the first half of the sessions. During the latter part of the session, the behavioral profile of curare-infused rats

Fig. 12. Dose-response effects of carbachol and atropine on activity. The overall motor activity of rats receiving bilateral hippocampal infusions of carbachol, atropine, or a combined solution of carbachol plus atropine is shown. Abscissa is base ten logarithm of the weightkolume concentration; ordinate is group mean of the total number of photobeam interruptions during the 40-min experimental session. Saline animals are a composite group.

121 was surprisingly similar to carbachol in that locomotor activity was increased and responsivity to novel stimuli was decreased. In pilot studies high doses of carbachol and curare were both found to produce seizure activity in the form of ‘wet dog’ shakes, in confirmation of previous EEG studies 1929.Blockade of nicotine receptors with high concentrations of nicotine failed to elicit any behavioral signs of epileptiform activity, so nicotinic receptor antagonism is an unlikely primary mechanism for these effects. The similarities between the effects of carbachol and curare might rather have been due to curare’s inhibition of acetylcholinesterase, which occurs at concentrations slightly above those necessary to block nicotinic receptorss1s4g. In both cases, then, increased locomotion and high dose seizures might have been due to muscarinic receptor stimulation. Some of the effects of the infusion of the high dose (3 mgjml) of carbachol, in particular the increase in floor pokes and decrease in rearing, were not blocked by coinfusion of atropine. These effects could not have been mediated via nicotinic receptors, since nicotine infusions did not produce comparable efffects. They were not more stable effects of carbachol, less sensitive to blockade and maintained in spite of the effectively lower dose of carbachol, since opposite effects on rearing were produced by lower doses of carbachoi. They cannot be attributed to a generalized dysfunctional state of the cholinergic receptor, because atropine alone had the opposite effect on floor pokes and decreased rearing by only 10%. These changes in rearing and holepoking activity therefore appear to have been non-specific high dose effects, perhaps not mediated at cholinergic receptors. This interpretation is supported by the evidence of undue seizure activity at the start of the session when this concentration of carbachol was infused. Localization of carbachol’s site of action to the dentate gyrus is supported by the results from the dye infusions. Spread of the infusate appears to have been restricted to the dentate gyrus of anterodorsal hippocampus. The lack of effects of control carbachol infusions into the lateral ventricle and alveus also delimits the probable locus of carbachol’s actions. Clearly, ventricular leakage cannot account for the effects observed at the 0.1 mg/ml dose. Nor does the CA1 region of the hippocampus proper appear to have been responsible for carbachol’s effects, since these control infusions permitted access of the infusate to the stratum oriens adjacent to the CAI pyramidal cell layer, where a putative cholinergic terminal field has been demonstrated by AChE histochemistrysO@. Furthermore, although the possibility of CA3 mediation cannot be eliminated, it should be noted that control and experimental infusion sites were approximately equidistant from that region. The ineffectiveness of infusions into the control site is not surprising since the highest activity levels of CAT12 and AChEas in the hippocampal formation are found in the dentate gyrus. The unilateral carbachol infusions were administered as a control for the possibility that carbachol’s effects were due to an inadvertent asymmetry between the distribution of the infusate in the dentate gyri on the two sides of the brain. If asymmetry of the infusion sites and spread of infusate were in fact a critical independent variable then the paired carbachol and saline infusions would have been expected to produce, in contrast to the bilateral carbachol infusions, greatly exagge-

122 rated effects, or else qualitatively different effects, or, perhaps, no effects at ah. The partial but qualitatively simitar effects seen with the uniJatera1 infusions indicate that experimental error-induced differential distribution of the infusate on either side of the brain was not a key determinant of the treatment effects seen with the bilateral carbachol infusions. The increased locomotion, rearing, and holepoking observed in the present study during carbachol infusions into the dentate gyrus thus indicate that cholinergic receptor stimulation in the dentate gyrus can lead to the release of motor activity. However, pharmacological studies utilizing learning paradigms have in general supported the hypothesis of cholinergic mediation of habituation7 and of the explicit involvement of the septo-hippocampat pathway in this processs. The time course of the increase in locomotor activity produced by the intermediate dose of carbachol, graphed in Fig. IA, could in fact be construed as a disruption of habituation to environmental stimuli. Three points argue against the adequacy of this interpretation. First, the higher-dose effect had a shorter latency of onset and resulted in a habituation curve steeper than the one seen at the intermediate dose. Second, the slope of the intermediate dose curve is positive between the third and fourth blocks, suggestive of an active process. This transition was frequently abrupt, as dramatically illustrated by the time course of the spatial distribution of the locomotor activity of the carbacholinfused rat depicted in the hard-copy x,y plots of Fig. 5. Third, the increase in spontaneous motor activity produced by carbachol, while explainable as maintained responding to environmental stimuli due to decreased habituation to those stimuli, in fact appears to have been associated with a decrease in the rats’ stimulus responsivity. The carbachol rats failed to respond to novel objects placed in three of the wall holes and also failed to show the generalized increase in poking and rearing of saline-infused rats to the introduction of odorous stimuli to the experimental chamber. The medial septum appears to act as the generator for a unique pattern of electroencephalographic activity in the hippocampus, rhythmical slow waves known as hippocampal ‘theta’. Electrical stimulation of the medial septai nucleus drives hippocampa1 theta and lesions of the septum, or of the fornix, which interconnects the two structures, abolishes itl511Q”. Furthermore, single units have been found in the medial septum and nucleus of the diagonal band that burst in phase with the theta rhythm”6337 and such rhythmic firing will continue, under certain conditions, in the absence of hippocampal thetas7+47*57.Evidence that the septo-hippocampal pathway utilizes ACh as a neurotransmitter has been discussed above. The behavioral correfates of theta have been heavily studied (see review by O’Keefe and Nadelss). Locomotion has been established as a sufficient condition for the appearance of hippocampal theta 50,~~.It also appears, however, in response to a novel stimulus, especially in the rabbit 16. jn the rat too, it has been recorded when the animal is not in motion, as during sniffing or during the period of immobility preceding a jumpts,5(). It is now generally agreed that theta in the rat can be behaviorally and pharmacologically divided into high frequency 7-10 Hz movement theta and low frequency 4-7 Hz non-movement theta21*51,52.The pharmacological distinction is based upon selective blockade by atropine of non-movement theta OnJY.

123 Thus there are non-cholinergic mechanisms that can generate theta during movement, perhaps via a non-cholinergic septo-hippocampal pathway. Hippocampal theta accompanies the high frequency, desynchronized cortical EEG by which arousal is defined and is also present during ‘paradoxical’ or ‘rapid eye movement’ sleep. This last form of non-movement theta is also blocked by atropine. The association of nonmovement theta with eye movements, sniffing, orienting, and subsequent locomotion, rearing, or jumping suggests that the cholinergic input to the hippocampus participates in the changing of the set of sensory stimuli to which the animal is responding and in the initiation of movement. The effects of high dose atropine infusions into the dentate gyrus upon the rats’ sequential patterns of locomotor activity observed in the present experiment are consistent with the hypothesis of a role for the cholinergic input to the hippocampus in the initiation of movement and the re-direction of responses. Atropine-infused animals moved hesitantly forward through the center, rapidly along the walls and only reversed direction after reaching a wall of the experimental chamber, rarely making short back-and-forth trips. Stimulus perseveration and deficient shifting of motor responding was also evident in a decrease in varied holepoke duration relative to repeated holepoke duration. In agreement with these findings, Schallert et al.3s have reported that rats administered systemic injections of attopine, unlike saline controls, failed to spontaneously turn around in a narrow, blind alley but instead became ‘ttapped.’ Changes in their direction of movement were strictly stimulus dependent, as demonstrated by their ability to follow a sensory stimulus, a blunt probe, out of the alley. Slow-motion split-screen videotape showed that control rats but not atropine rats generated hippocampal theta between successive movements in the open field, as during shifts in the direction of locomotion or before rearing. The results from this study would thus appear to indicate that the cholinergic input to the hippocampus plays an active role in movement initiation. In agreement with this conclusion is the finding that electrical stimulation of the dentate gyrus, only at parameters that disrupt hippocampal theta, produces increased latencies and suppression of jumping, swimming, and ambulatione. Nevertheless, the results from this study are not contradictory to the hypothesis of hippocampal participation in the process of habituation. The septal input to the hippocampus is subject to a strong cholinergic influence diffusely ascending from medial brainstem reticular formation2s*33936343, and appears to act as a priming stimulus signalling the animal’s level of arousal and the level of incoming sensory stimulation, but perhaps also conveying a limited number of contextual cues. This input seems to promote the release of motor responding and the processing of more specific and complex sensory information derived from tertiary associational cortex 35.53. This latter information reaches the hippo~mpus via the perforant path arising in entorhinal cortex, the other major afferent to the hippocampusd. The decrease in responding to both novel objects and novel odors by the carbachol-infused rats can perhaps be attributed to the independence of the experimental manipulation from the actual sensory input that the animal was receiving. The hippocampus has long been thought to be involved in the response to novelty since

124 animals with hippocampal lesions are less distracted than controls by the introduction of novel stimuIis,~,l”~18~1p~“s. Furthermore, electrophysiological studies have shown that some of the cells in CA1 specifically respond to the removal of a sensory cue from a familiar environment3~. In this ex~riment it would seem that the carbachol rats failed to respond to the novel sensory stimuli as control animals did because the exogenous stimulation of cho~inergic receptors in the h~ppocampus produced a nondiscriminant release of responding to ah environmental stimuli. In the same way the apparent lack of habituation in these animals might be attributed to carbachoi’s supersession of hippo~anlpo-septal feedback that normally produces decreased responding to familiar stimuh. Perhaps the endogenous choiinergic innervation of h~ppocampus has the function of targeting sensory stimuli in the environment so that in normal animals as well it faciiitates motor responding, but in that case they are responses to more appropriate stimuli. 5. SUMMARY

Awake, unrestrained rats in a holeboard/activity chamber received direct bilateral infusions of the mixed muscar~nic~~icotinic cholinergi~ agonist carbachol into the dentate gyrus during a 40-min experimental session. The computerized holeboard system measured the animals’ locomotor activity, stimulus responsivity, and response to novelty, and ~rmitted the reconstruction and analysis of their sequential patterns of movement. Carbachol infusions produced a dose-dependent increase in locomotor activity that was accompanied by increased holepoking and rearing. The response to novel objects and odors was depressed by carbachol. Carba~hoi-infused rats also exhibited dose-dependent changes in their patterns of locomotor activity, Hippocampal infusions of the mus~arinic antagonist atropine produced nons~gni~~ant decreases in locomotion and a ~rseverative pattern of locomotor activity and stimulus responding. Co-infusion of atropine blocked the effects of carbachol, whereas the effects of the nicotinic agonist nicotine and an~gonist curare were negligible. Ventricular infusions of an intermediate dose of carbachol failed to affect the animals’ behavior and unilateral infusions produced effects that were similar to but milder than the bilateral infusions. Dye infusions revealed that spread of the infusate was restricted to the dentate gyrus of the anterodorsal hippocampal formation. The locomotor activation and reduced stimulus responsivity caused by the carbachol infusions into the dentate gyrus were interpreted as indicative of a role for the cholinergic septohippocam~l pathway in the release of motor responding and in hippocampal processing of sensory information.

This work was supported in part by NIMH Grant MH 30914 to D. Janowsk~ and by NSF Grant BNS 04676. We grafetully acknowledge the assistance of Bernadette Allen and Alyson Hansen.

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