s2fieldsofstudy: Biology Psychology

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Visual relational (“categorical”) learning and performance by rats with temporal or partial striate cortical lesions

Previous research has shown that injuries of the inferotemporal cortex in monkeys result in impaired visual “categorical” perception. The goal of this project was to explore the possibility that a similar deficit might accompany injuries of the visual portions of the temporal cortex in rat, namely area TE2. Rats were prepared with bilateral TE2, partial striate, or sham cortical lesions and trained to discriminate between 17- (S+) and 3-cm (S−) diameter circles in a Y-shaped water maze. After criterion on this discrimination was reached, tests of relational perception were conducted with intermediate-size stimuli. The larger of the two stimulus pairs consistently cued the location of a submerged escape platform. Not only did rats with TE2 lesions learn the original discrimination as rapidly as normal rats and rats with partial striate lesions, but their performance paralleled that of the other groups on all tests of relational perception with intermediate stimuli. Several possible explanations for the discrepancy between the results observed here with rats and those observed with nonhuman primates are discussed.

It is now well established that two distinct cortical visual pathways exist in human (Haxby et aI., 1991;Horwitz et aI., 1992) and nonhuman (Macko et aI., 1982;Mishkin, Ungerleider, & Macko, 1983;Ungerleider & Mishkin, 1982) primates. The dorsal occipital-parietalprefrontal pathway is specialized for visuospatial perception, whereas the ventral occipital-temporal-prefrontal pathway is specialized for complex pattern and object identification. Lesions within these regions produce severe dissociable deficits in visual-spatial and visual-memory tasks, respectively. Regional cerebral blood flow measurement revealed by positron emission tomography while humans performed a face-matching task or dot-location task has supported studies based on lesion by showing that the occipitotemporal cortex is involved in the visualmemory task whereas the posterior parietal cortex (PPC) is involved in visual-spatial localization (Haxby et aI., 1991 ).
In a review of the scant literature on this topic in rat, Kolb (1990) hypothesized that a similar organization appeared to characterize the corticocortical projections emanating from the striate cortex of the rat. That is, an anterodorsal pathway arising from the striate and peri striate regions terminates in the PPC (Kolb & Walkey, 1987), whereas a more ventrorostral pathway terminates in the visual-temporal region labeled TE2 (specifically, the This research was supported in part by a Georgia College and State University faculty research grant to the second author. We thank David M. Compton for his assistance with several methodological and technical aspects of the project. Correspondence should be addressed to W. F. McDaniel, Department of Psychology, Georgia College and State University, Milledgeville, GA 31 061-0490 (e-mail: bmcdanie@mail. gcsu.edu). more caudal regions ofTE2; Shi & Cassell, 1997). Neurobehavioral investigations have revealed that the PPC in rat plays an important role in spatial navigation on the basis of visual cues (e.g., Kolb, Buhrmann, McDonald, & Sutherland, 1994;Kolb & Walkey, 1987;McDaniel & Thomas, 1978;McDaniel & Wall, 1988), and that the PPC plays different but complementary roles in the two hemispheres in processing such cues (McDaniel et aI., 1995;McDaniel, Williams, Attaway, & Compton, 1998). Fewer studies have focused on functions of TE2 in rat. Davis and McDaniel (1993) prepared rats with bilateral PPC, TE2, or sham cortical ablations and trained the animals on a visual matching-to-sample problem and on a nonconceptual visuospatial conditional problem with the order of exposure to the tasks counterbalanced within groups. Animals with either type of lesion were inferior to the sham control animals on both tasks, and a dissociation between the lesion groups failed to emerge. Thus, although animals with TE2 lesions performed poorly on the visual-memory task, so did the animals with PPC lesions. Nevertheless, Kolb et ai. (1994) conducted a similar study with what appears to be a simpler methodology and found, as is the case with primates, a dissociation between the influences of PPC and TE2 lesions on visualspatial and visual-memory functions. Animals with TE2 lesions were deficient in their visual matching-to-sample task, but not a spatial navigation task. The opposite was found with PPC-lesioned rats.
Recent investigations using electrophysiological methods and assays for Fos (i.e., the protein product of the immediate early gene c-fos) have supported the conclusion that area TE2 plays an important role in visual memory (Zhu, Brown, & Aggleton, 1995;Zhu, McCabe, Aggleton, & Brown, 1996). These investigations have shown that neurons in rat TE2 cortex are less responsive to familiar visual stimuli than they are to novel stimuli. Similar results had previously been described in primates (e.g., Sobotka & Ringo, 1993). Also, they have shown that there is less intraneuronal c-fos activity in area TE2 when the animal is exposed to familiar visual objects than when exposed to novel objects. Similar results were observed in the perirhinal cortex, but not in any of the other areas studied (e.g., superior colliculus, occipital association cortex, entorhinal cortex, hippocampus, anterior cingulate gyrus, amygdala, etc.).
It has been reported that the temporal association cortex of humans and the inferotemporal (IT) cortex of monkeys plays an important role in "categorical" or relational visual perception (see Kolb & Whishaw, 1990, p. 458). Wilson and DeBauche (1981) trained cynomolgous monkeys to discriminate between stimuli at two ends of a continuum in three separate problems. The problems consisted ofa length discrimination task (10 vs. 55 mm), a stripe orientation discrimination task (0° vs. 90°), and a texture discrimination task (3.0 vs. 23.0 lines per cm). The animals then received either IT or partial lateral striate (PS) cortical ablations, and they were tested with intermediate stimuli for both between-(e.g., with respect to the line length discrimination, 20 vs. 45 mm) and within-(e.g., 10 vs. 25 mm) category perception. Monkeys with IT lesions failed to demonstrate categorical perception, whereas normal control monkeys and monkeys with PS lesions demonstrated relatively good transfer of the preoperative habits to the postoperative discriminations with new stimuli. The authors concluded that "lacking IT cortex, monkeys tend to perceive visual stimuli more in terms of their physical values alone, and less in terms of their relationship to stimuli experienced previously in a perceptual domain" (p. 36).
The purpose of the present study was to explore the possibility that area TE2 in rat plays a necessary role in "categorical" or relational visual perception, as is the case in primates. Rats were prepared with bilateral TE2, PS, or sham cortical lesions and trained to discriminate a 17cm-diameter black circle (S+) from a 3-cm black circle (S-). In several subsequent phases, generalization ofthe previously learned response to novel stimuli with intermediate diameters (e.g., 13 vs. 7 cm) was assessed. If the rat temporal cortex is important in "categorical perception," as appears to be the case for primates, then rats prepared with TE-2 lesions should perform as well as normal rats while learning the initial visual discrimination. However, TE-2 injured rats should exhibit considerable behavioral dysfunction on tests of visual relational perception. Three months after categorical perception was examined, long-term retention was explored by retraining the rats on the original discrimination.
Rats with PS lesions served as lesion control group to parallel the design in the study by Wilson and DeBauche (1981), where monkeys with PS lesions served as controls. Rats with PS lesions generally perform relatively well in tests of pattern discrimination, and it has been shown that even when prepared with total striate ablations, the destriate rat eventually masters demanding visual discriminations (e.g., Hughes, 1977;McDaniel & Noble, 1984).

Subjects and Surgery
Fifteen male Long-Evans rats, bred in our colony and ranging in age from 7 to 11 months at the beginning of the experiment, were used as subjects. Five rats were randomly assigned to each of three surgical conditions: PS, TE2, or sham control. Rats were individually housed in stainless steel rodent cages and entrained to a reversed Iight-dark cycle with light offset at 8:00 a.m. and onset at 8:00 p.m. All surgical and behavioral procedures were conducted during the dark phase. The animals were maintained on an ad-lib schedule offood and water. All aspects of the methodology were reviewed and approved by the University's Institutional Animal Care and Use Committee.
Each animal was weighed and administered an intraperitoneal injection of atropine (.35 mg/kg) and sodium pentobarbital (45 mg/kg). After the scalp was shaved and scrubbed with a 10% povidone-iodine solution, the animal was placed in a Kopf stereotaxic instrument. Using clean surgical instruments, a midline incision was made, the temporalis muscle was retracted ventrolaterally with a periosteal elevator, and the dorsal cranium was exposed.
For animals assigned to the sham control group, surgery was terminated at this point. The wound was closed with wound clips, and Mycitracin triple antibiotic ointment with lidocaine was applied liberally to the incision site.
For animals receiving PS lesions, the skull was trephined at an area 3.0 mm anterior to the Lambdoid suture and 3.0 mm lateral to the sagittal suture. Rongeurs were used to expand the area to expose area 17 (Hughes, 1977), and the underlying cortical tissue was aspirated. For animals receiving TE2 lesions, the skull was trephined 5.0 mm posterior to the coronal suture and 5.5 to 6.5 mm lateral to the sagittal suture. The craniotomy was enlarged several millimeters along the AP plane with a microrongeur to expose area TE2. Under visual guidance from a jeweler's loop (3 X magnification), the dura was excised with a microdissection scalpel, and the target cortical mantle was aspirated through a glass micropipette. Following surgical procedures, the cavities were packed with Gelfoam and the wounds were closed as described for sham control animals. A 7-day postoperative recovery period was observed prior to initiating an evaluation of kinetic function. Training on the visual discrimination task began 13 days after surgery.

Histology
Upon completion of the behavioral procedures, PS and TE rats received an overdose of sodium pentobarbital, and they were perfused through the heart with 50 cc of isotonic saline followed by 50 cc 10% formalin. The brains were removed and stored in 10% formalin for 7 days prior to being photographed from the dorsal or lateral perspectives. Then they were transferred to a 30% sucrose solution in 10% formalin mixture and refrigerated for several days. After being blocked to include the lesion site and thalamus, brains were frozen in a Cryocut II microtome and sectioned into 60-llm slices through the coronal plane. Every fifth section was mounted on a slide and stained with cresyl-violet acetate. Sections were examined under a light microscope for evidence of retrograde degeneration in the thalamus, for accuracy of lesion placement, and for lesion depth.

Apparatus and Stimuli
The apparatus for examining visually guided behavior was a white, circular, galvanized steel water tank that measured 1.4 m in diameter and 0.6 m in height. The water tank was fitted with a Yshaped alley also constructed from galvanized steel. The Y maze consisted of a stem, 86 cm long and 30 cm wide, with two arms, each 61 cm long and 54 cm wide. The apparatus was filled with water to a depth of20 cm. Water temperature was maintained at 26° :': 1°C. Bromide tablets were added approximately every 10 days to the water to discourage microbiological growth. White nontoxic paint (Artistica II tempera, Hazleton, PA) was added to the water to obscure view of a white escape platform (26 x 17 x 17 cm) submerged 2 cm below the water surface. Throughout all behavioral procedures, the platform was consistently located beneath the larger of two visual stimuli, and it was positioned adjacent to the rear wall of either Y arm of the apparatus. The distance from the start position to the escape platform was 140 cm.
The visual stimuli consisted of eight, two-dimensional circles differing by increments of 2 cm (3,5,7,9,II,13,15,or 17 cm in diameter). Each circle was solid black and centered on white paper (33 X 25 cm). The circles were printed with nonwater soluble ink, and the stimuli were laminated. The stimuli were suspended from the rear wall of each of the two arms of the Y, and the bottom edges of the stimuli were approximately 2 cm above the water surface.

Rotating Rod Test
A wooden rod (3.5 cm in diameter and 180 cm long) wrapped with grip tape and rotating at a rate of five rotations per minute was used to assess gross locomotor proficiency. The rod was horizontal to the plane of the floor, and the area below the rod was covered with foam strips to cushion falls. Gross kinetic functions were assessed for 5 consecutive days. Each animal was rated for a I-min period (excluding the seconds between a completed fall and the replacing of the animal onto the rod). The number of falls observed during the I-min contact with the rod constituted the gross measure of kinesis.

Visual Learning and Performance
Discrimination training. Following a 13-day postoperative recovery interval, training began on the 3-cm-diameter circle (S -) versus the 17-cm-diameter circle (S+) discrimination task. Each trial began by placing the rat in the start location with its head facing the front wall of the apparatus. The left-right alley positions of the S-and S+ stimuli and the escape platform were randomized using a Fellows (1967) series. Correct responses were defined as entry into the arm of the Y containing the larger stimulus and subsequent escape onto the submerged platform. Errors were defined as entry into the alley containing the smaller diameter circle to the point that the root of the tail crossed the entrance to the arm. Animals were permitted to self-correct incorrect choices, and they were allowed to remain on the platform for approximately 10 sec. During each intertrial interval (IT!) of approximately 20 sec, the animal was placed in the home cage while the experimenter arranged the stimuli and positioned the escape platform for the next trial. The animals were trained on 10 trials per day, and the criterion to demonstrate acquisition was defined as one session of nine correct trial choices within a sequence of 10 trials. Upon attaining criterion, each animal proceeded with the procedural phases designed to examine relational perception.
Relational discrimination. The probes of relational perception consisted off our phases. As was the case in discrimination training, the escape platform was located beneath the larger of two stimuli in each trial of each phase. In Phase I, the animals were required to discriminate between stimuli from different categories, wherein the diameters of the circles were reduced from 3 cm and 17 cm to 5 cm and 15 cm, respectively. In the second phase, the stimuli used in training (i.e., the original discrimination) and in Phase I were used. Each session required the rat to discriminate between a 3-cm circle (S-) and a 17-cm circle (S+), or between a 5-cm circle (S-) and a 15-cm circle (S+) for an equal number of trials within each session.

VISUAL RELATIONAL LEARNING 343
Again, the criterion to demonstrate acquisition was defined as one session of nine correct choices within a session of 10 trials. Phase 3 was composed of two subphases. First, across five sessions composed of 50 trials total, animals were required to discriminate between two stimuli in which the diameters of the circles were reduced to 7 cm (S-) and 13 cm (S+). Next, across another five sessions composed of 50 total trials, animals were required to discriminate between two stimuli in which the diameters of the circles were reduced further to 9 cm (S -) and 11 cm (S+). After testing occurred for 10 sessions, animals completed Phase 3 and then entered Phase 4, the final relational discrimination phase. In Phase 4, across seven 18-trial sessions (126 total trials), animals were presented with assorted-paired comparisons, in which both cues originated from within the same category. Hence, each pair contained either only previously negative (S-) circles (3, 5, 7, or 9 cm), or only previously positive (S+) circles (1 I, 13, 15, or 17 cm).
Specifically, the animals were required to discriminate between two previously negative circles wherein the larger stimulus was 2, 4, or 6 cm larger in diameter than the smaller stimulus (e.g., 3 vs. 5 cm, 3 vs. 7 cm, or 3 vs. 9 cm). The correct choice was the larger of the two previously negative circles. Similarly, animals were required to discriminate between two previously positive circles wherein the larger stimulus was 2, 4, or 6 cm larger in diameter than the smaller stimulus (e.g., 15 vs. 17 cm, 13 vs. 17 cm, or II vs. 17 cm). Thus in this phase, all trials involved the execution of within-category discriminations. The correct choice was the larger of the two previously positive circles. Within each session of 18 trials, each set of 6 trials was composed of paired comparisons with differences of2, 4, or 6 cm. The order of the assorted comparisons was presented in a quasi random manner (i.e.,17 vs. 11,9 vs. 3,17 vs. 15,15 vs. II,7 vs. 5,17 vs. 13,9 vs. 7,9 vs. 3,17 vs. 11,9 vs. 5,5 vs. 3,13 vs. II,7vs.3,15vs.13,15vs.ll,17vs.II,9vs.5,and9vs.3).Following the completion of seven sessions in Phase 4, procedures were terminated temporally for all animals.
Retention. Following a 3-month hiatus, long-term retention was explored by retraining the animal through criterion on the original discrimination.

Lesion Analysis
Representative photographs of PS lesions (observed from the dorsal perspective) and TE2 lesions (from the lateral perspective) are displayed in Figure 1. The location of the lesions appeared to be consistent with those depicted in previous investigations (Davis & McDaniel, 1993;Kolb et aI., 1994;McDaniel & Skeel, 1993;Meyer, Meyer, & Cloud, 1986).
Microscopic examination of the sectioned brains revealed that PS lesions removed variable amounts ofZilles's (1985) Oc 1 M and Oc2L, and large portions of Oc2MM and Oc2ML. The anatomical alterations induced by PS lesions are displayed in reconstructions of coronal sections of typical PS lesions in Figure 2. All PS lesions invaded portions of retrosplenial agranular cortex, and 1 PS animal had slight damage to the corpus callosum. Patchy gliosis, cell loss, and cell shrinkage in the lateral geniculate nucleus accompanied PS lesions.
The topography and anatomical alterations induced by TE2 lesions appeared to be similar to those reported in several previous studies (Davis & McDaniel, 1993;Kolb et aI., 1994;McDaniel & Skeel, 1993) Zilles's (1985) Te2, variable amounts ofTe3, the posterior region of Te I, and a modest portion of the lateral border oflateral peri striate cortex (Oc2L). Additionally, lesions in 2 TE2-damaged animals included minor invasion of the perirhinal cortex, a finding also reported by Kolb et al. (1994). In cases of TE2 lesions, as has been described in other studies (Davis & McDaniel, 1993;Meyer et aI., 1986), there was no obvious evidence of necrosis or gliosis within the thalamus. Although there appeared to be no significant subcortical or callosal damage, 1 TE animal did sustain superficial unilateral damage to the ventrolateral portion of the hippocampus.

Rotating Rod Test
An analysis of variance (ANOVA) for a 3 X 5 splitplot factorial (SPANOVA) confirmed that there were no overall differences among the three lesion groups across assessments in terms of number of falls [F(2,12)  Numerals concern the approximate posterior location with respect to bregma. and balance as a function of exposure to the task and as a function of recovery time for all groups.

Discrimination Training
A one-way ANOVA was used to evaluate the number of errors through criterion committed by the animals with different lesions while learning the original discrimination (i.e., 3 vs. 17 cm). As is apparent in Figure 4, the analysis revealed that all groups learned the initial discrimination task with nearly equivalent proficiency [F(2,12) = 0.40,p > .05].
Relational discrimination. A two-way ANOVA was used to assess the number of errors committed through criterion on the original discrimination relative to the number of errors on the discrimination used in Phase I of relational performance testing. The analysis revealed that the number of errors committed on the second discrimination was significantly lower than that observed for the original task [F(I,12) = 101.29,p < .0001]. This result, displayed in Figure 4, shows that the animals transferred from the first pair of stimuli to the second pair of stimuli with relative ease. The interaction between lesion and discrimination problems was nonsignificant [F(2,12) = 0.33, p > .05]. This finding reveals that the transfer of training was equivalent for the three lesion groups.
A one-way ANOVA was used to examine errors through criterion across groups when paired stimuli, differing by 14 cm, were interspersed with an equal number of trials of paired stimuli differing by 10 cm (e.g., Phase 2). Shown graphically in Figure 4, satisfactory performance by all animals was found on this problem. No differences among the lesion groups were observed [F(2, 12) = 0.66,

p> .05].
Next, a 3 X 2 SPANOVA was used to analyze the proportion correct responses for when the difference between paired stimuli was reduced by two more steps. That is, 50 trials were administered in which stimuli differed by 6 cm (i.e., 7 vs. 13 cm); then, 50 trials were administered in which stimuli differed by 2 cm (i.e., 9 vs. II cm). As before, no differences among the lesion groups emerged on these discriminations [F(2,12) = 1.01,p > .05]. However, greater choice accuracy was observed on the 7-vs. 13-cm discrimination (6-cm difference between stimuli; 80% choice accuracy for all groups) than on the 9-vs. II-cm discrimination (2-cm difference between stimuli; range of 55% to 60% correct for all groups) Finally, a two-way SPANOVA (3 lesion groups X 3 steps of difference between paired stimuli) was used to assess the proportion correct responses among all lesion groups across 126 trials defined earlier as Phase 4, wherein stimuli were randomly presented from within one category or the other. Specifically, animals were given an equal number oftrials in which stimuli paired from within the same category differed by 6 cm (e.g., 3 vs. 9 cm and II vs. 17 cm), 4 cm (e.g., 13 vs. 17 cm, II vs. IS cm, 5 vs. 9 cm, and 3 vs. 7 cm), and 2 cm (e.g., IS vs. 17 cm, 13 vs. IS cm, II vs. 13 cm, 7 vs. 9 cm, 5 vs. 7 cm, and 3 vs.   Wilson and DeBauche (1981) reported that monkeys with bilateral lesions of the IT cortex, when presented with between-category discriminations, exhibited greater performance deficits than did normal monkeys or monkeys with PS lesions. As was the case in the Wilson and DeBauche investigation, the original discrimination of the present experiment involved comparing the extreme values from a given continuum of stimuli. Circles ranged from 3 to 17 cm in diameter by increments of 2 cm. The continuum was, in essence, divided into two categories wherein stimuli with diameters greater than 10 cm were considered to be similar to the original positive stimulus. Likewise, stimuli with diameters smaller than 10 cm were considered to be more closely related to the original negative stimulus. Thus, between-category discriminations involved 7-versus 13-cmand 9-versus II-cm-diameter circles. In contrast, within-category judgments utilized stimuli such as II versus 17 cm or 3 versus 5 cm, where both members of the paired comparison originated from the same side of the continuum. Although the same physical step differences between compared stimuli (2-cm step difference and 6-cm step difference) were represented by both judgment types, the distinction between the judgment types was that the between-category judgment compared a stimulus that approximated or was equivalent to the original positive cue, with another stimulus that was similar to the previously experienced negative stimuli. On the other hand, the within-category judgment involved the comparison of two stimuli, both of which were near or equivalent to either the original positive cue or the original negative cue.

Microanalysis of Relational Discriminations
In order to compare discrimination performances among groups on between-category stimuli with discrimination performances among groups on within-category stimuli, data from training and performance testing were combined to form between-category and within-category problems. Further, in both considerations, the diameters of the stimuli differed by either 6 or 2 cm. In order to evaluate relational learning, the pairs of stimuli included in the statistical analysis were those in which performance on a given physical step difference could be assessed for both types of judgment for all animals. (In other words, 4-cm differences were not examined in the phases of training and testing that involved between-category judgments.) A comparison on within-versus between-category performance was central to the analyses conducted by Wilson and DeBauche (1981), and it was on the betweencategory discriminations where significant deficits were observed in the IT-Iesioned monkeys. Importantly, all animals performed poorly on within-category discriminations, as indicated by the quote: "Finally, it should be noted that the animals with IT lesions performed as well (or as poorly) on within-category discriminations as animals in the other groups" (p. 35).
A three-way SPANOVA with two repeated measures was used to examine the proportion of correct responses of animals as a function oflesion condition (i.e., 3 lesion groups X 2-vs. 6-cm physical step differences X betweenvs. within-category problems). This analysis failed to reveal influences that could be ascribed to the lesions (i.e., for the main effects and all interactions involving the lesion condition,ps > .05). However, as expected, a significant main effect was found for the physical step difference between stimuli [F(l,12) = 92.38,p < .0001]. That is, all groups performed much more accurately on trials in which stimuli differed by 6 cm (M = .74, SD = .02) than on trials in which stimuli differed by 2 cm (M = .52, SD = .02). The results are illustrated in Figure 6. It is apparent that there is a floor effect wherein the performance of all groups plummeted to chance when animals were required to discriminate stimuli differing by a 2-cm diameter. This is probably due to the rat's limited visual acuity relative to some animals (Dean, 1990). In addition, a significant main effect was observed for between-category versus within-category judgments [F(I,12) = 9.00, p = .0111]. This result showed that, regardless oflesion group, overall the animals performed more accurately when discriminating between pairs involving stimuli from between categories (M = .66, SD = .0 I) than when discriminating between pairs involving cues from within the same category (M = .61, SD = .02). The category problem X step difference interaction was not significant.

Retention
A one-way ANOVA was used to analyze the savings scores across lesion groups when the animals were retrained on the original visual discrimination (i.e., 3 vs.

DISCUSSION
The influences of lesions within the visual region of the rat's temporal cortex (TE2) or striate cortex on visual relational perception were investigated. Initially rats were trained to discriminate between a 17-cm-diameter circle and a 3-cm-diameter circle. Consistent with previous results (Davis & McDaniel, 1993;Meyer et ai., 1986), rats with TE2 lesions learned this simple visual discrimination as rapidly as normal controls. A similar result has been observed in studies with IT-injured monkeys. That is, although monkeys with IT lesions must relearn visual discriminations acquired prior to brain damage (Dean & Cowey, 1977;Dean & Weiskrantz, 1974), they learn and perform simple visual discriminations learned postoperatively as well as normal control animals (e.g., Butter & Hirtzel, 1970;Ungerleider & Pribram, 1977). With respect to the present study, once the initial discrimination was learned, the difference in diameter between the two cues was reduced in a series of steps. Thereafter, the animals were tested for their abilities to discriminate other pairings of stimuli in accordance with a relational rule (i.e., navigating toward a larger circle led to escape, whereas navigating toward a smaller circle did not). Contrary to monkeys with IT lesions (Wilson & DeBauche, 1981), in the present study rats with TE2 lesions trans- . Mean and standard error percentage correct responses, pooled across between-category and within-category judgments for stimuli differing by 6 and 2 cm. Asterisks indicate significantly better performance on trials with stimuli differing by 6 cm. ferred relatively well across all between-category problems demanding relational perception. Consistent with the Wilson and DeBauche study with monkeys, we found that performance was significantly better for all groups when challenged with between-category discriminations than with within-category discriminations. It was on between-category judgments that monkeys with IT lesions performed poorly relative to controls and, as indicated earlier, both brain-injured and normal monkeys performed poorly on the within-category discriminations. Finally, following a 3-month interval, the rats were retrained on the original discrimination to examine retention. Both the normal controls and the brain-injured animals showed good savings. This result is similar to that of Myhrer (1992), who found, as has been demonstrated with IT-injured primates, that rats with temporal cortex lesions have retrograde rather than anterograde retention impairments. As expected, rats with PS lesions performed well on all problems. Similar results were reported for monkeys with partial PS lesions (Wilson & DeBauche, 1981).
There are several possible reasons why the rats with TE2 lesions performed well in the present study, but monkeys with IT lesions in the Wilson and DeBauche (1981) study performed poorly with the relational or categorical aspects of testing. First, Wilson and DeBauche trained their animals on the discrimination prior to surgery and then retrained them on the same discrimination postsurgically prior to testing them with combinations of intermediate stimuli. All training in the present study was conducted postsurgically. Second, and perhaps more importantly, after learning the original discrimination, our animals were presented with a series of transfer trials with stimuli differing from the original stimuli by a small amount. Specifically, the 17-versus 3-cm discrimination was shifted to a 15-versus 5-cm discrimination, and after attaining criterion with these stimuli, the animals experienced trials involving both pairs of stimuli. This experience may have facilitated the use of a relational rule by the rats in the present study. In contrast, Wilson and De-Bauche presented the trials with numerous intermediate stimuli immediately after retraining the animals on the original discrimination postsurgically. Results more consistent with those observed by Wilson and DeBauche may have been observed if we had used an identical training protocol. We believed that the result observed in monkeys was robust. Thus, we expected that similar results would be observed in rats prepared with TE2 lesions despite the use of behavioral procedures that had been developed to optimize transfer of training.
Another plausible explanation can be found in a recent review of the literature concerning the dual cortical visual system hypothesis from a comparative neurological perspective (Ellard, 1998; see especially p. 179 for the quotations below). Ellard reported that in primates some regions of the ventral "stream" (i.e., posterior temporal cortex) are specialized for "movement-based imagery." That is, some temporal regions playa prominent role in "viewpoint-invariant representation of threedimensional objects." Given the results in our lab (Davis & McDaniel, 1993) and Kolb's lab (Kolb et aI., 1994) showing that deficits in visual memory accompany TE2 lesions in rats, we suspect that this may apply to two-dimensional forms as well. Ellard also postulated that other temporal association areas "are perhaps more concerned with more abstract, cognitive representations of objects." Ellard speculated that the ventral stream identified in nonprimate species subserves only the former function, and that it is only in primates that the latter functions have been subsumed by the temporal cortex. The data from the present study are consistent with Ellard's hypothesis.
Obviously more research is needed to resolve this topic, but it is clear that in the present study rats with TE2 lesions generalized a relational perceptual rule relatively well across the various pairs of circular stimuli with intermediate diameters. It may be the case that perceptuaL categorization of the type studied here is subserved by other visual cortical territories in animals with less complexly organized temporal lobes. A likely candidate region mediating such basic functions in nonprimate mammals would be the one ofthe many retinotopically organized regions adjacent to TE2, namely the lateral peristriate cortex (see, e.g., Montero, 1993). Although the influences of total lateral peri striate cortex lesions on visual discrimination have been studied (Gallardo et aI., 1979;Mc-Daniel, Coleman, & Lindsay, 1982;McDaniel & Wall, 1988), to our knowledge, neurobehavioral studies of distinct subterritories within the lateral peri striate cortex have not yet been conducted.
It is hoped that the results of this study will stimulate other investigations of the roles played by rodent TE2 in higher perceptual functions. We plan to replicate this study using stimuli that control luminous flux as potential discriminanda and behavioral protocols parallel to the relational perception testing methods used by Wilson and DeBauche (1981).


G Protein α Subunits Activate Tubulin GTPase and Modulate Microtubule Polymerization Dynamics*

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein α subunits Gi1α, Gsα, and Goα are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Giα inhibited polymerization of tubulin-GTP into microtubules by 80–90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that Gi1α increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Gα may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals.

Microtubules, a major component of the cytoskeleton, are involved in a variety of cellular functions including chromosome movements during mitosis, intracellular transport, and the modulation of cell morphology. In general, the biological function of microtubules is based in significant part on the ability of tubulin to polymerize and depolymerize. In living cells, microtubules exist in both dynamic and stable populations, with each population called upon to carry out distinct cellular functions (1,2). Proper control of microtubule dynamics is essential for many microtubule-dependent processes.
Microtubule ends can interconvert between slow elongation and rapid shortening, a process called dynamic instability, because of the presumed gain and loss of a small region of tubulin-liganded GTP at the microtubule end (3)(4)(5). Tubulin dimers bind 2 mol of GTP/mol of tubulin, one exchangeable (the E-site 1 in ␤-tubulin) and the other nonexchangeable (in ␣-tubulin). GTP bound to the exchangeable site becomes hydrolyzed upon incorporation of the tubulin into the microtubule. This hydrolysis creates a microtubule consisting largely of GDPtubulin, but a small region of GTP-liganded tubulin, called a "GTP cap," remains at the end. The loss of the cap results in a transition from growth to shortening (called a catastrophe), whereas the reacquisition of the GTP cap results in a transition from shortening to growing (called a rescue) (6). The GTPase activity of tubulin is normally low, and hydrolysis of the E-site GTP requires activation. This activation normally occurs when the tubulin dimer binds to the end of a growing microtubule. It is thus suggested that one tubulin dimer might act as a GTPase activator for another during polymerization (7).
Several microtubule-associated proteins are known to regulate microtubule dynamics by stabilizing microtubules (8,9). Stabilization of microtubules by microtubule-associated proteins is achieved, in part, by suppressing the rate and extent of microtubule shortening and by suppressing the catastrophe frequency and increasing the rescue frequency (6, 10 -12). It is noteworthy that the catastrophe frequency observed in cells is much higher than that observed in vitro with microtubules composed of pure tubulin (13), suggesting the possible control of the process by additional cellular factors (14 -17).
Studies have demonstrated that microtubule polymerization and stability are also affected by second messenger-activated protein kinases, suggesting the possibility that microtubule dynamics may be regulated by extracellular signals through G proteins (for review see Ref. 18;also Refs. 19 and 20). G proteins act as arbiters of cellular signaling, and they may associate in cells directly with microtubules (21)(22)(23)(24)(25)(26). Heterotrimeric G proteins are composed of ␣ and ␤␥ subunits. G␣ subunits bind GTP and display various levels of intrinsic GTPase activity. Certain G protein ␣ subunits (G i1 ␣, G s ␣, and G q ␣) bind to tubulin with high affinity (27)(28)(29)(30). This binding appears to activate the G proteins in association with a direct transfer of GTP from the E-site in tubulin to G␣ (transactivation) (29,31). In addition to activating G␣, the association between G␣ and tubulin induces a GTPase activity in tubulin similar to that seen after the self-association of tubulin dimers during the formation of a microtubule (32). Recent studies have also shown that G␤ 1 ␥ 2 binds to microtubules and promotes microtubule assembly in vitro (26). These studies indicate that G proteins may modulate microtubule polymerization dynamics and cytoskeletal organization or function. In the present study, the modulation of microtubule assembly and dynamics by G protein ␣ subunits was investigated. We report here that ␣ subunits of G proteins activate the intrinsic GTPase of tubulin (i.e. they act as a GTPase activating protein for tubulin), and the GTP hydrolysis modulates microtubule assembly and dynamics in vitro.

EXPERIMENTAL PROCEDURES
Tubulin Preparations-Tubulin for all studies except the dynamic instability analysis was purified from fresh sheep brain by cycles of assembly and disassembly (33) followed by phosphocellulose chromatography (34). The resulting tubulin preparations were more than 97% pure as determined by Coomassie Blue staining of SDS-polyacrylamide * This work was supported by Public Health Service Grants MH 39595 and AG 15482 (to M. M. R.) and NS13560 (to L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  gels (not shown). The tubulin was stored in liquid nitrogen and used within 2 weeks. Bovine brain tubulin was used for dynamic instability analyses as described elsewhere (10). Tubulin liganded with GTP, GppNHp, or [␣-32 P]GTP was prepared by removing exchangeable nucleotide from the tubulin by charcoal treatment followed by incubation with 0.5 mM GTP, 0.5 mM GppNHp, or 0.1 mM [␣-32 P]GTP (31). The samples were then desalted twice on centrifugal gel filtration columns using P6-DG resin (Bio-Rad) as described previously (31). After desalting, 0.5-0.8 mol of guanine nucleotide was bound/mol of tubulin. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (35).
G Protein Purification-Recombinant G i1 ␣, G s ␣, or G o ␣ were produced in Escherichia coli using constructs provided by Dr. Maurine Linder (Washington University, St. Louis, MO). The vector used contained G i1 ␣, G s ␣, or G o ␣ cDNA preceded by a nucleotide sequence encoding a His 6 -amino acid stretch as an affinity tag under the control of a T7 promoter. E. coli was grown and harvested, and G proteins were purified over a Qiagen nickel column with a subsequent MonoQ high pressure liquid chromatography step (36). The Q204L mutant of G i1 ␣, a generous gift from Drs. J. Hepler and A. G. Gilman (University of Texas Southwestern Medical Center, Dallas, Texas), was expressed in E. coli and purified as described (36). Bacteria containing myristol transferase and G i1 ␣ (a gift from Dr. M. Linder) were used to express myristoylated G i1 ␣, which was then purified as described earlier (37).
GTP Hydrolysis-Tubulin was allowed to bind [␣-32 P]GTP, and unbound nucleotide was removed by gel filtration using a P6-DG column (Bio-Rad). The samples were then incubated with or without G␣ at 30°C for 30 min and treated with 1% SDS at room temperature for 15 min. Nucleotide analysis was done by thin layer chromatography on polyethyleneimine cellulose plates (32,38). Two l of a 10 mM solution of GTP and GDP were spotted 1.5 cm apart on a polyethyleneimine cellulose thin layer plate, followed by 2-5 l of each sample. The chromatograms were developed in 0.35 M NH 4 HCO 3 . The spots containing GTP or GDP were visualized with a UV lamp, and plates were exposed to film for autoradiography. Quantitative analysis was done using a Molecular Dynamics PhosphorImager system.
Microtubule Assembly-Tubulin-GTP or tubulin-GppNHp in PEM buffer (100 mM PIPES, 2 mM EGTA, 1 mM MgCl 2, pH 6.9) was preincubated with or without G␣ at 30°C for 30 min. Polymerization was then initiated by adding 30% glycerol and an additional 2 mM MgCl 2 and incubating at 37°C for 45 min to 1 h. The extent of microtubule assembly was quantified after pelleting the microtubule polymers by centrifugation at 150,000 ϫ g for 20 min at 37°C. Pellets were resuspended in 4°C PEM buffer, and protein concentrations in the pellet and supernatant fractions were determined (35). Before testing the effect of G proteins on microtubule assembly, free nucleotide was separated from G protein ␣ subunits, and the buffer was changed to PEM by passage of the proteins through a rapid spin column (Bio-Gel P6DG, Bio-Rad). Alternatively, when G␣ concentrations were low, a buffer control was performed to avoid a reduction in protein concentration by gel filtration.
Electron Microscopy-Fifteen l of the microtubule sample was placed on a Formvar-coated nickel grid. After 10 -15 s, the grids were rinsed with 10 drops of 2% uranyl acetate for negative staining, blotted dry with a filter paper, and viewed in a JEOL 100S electron microscope.
Microtubule Dynamics by Video Microscopy-Tubulin (12 M) was mixed with Strongylocentrotus purpuratus flagellar seeds in 80 mM PIPES, 0.8 mM Mg 2ϩ , 1 mM EGTA, pH 6.8 (PME buffer), containing 275 M GTP in the absence or presence of G i1 ␣ and incubated for 25 min at 37°C for assembly to reach steady state. The seed concentration was adjusted to achieve 3-6 seeds/microscope field. 2.5 l of the microtubule suspension was prepared for video microscopy, and the dynamics of individual microtubules were recorded at 37°C as described previously (10). Under the experimental conditions used, microtubule growth occurred predominantly at the plus ends of the seeds as determined by the growth rates, the number of microtubules that grew, and the relative lengths of the microtubules at the opposite ends of the seeds (6, 10, 39 -41). Microtubule length changes were measured in real time at 3-6 s intervals until microtubules underwent complete depolymerization to the axoneme seed or until the microtubule end became obscured. The length changes undergone by a particular microtubule as a function of time were used to create a "life history" plot. The growing and shortening rates were determined by least squares regression analysis of the data points for each growing or shortening phase. The reported mean growing and shortening rates represent the mean values for all growing and shortening events observed for a particular reaction condition. We considered a microtubule to be in a growing phase if the microtubule increased in length by Ͼ0.2 m at a rate Ͼ0.15 m/min and in a shortening phase if the microtubule shortened in length by Ͼ0.2 m at a rate Ͼ0.3 m/min. Length changes equal to or less than 0.2 m over the duration of 6 data points were considered as attenuation phases. A total of 22-25 microtubules was analyzed for each experimental condition. The catastrophe frequency was determined by dividing the number of catastrophes by the sum of the total time spent in the growing plus attenuated states for all microtubules for a particular condition. The rescue frequency was calculated by dividing the total number of rescue events by the total time spent in the shortening states for all microtubules for a particular condition.

RESULTS
G i1 ␣ Activates the Intrinsic GTPase of Tubulin-Tubulin binds to G i1 ␣ and G s ␣ with a K d of approximately 130 nM coupled with a transactivation of G␣ in which 25-50% of E-site tubulin-bound GTP is transferred directly to the G␣ (28,29). G i1 ␣ binding to tubulin in vitro also activates GTP hydrolysis (32). Both tubulin and G i1 ␣ have intrinsic GTPase activities. Because the intrinsic GTPase activity of tubulin is very low, two possibilities exist to explain the higher rate of GTP hydrolysis. One possibility is that G i ␣ hydrolyzes the E-site-bound GTP after transfer to the G i ␣. The second possibility is that G i ␣ The samples were then treated with 1% SDS and subjected to thin layer chromatography on polyethyleneimine cellulose plates. One of three similar experiments is shown.

TABLE I
Comparison of the effect of G i1 ␣ on microtubule assembly induced by GTP or GppNHp Tubulin-GTP or tubulin-GppNHp was preincubated with or without G i1 ␣ (as described in the Fig. 2 legend) followed by polymerization in the presence of GTP or GppNHp as indicated. Assembly was quantified by centrifuging the polymer at 150,000 ϫ g and represented as % of control (assembly in the absence of G i1 ␣ was considered 100%). Values represent mean Ϯ S.E. of at least three experiments.

Samples
Microtubule assembly Samples Microtubule assembly

% control % control
Tubulin-GTP (control) 100 Tubulin-GppNHp (control) 100 activates the GTPase of tubulin by inducing a conformational change in the tubulin, similar to the way in which tubulin dimers activate neighboring GTPase activity during microtubule polymerization.
To distinguish between these possibilities, we used a mutated form of G i1 ␣ with a single amino acid substitution, Gln 204 3 Lys (Q204LG i1 ␣), with incapacitated GTPase activity. However, the ability of the mutated Q204LG i1 ␣ to bind GTP is unaltered (42). The mutated Q204LG i1 ␣, or wild-type G i1 ␣, was incubated with tubulin-[␣-32 P]GTP under conditions in which tubulin does not polymerize, and the extent of GTP hydrolysis was determined by thin layer chromatography. As shown in Fig. 1, the tubulin-bound [␣-32 P]GTP was poorly hydrolyzed in the absence of G i1 ␣ (10.1 Ϯ 1.9%, n ϭ 10). In the presence of G i1 ␣, 71.8 Ϯ 3.4% (n ϭ 10) of the E-site-bound GTP was hydrolyzed. This hydrolysis could be a combination of that occurring in the tubulin E-site and in G i1 ␣. When Q204LG i1 ␣ was added to the tubulin, 49 Ϯ 3% (n ϭ 3) of the bound GTP was hydrolyzed. Because Q204LG i1 ␣ cannot hydrolyze GTP, the tubulin must have been responsible for the GTP hydrolysis. Because in the presence of G i1 ␣, 71.8% of the E-site GTP was hydrolyzed, approximately 23% of the GTP must have been hydrolyzed by G i1 ␣. Myristoylated G i1 ␣ was also tested for its ability to activate tubulin GTPase. The amino terminus of G i1 ␣ is myristoylated in vivo, a modification that is important for association of G i1 ␣ with membranes and G␤␥ (43). We found that 80.4 Ϯ 2.3% (n ϭ 7) of the tubulin-bound [␣-32 P]GTP was hydrolyzed by myristoylated G i1 ␣. The slightly increased potency of myristoylated G i1 ␣ to activate tubulin GTPase as compared with G i1 ␣ may suggest an enhanced ability of myristoylated G i1 ␣ to bind to tubulin. The results indicate that G i1 ␣ may act as a GTPase activating protein for tubulin.
G i1 ␣ Inhibits Microtubule Assembly in a GTP-dependent Manner-G i has been shown previously to inhibit microtubule (B, C, E, and F). A-C, assembly carried out in the presence of GTP. D-F, assembly carried out in the presence of GppNHp. Note that in D, some microtubule bundling occurred. Tub, tubulin.

TABLE II
Effect of GTP or GppNHp on G i1 ␣-mediated inhibition of microtubule assembly Experimental protocol was similar to that as described in Table I except that GTP (at indicated concentrations) was added to the samples in which tubulin-GppNHp was preincubated with G i1 ␣, whereas GppNHp was added to G i1 ␣-preincubated tubulin-GTP samples. Samples were subjected to polymerization at 37°C and quantified as in Table I Tubulin-GppNHp (control) 100 Tubulin-GTP 100 polymerization (44). This inhibition might occur by binding of the G i to tubulin and sequestering it, making the tubulin unavailable for polymerization. Tubulin with GTP in the E-site (1.5 mg/ml) polymerizes into microtubules in the absence of exogenous GTP as shown in Table I. Assembly of the tubulin-GTP in the presence of 0.75 mg/ml of G i1 ␣ resulted in ϳ85% inhibition of assembly, and exogenous GTP overcame the ability of G i1 ␣ to inhibit assembly in a GTP concentration-dependent manner. To determine whether inhibition of microtubule assembly by G i1 ␣ was the result of hydrolysis of the E-site GTP by G i1 ␣, we prepared tubulin with GppNHp (a hydrolysisresistant GTP analog) in the E-site. As also shown in Table I, in the absence of exogenous nucleotide, G i1 ␣ reduced the extent of microtubule polymerization by approximately 85%, and exogenous GppNHp did not restore microtubule polymerization.
Thus, it appears that GTP hydrolysis resulting from the association of tubulin and G i1 ␣ plays a critical role in modulating microtubule assembly. When the microtubule pellet was analyzed by SDS-gel electrophoresis, Coomassie Blue staining did not reveal incorporation of G i1 ␣ in microtubules. However, some incorporation of G␣ into the microtubule fraction was observed by Western blotting using a G i1 ␣ antibody (data not shown). Tubulin Exchanges Nucleotide in the G␣-Tubulin Complex-Addition of exogenous GTP to the tubulin-G i1 ␣ complex with either GTP or GppNHp in the E-site reversed the ability of G i1 ␣ to inhibit polymerization (Table II). Furthermore, addition of exogenous GppNHp to the tubulin-G i1 ␣ complex with GTP in the E-site, inhibited microtubule polymerization. These results indicate that exogenous GTP and GppNHp can exchange with either GppNHp or GTP in the tubulin E-site when complexed with G i1 ␣. The GTPase-deficient G i1 ␣ variant, Q204LG i1 ␣, also inhibited microtubule polymerization in a manner similar to G i1 ␣ (by 74.5 Ϯ 9.5%), suggesting that GTP hydrolysis in G␣ does not cause the inhibition of microtubule assembly.
Microtubules Polymerized in the Presence of G i1 ␣ Have Typical Morphology-Electron microscopic analysis of the polymers formed in the presence of G i1 ␣ and excess GTP or Gp-pNHp indicated that they were normal microtubules. G i1 ␣ blocked the formation of microtubules regardless of the nucleotide bound to the tubulin (Fig. 2, B and E). The addition of 50 M GTP reversed the G i1 ␣-mediated inhibition of microtubule assembly, and microtubules were formed (Fig. 2C), whereas the addition of 50 M GppNHp did not (Fig. 2F).
Specificity of G Protein ␣ Subunits for Inhibition of Microtubule Assembly and GTPase Activity-G s ␣ binds to tubulin with an affinity similar to that of G i1 ␣ (28). Thus, it was predicted that G s ␣ would also inhibit microtubule assembly. In the presence of G s ␣, microtubule assembly was reduced to 22% (21.8 Ϯ 10.5%) of the control (Fig. 3A). Although G o ␣ does not bind to tubulin with an affinity as high as that of G i1 ␣ or G s ␣ (28), G o ␣ inhibited microtubule polymerization similarly to G i1 ␣ and G s ␣ (by 85%). These results are consistent with the possibility that there is a preferential interaction of G o with oligomeric tubulin or microtubules as compared with dimeric tubulin (44). The retinal G protein transducin (G t ␣), which does not bind to tubulin or microtubules, did not inhibit microtubule assembly (Fig. 3A). Furthermore, the GTPase activity of tubulin was activated by G s ␣ (73.8 Ϯ 3.8%) and G o ␣ (93 Ϯ 2.7%) but not by G t ␣ (28.5 Ϯ 2.5%) (Fig. 3B). The activation of tubulin GTPase by G i1 ␣ was maximal at a G␣:tubulin ratio of 1:1.
G i1 ␣ Increases Microtubule Dynamic Instability by Increasing the Catastrophe Frequency-In an effort to determine how G␣ modulates microtubule polymerization dynamics, we measured the dynamics of individual microtubules at steady state in vitro, in the presence or absence of G i1 ␣, by video microscopy.
Microtubules can alternate between phases of growing and shortening and also spend a small fraction of time in an attenuated (paused) state, neither growing nor shortening detectably, a behavior called dynamic instability (10). The transition frequencies among the growing, shortening, and attenuated states are thought to be important in the regulation of microtubule dynamics in cells (13,14,45). Life history traces of several microtubules in the absence (panel A) or presence (panel B) of G i1 ␣ are shown in Fig. 4. Addition of G i1 ␣ (4 M) visually increased the catastrophe frequency. The dynamic instability parameters were determined quantitatively from such life history plots. As shown in Table III, G i1 ␣ did not alter the rates of microtubule growth or shortening. However, 4 M G i1 ␣ significantly reduced the average length that microtubules grew per individual growth event (1.5 Ϯ 0.2-0.9 Ϯ 0.2 m). G i1 ␣ also reduced the percentage of total time that microtubules spent in the growing phase and increased the percentage of total time they spent in the shortening phase. G i1 ␣ significantly increased the catastrophe frequency (by 2.6-fold in the presence of 4 M G i1 ␣). The catastrophe frequency per micrometer of length grown was determined by dividing the total number of catastrophic events by total length increase during growing events. G i1 ␣ also increased the catastrophe frequency per micrometer of length grown. G i1 ␣ had no effects on the rescue frequency (transition from shortening to the growing or attenuated state) per unit of time or per unit of length shortened (Table III). Dynamicity is a parameter that reflects the overall dynamics of the microtubules (the total detectable tubulin dimer addition and loss at a microtubule end including the time spent in the attenuated state) (39). G i1 ␣ (4 M) increased the dynamicity by 44%. Thus, G i1 ␣ increases the dynamic behavior of the microtubules primarily by increasing the catastrophe frequency. DISCUSSION In the present study, the ␣ subunits of G proteins (G i1 , G s , and G o ) were shown to activate the GTPase activity of tubulin, indicating that G␣ may serve as a GTPase activating protein for tubulin. In addition, G␣ inhibited microtubule assembly and increased microtubule dynamic instability in vitro. The assembly of tubulin into microtubules was blocked by G␣ (80 -90%), regardless of whether GTP or GppNHp was bound in the tubulin E-site. In addition, the addition of exogenous GTP, but not the addition of the hydrolysis-resistant GppNHp, overcame the inhibition of microtubule polymerization by G␣. A model for how G␣ might interact with tubulin and how exogenous GTP might overcome the interaction is presented in Fig. 5A. In this model, G␣ is suggested to bind to tubulin and activate the intrinsic GTPase of tubulin in a manner similar to that in which GTP hydrolysis occurs in tubulin during formation of a microtubule. However, unlike the formation of microtubules from tubulin dimers, G␣ dissociates from the tubulin-G␣ complex subsequent to GTP hydrolysis. G i1 ␣ altered microtubule dynamics by increasing the catastrophe frequency (the frequency of switching from growing to shortening; see Table III). Microtubules are composed of an unstable tubulin-GDP core and a stable tubulin-GTP or tubulin-GDP-P i cap at the microtubule ends (46,47). Microtubules grow for as long as they maintain a GTP cap, but loss of the cap exposes the labile tubulin-GDP core, and the microtubules rapidly shorten. These data are consistent with the possibility that G i1 ␣ activates tubulin GTPase at the microtubule ends, thus resulting in loss of the GTP cap (Fig. 5B). Alternatively, G i1 ␣ might increase the catastrophe frequency by reducing the effective tubulin concentration, thereby binding to and sequestering soluble tubulin. However, this sequence appears unlikely because G i1 ␣ did not reduce the individual microtubule growth rate (Table III).
It is suggested that G␣ is released from microtubules after binding and subsequent hydrolysis of the E-site GTP. The   5. Model for the effects of G␣ on microtubule assembly and dynamics. A, a scheme for tubulin-G␣ interaction for the regulation of microtubule assembly. The binding of G␣ to tubulin-GTP inhibits microtubule polymerization and promotes GTP hydrolysis, suggesting that the binding of G␣ to tubulin induces a conformation in tubulin similar to that occurring during microtubule formation. G␣ dissociates from the tubulin-G␣ complex after GTP hydrolysis. Addition of exogenous GTP, but not GppNHp, restores microtubule polymerization, indicating that the formation of the tubulin-G␣ complex is required for the inhibition of microtubule polymerization. B, possible mechanism for the regulation of microtubule dynamics in vivo by G␣. The binding of G␣ to the end of a microtubule induces hydrolysis of GTP and subsequent loss of the stabilizing cap, resulting in the transition to microtubule depolymerization (a catastrophe). GTP hydrolysis destabilizes the tubulin-G␣ complex, and G␣ dissociates from tubulin and is now ready for another cycle of interaction with the microtubule ends. released G␣ could be recycled for further interaction with newly growing microtubules, reducing the G␣ concentration required to exert this effect. In fact, 4 M G i1 ␣, a concentration 3-fold lower than the tubulin concentration (12 M), increased the catastrophe frequency 2.6-fold (Table III).
Although G proteins are usually confined to the plasma membrane, translocation of activated G␣ from the membrane to the cytosol has been observed (48 -51). Furthermore, whereas G proteins are normally associated with second messenger-generating enzymes, or ion channels, results from several laboratories suggest that G proteins may be involved in cell growth and differentiation, perhaps through their association with cytoskeletal components (21)(22)(23)(24)(25)(26). For example, an association of G␣ and G␤␥ with the microtubule cytoskeleton has been reported (21, 24 -26). Furthermore, an association of G o ␣ and -␤ (or -␤␥) with spindle microtubules suggests that G protein subunits may play some role in regulating the assembly and disassembly of the mitotic spindle (23,24). The ␤-adrenergic receptor kinase (known as ␤ARK or GRK2), which mediates agonist-dependent phosphorylation and desensitization of G protein coupled receptors, has been shown to associate with microtubules and to phosphorylate tubulin in an agonist-dependent manner (19,20). Taken together, these data suggest a link between microtubules and G protein-mediated signaling that may regulate cell division and differentiation.
G proteins, particularly G o ␣ and G␤, are abundant at the growth cone membrane of neurons (52). Growth cones at the growing tips of developing neurites are highly specialized organelles that respond to a variety of extracellular signals to achieve neuronal guidance and target recognition. Coordinated assembly of microtubules in concert with actin filaments and neurofilaments is required for growth cone motility and neurite outgrowth (53,54). Activation of a G protein coupled receptor has been shown to collapse the growth cone cytoskeleton (55).
Because some G␣ appears to be released from the membrane subsequent to hormone or neurotransmitter activation (48 -51), it is possible that these proteins participate in localized regulation of the cytoskeleton. Thus, microtubule dynamics at growth cones could be mediated by G o ␣ and G i ␣. Based on observations in this report as well as the emerging results from various laboratories, it is reasonable to postulate that extracellular signals orchestrate G proteins (both G␣ and G␤␥) and mobilize them to bind to microtubules. Such a process is likely to provide a venue by which extracellular signals modify cell form and growth.