Behavioral Neuroscience 2015, Vol. 129, No. 2, 113-128

© 2015 American Psychological Association 0735-7044/15/$ 12.00 http://dx.doi.org/10.1037/bne0000047

NMDA Blockade Inhibits Experience-Dependent Modification of Anterior Thalamic Head Direction Cells Laura E. Berkowitz, Isaac Ybarra, Jessicah A. Jones, Michele E. Amato, Annette M. Rodriguez, and Jeffrey L. Calton California State University-Sacramento Head Direction (HD) cells of the rodent Papez circuit are thought to reflect the spatial orientation of the animal. Because NMDA transmission is important for spatial behavior, we sought to determine the effects of NMDA blockade on the basic directional signal carried by HD cells and on experiencedependent modification of this system. In Experiment 1, HD cells were recorded from the anterior dorsal thalamus in female Long-Evans rats while they foraged in a familiar enclosure following administration of the NMDA antagonist CPP or saline. While the drug produced a significant decrease in peak firing rates, it failed to affect the overall directional specificity and landmark control of HD cells. Experiment 2 took place over 2 days and assessed whether the NMDA antagonist would interfere with the stabilization of the HD network in a novel environment. On Day 1 the animal was administered CPP or saline and placed in a novel enclosure to allow the stabilization of the HD signal relative to the new environmental landmarks. On Day 2 the animal was returned to the formerly novel enclosure to determine if the enclosure specific direction-dependent activity established on Day 1 was maintained. In contrast to HD cells from control animals, cells from animals receiving CPP during the initial exposure to the novel enclosure did not maintain the same direction-dependent activity relative to the enclosure in the subsequent drug-free exposure. These findings demonstrate that plasticity in the HD system is dependent on NMDA transmission similar to many other forms of spatial learning. Keywords: CPP, experience-dependent plasticity, spatial navigation, place cells, rat

The ability to accurately navigate through an environment is an important behavior found in many animal species and sev­ eral cellular components have been implicated in the processes underlying this behavior. Head direction (HD) cells, found in various areas of the rat limbic system, are thought to represent the directional orientation of the animal. Each HD cell fires maximally when the animal is facing a particular direction within the horizontal plane, the preferred direction of that cell. Previous work has demonstrated that the preferred direction of a HD cell can be affected by the position of visual landmarks within a given environment (Taube, Muller, & Ranck, 1990a), and that this control of the HD system by a landmark can be established and maintained following only a brief exposure (Goodridge, Dudchenko, Worboys, Golob, & Taube, 1998). As of yet, however, the underlying cellular mechanisms by which environmental landmarks develop control of the HD system have not been elucidated. Investigations of the neurochemical basis of spatial behavior have implicated a critical role for the A-methyl-D-aspartate

(NMDA) subtype of the glutamate receptor. In one of the earliest reports of the involvement of this receptor in spatial navigation, Morris, Anderson, Lynch, and Baudry (1986) dem­ onstrated that rats chronically infused with the NMDA receptor antagonist AP5 were impaired in learning the position of a hidden platform in a pool of water but were not impaired at learning a simple visual discrimination. At that time, the find­ ings of Morris et al. complemented the emerging lesion data in supporting the view that the hippocampus is an important brain structure for spatial memory processes (Morris, Garrud, Rawl­ ins, & O’Keefe, 1982; Olton, Walker, & Gage, 1978) and were among the first to provide evidence for the idea that NMDA receptor-mediated plasticity in the hippocampus may be the cellular mechanism of memory formation (e.g., Bliss & Collingridge, 1993). Closely related to HD cells, place cells, found primarily in the hippocampal formation, display discreet firing fields (place fields) that appear to signal the location of the animal in an environment (O’Keefe & Dostrovsky, 1971). Given the afore­ mentioned relationship between NMDA transmission and spa­ tial behavior, several experiments have examined the effects of NMDA blockade on place cell functioning (Bett et al., 2013; Faust, Robbiati, Huerta, & Huerta, 2013; Kentros et al., 1998). In the first of these studies, Kentros et al. found that systemic administration of the competitive NMDA antagonist RS-3-(2Carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP) pro­ duced no obvious effect on hippocampal CA1 place cell firing characteristics in a familiar environment but place fields that were expressed in a novel environment during NMDA blockade

Laura E. Berkowitz, Isaac Ybarra, Jessicah A. Jones, Michele E. Amato, Annette M. Rodriguez, and Jeffrey L. Calton, Department of Psychology, California State University-Sacramento. This research was supported by NIH Grant 1R15NS071470-01 and by UEI Grant 160284. Correspondence concerning this article should be addressed to Jeffrey L. Calton, AMD 350, Department of Psychology, 6000 J Street, Sacramento, CA 95819. E-mail: [email protected] 113

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often changed their position the next day when the animals were returned to the same environment in the absence of the drug. The authors interpreted this finding to suggest that NMDA receptor-mediated activity is critical for long-term stabilization of place cell activity relative to a given environment. Other studies have used selective gene knockout of the NMDA receptor to examine the role of this neurotransmitter system in the functioning of place cells. For instance, McHugh, Blum, Tsien, Tonegawa, and Wilson (1996) found that CA1 place cells from mice with selective deletion of NMDA receptors in that hippocam­ pal subregion exhibited larger and more ambiguous place fields, less directional sensitivity in a linear task, and less coactivation between other place cells with overlapping firing fields; an effect that might have explained the water maze deficits shown by CA1 NMDA knockout mice in a companion paper (Tsien, Huerta, & Tonegawa, 1996). In a follow-up study, CA3 NMDA receptor deletion led to CA1 place cells with degraded spatial signals in novel but not familiar environments (Nakazawa et al., 2003) supporting the notion that these receptors play a critical role in the incorporation of novel information into hippocampal spatial rep­ resentations. Little has been done to examine the role of the NMDA receptor in the functioning of HD cells. As in place cells, the spatial signal displayed by the HD system seems to be driven by experience. For instance, Goodridge et al. (1998) demonstrated that when a new environmental landmark is presented to an animal, it takes several minutes of exposure before the landmark develops the ability to control the HD system. On a similar note, the pre­ ferred direction that a HD cell exhibits in a new environment appears to be determined during the initial exposure of the animal to that environment. When an animal walks from a familiar environment into a novel environment the recorded cell will usually maintain the same preferred direction as in the familiar environment (Taube & Burton, 1995). In contrast, when the animal is manually moved between a familiar and novel environment, the HD cell will often show a different preferred direction in the new environment (Dudchenko & Zinyuk, 2005; Taube, Muller, & Ranck, 1990b). In either case, assuming that the initial exposure to the landmarks in the novel environment leads to the landmarks gaining the ability to con­ trol the HD system (Goodridge et al., 1998; Knierim, Kudrimoti, & McNaughton, 1995), the cell can be expected to show the same preferred direction in subsequent exposures to the formerly novel environment as initially established in the first exposure. The present investigation sought to characterize the effects of NMDA blockade on the basic directional characteristics of the HD system (Experiment 1), as well as on the experience-dependent stabilization of this system that occurs when the animal is placed in a new environment (Experiment 2). Based on the previous evidence found in place cells (Faust et al., 2013; Kentros et al., 1998) and the well documented relationship between NMDA transmission, neuroplasticity, and spatial behavior, we hypothe­ sized that NMDA blockade would exert only mild effects on the basic directional-dependent activity in a familiar environment but would produce larger effects on the stabilization of the HD system in a novel environment.

General Method Subjects and Surgical Procedures Over the two experiments, 38 female Long-Evans rats (Simonsen Laboratories, Gilroy, CA) weighing 250-300 g served as subjects. The rats were singly housed in Plexiglas cages and were exposed to a 16:8-hr light-dark cycle. Following surgery, food was restricted to maintain a body weight of 85-90% of the free feeding weight. Water was provided ad libitum. All care and treatment of the animals was approved by the CSUS Institutional Animal Care and Use Committee and adhered to the APA ethical principles of animal use. Each rat was surgically implanted with a recording electrode that was moveable in the dorsal/ventral axis. In brief, animals were anesthetized using a cocktail containing ketamine (30 mg/kg), xylazene (6 mg/kg), and acepromazine (1 mg/kg). The scalp was disinfected with an iodine-based solution and an incision was made from the bridge of the nose to the base of the occipital plate to expose the skull. The electrode was implanted just above the anterior dorsal thalamic nucleus using the following coordinates from Paxinos and Watson (1998): A/P—1.5 mm relative to bregma, M/L—1.4 mm relative to bregma and D/V—3.7 mm relative to the top of the brain). The electrode was fixed in place with jeweler screws and orthopedic cement. Following surgery, animals were allowed 7 days to recover prior to initiating record­ ing procedures.

Recording Apparatus Most recordings occurred with the animal inside a wooden cylindrical enclosure (51 cm tall; 76 cm in diameter). This enclo­ sure was painted gray, except for an approximate 100° arc of the inner wall that was painted white from the bottom of the enclosure to the top to provide a visible landmark. A second recording enclosure was utilized for some sessions of Experiment 2. This wooden enclosure was shaped in the form of an equilateral triangle with walls 51 cm high and 99 cm long. The triangular enclosure was painted gray except for one comer that featured alternating black and white vertical stripes to provide a prominent visual landmark that was different from the white landmark utilized in the cylindrical enclosure. Because the cylindrical enclosure was uti­ lized when searching for cells (see below) it was very familiar to the animals by the time data collection occurred in either experi­ ment. In contrast, the triangular enclosure served as the novel environment in Experiment 2, and so the animals were not exposed to this enclosure until that particular recording session. During all experimental sessions, the recording enclosure was surrounded by a black curtain extending from the ceiling to the floor to hide visible landmarks outside of the recording environment. A white noise generator masked potential auditory cues, and four lights arranged symmetrically on the ceiling above the recording enclo­ sure provided illumination. Nonreflective photo backdrop paper served as the floor of the enclosure and was changed between sessions to hide potential olfactory cues. A pellet dispenser mounted on the ceiling dropped sucrose pellets (bio-serve; Frenchtown, NJ) at random intervals to encourage foraging behavior. A data acquisition system (Neuralynx; Bozeman, MT) was used to monitor and record electrical signals from the brain and also to

HD CELLS FOLLOWING NMDA BLOCKADE

track the head position of the animal by monitoring the position of red and blue LEDs attached to the headstage. The electrodes consisted of a bundle of 16 25-p.m diameter insulated nichrome wires wrapped around the pins of a custom connector that was potted in acrylic. The recording headstage (Neuralynx HS-18; Bozeman, MT) was connected by a recording cable to a motorized commutator that relayed the signal to the data acquisition system located in an adjoining room. Spikes on individual wires were amplified (20-50K), bandpass fdtered (600-6000 Hz), and stored for offline sorting based on spike shape using custom software. Daily screening for HD cells involved plugging the headstage onto the animal, placing the animal in the cylindrical enclosure, and examining the electrical signal for direction-dependent cellular activity indicative of an isolated HD cell. If no HD cell was identified, the electrode was lowered by 25-50 pm and the animal was returned to its home cage. If a HD cell was found, the recording apparatus was prepared for data collection. No attempt was made to disorient the animals (see below) prior to these screening sessions, and animals typically received 10-20 of these sessions total.

Drug Exposures During experimental sessions, animals were administered the competitive NMDA antagonist RS-3-(2-Carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) at a dose of 10 mg/kg or isotonic saline. The drug was obtained from R&D Systems (Ellisville, MO), and dissolved in isotonic saline. All injections were admin­ istered intraperitoneally at a final injection volume of 2 ml/kg.

Analysis of Cellular Data The directional specificity of recorded cells was qualitatively examined by constructing polar plots of firing rate (FR) by HD for each session. This was accomplished by dividing the 360° direc­ tional range into 60 6° bins and then calculating the average firing rate of the recorded cell during the periods in which the head of the animal was oriented within each directional bin. The effects of drug injection were determined on the following basic directional characteristics of recorded cells: peak firing rate, background firing rate, signal to noise, and directional information content. The peak firing rate (FRpeak) was the average firing rate corresponding to the preferred direction (i.e., the directional bin with the highest average firing rate across the session). The back­ ground firing rate (FRbgnd) was the average firing rate of the three directional bins centered at 180° opposite the preferred direction. Signal to noise was calculated using the ratio: (FRpeak - FRbgnd)/ FRpcak, producing scores that ranged between 0 and 1. This formula was utilized instead of the more traditional FRpeak/FRbgnd, because several cells had very low background rates and others had back­ ground rates of zero making the statistic fluctuate wildly and impos­ sible to calculate for some cells using the latter formula. Directional information content (DIC) is a measure of how many bits of HD information are conveyed by each spike (Skaggs, McNaughton, Gothard, & Markus, 1993) and was calculated by the following formula: 2 p t (X,/X) log2 (X;/X), where p t is the probability that the head pointed in the ith directional bin, X; is the mean firing rate for bin i, and X is the mean firing rate across all directional bins. In addition, data from individual cells were subjected to Rayleigh tests (Batsche-

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let, 1981) using the CircStat MATLAB Toolbox for Circular Statistics (Berens, 2009) to determine if spikes were distributed significantly nonrandomly relative to HD, as would be expected if a cell was directionally tuned. The critical statistic of the Rayleigh test is the mean vector length, r, which varies between 0 and 1, with higher values indicating that the distribution of spike directions is clustered, that is, distributed in a nonrandom fashion. The stability of the FR by HD tuning curves between sessions provided an index of whether the landmark cues in the recording environment effectively controlled the directional specificity of the recorded cells. This analysis was performed by using the average FR at each HD to calculate the mean angle of the firing activity (Batschelet, 1981) and then the difference between the mean angles of the two sessions was taken as the preferred direction shift between the sessions. The preferred direction shifts by the cells within each condition were then subjected to Rayleigh tests (Batschelet, 1981) to determine if the preferred direction shifts in each condition were distributed randomly (as would be the case if drug administration disrupted the stability of the HD system) or if the preferred direction shifts tended to cluster in a predictable direction (as would be the case if the preferred directions were controlled by the apparatus landmarks). Also calculated from the sample of preferred direction shifts in each condition was the mean vector length, r, and the mean angle of the sample, m, (Batschelet, 1981). Closely related to the Rayleigh test is the V-test, which determines whether angular values in a sample are nonrandomly distributed around a predicted angle (Batschelet, 1981). Finally, a variation of the Wilcoxon-Mann-Whitney test (Batschelet, 1981) was used to determine if the amplitude of the preferred direction shifts relative to the enclosure (absolute mean angles) between sessions were significantly different between the two drug condi­ tions.

Histology At the completion of the experiments, animals were anesthetized deeply and a small anodal current (20 p,A, 20 sec) was passed through one electrode wire to conduct a Prussian blue reaction. The animals were then perfused transcardially with saline followed by 10% formalin in saline and the brains were removed and placed in 10% formalin for at least 48 hr. The brains were then placed in a 10% formalin solution containing 2% potassium ferrocyanide for 24 hr and then reimmersed in 10% formalin (24 hr) before being sectioned (40 pm) in the coronal plane, stained with cresyl violet, and examined microscopically for localization of the recording sites. All recording electrodes were localized to the ADN or having passed through ADN.

Experiment 1 In this experiment the effects of CPP administration were as­ sessed to determine whether NMDA blockade would affect the basic directional characteristics of HD cells and the ability of a familiar environment to control the HD network. Given the fact that NMDA antagonists seem to impair spatial behavior, one possible mechanism of this effect would be a profound distortion of the directional signal carried by the HD network.

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Method Figure 1 presents the procedure of this experiment. Each cell was recorded over four 8-min recording sessions in the familiar cylindrical enclosure. Before and after each session, the animal received disorientation treatment by being placed in a cardboard box which was rotated for 1-2 minutes by an experimenter as it was carried around the perimeter of the enclosure. In the first session (Preinjection/Standard), the HD cell was recorded with the white cue positioned at the North position o f the enclosure. Fol­ lowing this, the animal was removed from the enclosure and placed in the cardboard box, the enclosure was rotated 90° in the counterclockwise direction to position the cue on the west wall, and the animal was returned for recording in the Preinjection/ Rotated session. The animal was then moved to another room, injected with either CPP or isotonic saline and returned to the home cage. After allowing 30 minutes for drug absorption, the animal was returned to the enclosure for the Postinjection/Standard session with the cue positioned at the North location. Finally, in the Postinjection/Rotated session the animal was removed and placed in the box, the enclosure was rotated 90° in the counter­ clockwise direction, the animal was returned and the cell was recorded.

Results and Discussion The results of Experiment 1 are based on recordings o f 16 cells recorded from 13 rats, with eight cells recorded from six rats in the Saline condition and eight cells recorded from seven rats in the CPP condition. Figure 2 presents polar plots of FR by HD tuning curves for several example cells from each condition across each session. In the Preinjection sessions all cells exhibited directional specificity as indicated by an elevated average firing rate at a specific preferred direction with increasingly lower firing rates moving away from that direction. In addition, this baseline direc­ tional specificity was controlled by the landmarks within the recording enclosure, as the preferred direction of cells shifted in concert with the enclosure in the Preinjection/Rotated sessions. As Figure 2 indicates, following administration of saline or CPP, all cells the Saline and most in the CPP condition continued to exhibit stable directional specificity that was controlled by the landmarks in the recording enclosure.

The effects of drug administration on basic tuning characteris­ tics were assessed by comparing Preinjection/Standard and Postin­ jection/Standard sessions on the measures of Peak FR, Background FR, DIC, and Signal to Noise. Table 1 presents mean preinjection and postinjection values on these measures across each condition. As a whole, injection o f the drug produced little change in direc­ tional specificity of recorded cells. The exception was Peak FR, which seemed to decrease following drug administration. A 2 X 2 (Session [preinjection, postinjection)] X (Condition [saline, CPP]) anal­ ysis of variance (ANOVA) performed on this measure found a significant effect of Session [F (l, 14) = 10.73, p = .006], no significant effect o f Condition [F (l, 14) = 0.12, p > .05], but a significant Ses­ sion X Condition interaction [F (l, 14) = 13.8, p = .002. Exam­ ination of the means and tests of the simple main effect of Session on each condition (see Table 1) indicates that Peak FR remained unchanged following injection of Saline, but significantly de­ creased following injection o f CPP. No other significant changes were found in the other basic directional measures following CPP administration. The ability of landmark cues to control the preferred direction of HD cells following NMDA blockade was examined by calculating the directional shift in the FR by HD tuning curves between sessions. Comparing the directional tuning between Preinjection/ Standard and Postinjection/Standard sessions provides an indica­ tion o f whether drug injection changed the preferred directions of recorded cells in the standard sessions. Figure 3A displays polar scatterplots of the directional shifts between these sessions. Ex­ amination of these directional shifts indicates that HD cells re­ corded from both saline and CPP animals showed stable direc­ tional specificity between Preinjection/Standard and Postinjection/ Standard sessions, as indicated by the finding that the directional shifts appeared centered around 0° in both conditions. Rayleigh tests o f these angular shift values supported this observation, showing that the angular shift values for both Saline and CPP conditions w ere significantly nonrandom ly distributed (m ean vector lengths (r) of 0.99 and 0.99 and m ean angles (m) o f —2.30° and —5.27°, for Saline and CPP conditions, respec­ tively; ps < .05). In addition, V -tests o f these angular shift values show ed that both Saline and CPP shifts w ere signifi­ cantly clustered around 0° (Vs of 7.34 and 7.03 for Saline and CPP, respectively; p s < .01).

Postinjection/Std

Preinjection/Std

Preinjection/Rot

Figure 1.

Overview of the procedure of Experiment 1.

Postinjection/Rot

HD CELLS FOLLOWING NMDA BLOCKADE

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Example Saline Cells P reinjection/S tandard

P reinjection/R otated

P ostinjection/S tandard

P ostinjection/R otated

90°

Ti P reinjection/S tandard

Preinjection/R otated

Cell BL35.c2 90°

-

P reinjection/S tandard

P ostinjection/S tandard

P ostinjection/R otated

90°

O

90°

-

-

P reinjection/R otated 90°

P ostinjection/S tandard

o

P ostinjection/R otated

90°

Example CPP Cells P reinjection/S tandard

Preinjection/R otated

P ostinjection/S tandard

CellBL17.c2 90°

il

90°

a 270°

P reinjection/S tandard Cell BL14.C1 90°

P ostinjection/R otated

P ostinjection/S tandard

270°

P ostinjection/R otated

90°

Figure 2. Representative FR X HD tuning curves in the form of polar plots from example cells in each condition of Experiment 1. The frequency shown in the bottom left of each panel is the peak firing rate of the cell for that session.

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Table 1 Effect o f Injection on Basic Directional Characteristics o f HD Cells in a Familiar Environment (Experiment I)

Saline Peak FR (Hz) Background FR (Hz) DIC Signal to noise Mean vector Length CPP Peak FR (Hz) Background FR (Hz) DIC Signal to noise Mean vector Length Note.

Preinjection

Postinjection

Pre vs. Post

62.0 (±8.9) 6.5 (±2.6) 0.76 (±.18) 0.88 (±.05) 0.57 (±.08)

63.5 (±10.1) 4.7 (±1.5) 0.80 (±.15) 0.88 (±.06) 0.63 (±.09)

1(7) 1(7) t(7) t(7) K7)

= = = = =

0.49, p 0.77, p 0.23, p 1.16, p 1.51, p

= = = = =

.639 .464 .822 .283 .175

70.8 (±6.3) 3.1 (±1.3) 0.92 (±.16) 0.95 (±.02) 0.68 (±.05)

47.5 (±3.7) 4.3 (±1.9) 0.49 (±.08) 0.91 (±.04) 0.58 (±.06)

K7) K7) 1(7) K7) 1(7)

= = = = =

3.90, p 0.56, p 2.19, p 1.16, p 1.24, p

= = = = =

.006 .591 .065 .283 .255

Values are means (±1 SEM). Pre vs. Post = paired two-tailed t test.

Comparison of angular shifts between Postinjection/Standard and Postinjection/Rotated sessions provides additional indication of whether the HD cells remained controlled by the landmarks within the recording enclosure after drug administration. Given that the enclosure was rotated 90° in the counterclockwise direc­ tion between these two sessions, we would expect that the pre­ ferred directions of the cells would shift a similar amount between these sessions if normal landmark control was exerted. The shift values displayed in Figure 3B indicate that all of the HD cells from saline-injected animals and all but one of the cells from CPPinjected animals shifted in concert with the landmarks of the recording enclosure. The one cell that did not seem to be controlled by the landmark following CPP administration exhibited periodic drifts in preferred direction during the postinjection sessions. This effect was apparently an aberration, as no other cells in this or the next experiment showed unstable preferred directions within a given session after CPP administration. The observation that most cells remained controlled by the rotated landmarks was supported by Rayleigh tests showing that shifts from both saline and CPP conditions were distributed nonrandomly (rs of 0.98 and 0.88 and ms of 91.7° and 99.2° for saline and CPP conditions, respectively; ps < .05), and V-tests showed that both distributions were signif­ icantly clustered around the expected 90° shift direction (Vs = 6.15 and 4.07, ps < .01 and p = .014 for saline and CPP conditions, respectively). Because the movement state of the animal may affect the signal carried by the HD system (Taube, 1995; Zugaro, Tabuchi, Fouquier, Berthoz, & Wiener, 2001) we examined the behavior of the animals to determine if CPP administration produced locomotor side effects. This question is of special significance because previous work has consistently shown that systemic administration of another NMDA antagonist, dizocilpine (MK-801), at higher levels produces gross motor distur­ bances including hyperactivity, ataxia, and circling behavior (Lopez Hill & Scorza, 2012; Koek, Woods, & Winger, 1988; Murata & Kawasaki, 1993). Close observation of the animals before and after injections detected no obvious drug-induced effects on locomotor behavior. While the drug may have pro­ duced a slight sedative effect on some animals, they continued to forage for food pellets following drug administration. These

qualitative observations are illustrated in Figure 4A, which displays example movement trajectories of the first two minutes of standard sessions before and after drug or saline administra­ tion. To quantitatively assess the movement activity of the animals before and after drug administration, the recording enclosure was divided into eight pie-slice-shaped regions and the average number of instances per minute the animal’s head crossed between regions (crossings/min) was computed. Figure 4B displays average crossings/min of Preinjection/Standard and Postinjection/Standard sessions for both Saline and CPP con­ ditions. Supporting our subjective observation that the drug may have produced a slight sedative effect on the animals, this measure of movement activity did seem to decrease for CPP animals following injection; however performing a Session X Condition ANOVA on this measure found no significant effects of Session [F (l, 14) = 3.39, p = .087], or Condition [F < 1], and the Session X Condition interaction was also not significant [F (l, 14) = 1.61, p = .225], To summarize, Experiment 1 demonstrated that stable directional-specific activity of the HD network in a familiar environment does not depend on NMDA receptor mediated transmission. While HD cells did show a mild degradation of directional activity during NMDA blockade, recorded cells remained directionally sensitive and usually exhibited stable activity relative to the landmarks in the apparatus following drug administration.

Experiment 2 The cellular correlates of spatial behavior have been shown to change as a result of experience and NMDA receptor-mediated cellular plasticity is a plausible candidate for these processes. In support of this notion, Kentros et al. (1998) found that the environmental-specific place cell signal, which normally becomes established during the initial exposure to a new environment, failed to stabilize when the animal was administered an NMDA antag­ onist during the initial exposure. As in the case of place cells, the spatial signal displayed by HD cells in a given environment is likely determined during the initial exposure to that environment (Dudchenko & Zinyuk, 2005; Goodridge et al., 1998), providing

HD CELLS FOLLOWING NMDA BLOCKADE

A

Preinjection/Standard vs Postinjection/Standard

119

to the h om e cag e fo r 2 4 ho u rs to allow the d rug e ffe c ts to d issip a te . O n D ay 2, the cell w as recorded in the triangular enclosure to determ ine if the preferred direction relative to the enclosure e x ­ hibited during the initial exposure o n the previous day w as m ain­ tained in subsequent exposures. In the first test session (R otated T riangle T est), the cell w as recorded w ith the enclosure rotated 120° in the counterclockw ise direction relative to the initial expo­ sure. T he purpose o f rotating the enclosure for this test session w as to determ ine if the preferred direction displayed on D ay 1 w as m aintained relative to the landm arks w ithin the triangular record­ ing enclosure (rather than the surrounding room ). In the subse­

B

Postinjection/Standard vs Postinjection/Rotated

Figure 3. Scatter diagrams showing the amount of angular shift of directional activity between sessions for cells in each condition of Exper­ iment 1. (A) The distribution of angular shifts between Preinjection/ Standard and Postinjection/Standard sessions demonstrate that cells in both saline and CPP conditions maintained a consistent directional preference following injection. (B) Angular shifts between Postinjection/Standard and Postinjection/Rotated sessions indicate that cells in the saline condition and most cells in the CPP condition remained controlled by the landmarks within the enclosure following injection. For this and upcoming scatter diagram figures, circles in each panel represent the amount of angular shift of each recorded cell in the second session relative to the first session. The dotted line in each panel indicates the shift predicted if the cells are perfectly controlled by the landmarks in the enclosure. The angle of the arrow in each panel represents the mean angle (m) of the shifts, and the length of the arrow denotes the mean vector length (r) of the shifts.

evidence for experience dependent plasticity in the H D system . E xperim ent 2 considered w hether blockade o f the N M D A receptor disrupts the ability o f a new environm ent to develop landm ark control o ver the H D system .

Method F ig u re 5 d isp la y s th e p ro c e d u re o f E x p e rim e n t 2. E ac h cell w as re c o rd e d o v e r 2 d a y s w ith tw o re c o rd in g se ssio n s on D ay 1 a n d th ree re c o rd in g se ssio n s on D a y 2. O n D ay 1, the an im al w as first re c o rd e d in the fa m ilia r c y lin d ric a l e n c lo su re (C y lin ­ d e r 1), to o b ta in b a se lin e c e llu la r a ctiv ity . T h e a n im a l w as then re m o v e d , tak e n to a n o th e r ro o m , and in je c te d w ith e ith e r C P P o r iso to n ic sa lin e a n d re tu rn e d to the h o m e cage. A fte r a llo w in g 30 m in u te s fo r d ru g a b so rp tio n , the a n im a l w as p la c e d in the n o v e l tria n g u la r e n v iro n m e n t (T ria n g le L ea rn in g ) a n d th e c ell w as re c o rd e d to d e te rm in e the d ire c tio n a l-d e p e n d e n t a c tiv ity in th e n e w e n v iro n m e n t. F o llo w in g th is, the ra t w as th en re tu rn e d

quent session (Standard T riangle T est) the cell w as recorded w ith the enclosure rotated b a ck to the original orientation o f D ay 1. Lastly, the cell w as recorded in the fam iliar cylindrical enclosure (C ylinder 2). T his final cylinder session w as used to com pare the preferred directions betw een the cy linder sessions o f D ay 1 and D ay 2 to provide som e verification that the sam e cell w as recorded over b oth days, as previous w ork has show n th at H D cells w ill show the sam e preferred directions in a given environm ent even if recorded over m ultiple days (T aube et al., 1990a). A ll recording sessions w ere 12 m inutes in duration and the flo o r pap er w as changed p rior to the start o f each session. W ith the exception o f the T riangle L earning session, the anim al w as given disorientation treatm ent in the cardboard box as described in E x perim ent 1 prior to every session on b oth days to disrupt the internal sense o f direction and encourage the anim al to use the landm arks in the enclosure for directional orientation. D isorientation treatm ent was not given prior to the T riangle L earning session because previous w ork has indicated that disorientation m ay inhibit the ability o f a new environm ent to acquire control o f the H D and place cell system s (K nierim et al., 1995).

Results and Discussion T h e fin d in g s o f E x p e rim e n t 2 are b a se d o n a to ta l o f 19 cells each re co rd e d fro m se p a ra te ra ts, w ith 10 c e lls re c o rd e d in th e salin e c o n d itio n and n in e c ells re c o rd e d in th e C P P c o n d itio n . F ig u re 6 p re sen ts re p re se n ta tiv e tu n in g c u rv es a cro ss the fiv e se ssio n s in each co n d itio n . A s in th e p re v io u s e x p erim e n t, re c o rd e d c ells w ere e x a m in e d fo r ch an g e s in b a sic m e a su re s o f d irec tio n a l sp e c ific ity fo llo w in g d ru g a d m in istra tio n , in th is case b y c o m p a rin g c e llu la r a c tiv ity b e tw e e n th e C y lin d e r 1 and T ria n g le L e a rn in g se ssio n s. T a b le 2 p re sen ts th e m ea n re su lts o f th ese c o m p a riso n s. In c o n tra st to th e p re v io u s e x p erim e n t, sig n ific a n t a tte n u a tio n o f th e se m e a su re s fo llo w in g in je c tio n o f C P P b u t n o t sa lin e led to sig n ific a n t S e ssio n X C o n d itio n in te ra c tio n s fo r n e arly all o f th e m ea su re s in clu d in g B a ck ­ g ro u n d FR [ F ( l , 17) = 7 .0 1 , p = .0 1 7 ], D ire ctio n a l IC [ F ( l , 17) = 5.83, p = .027], and M ean V e c to r L en g th [ F ( l , 17) = 14.05, p = 002]. O n a c ell by c e ll b a sis, w hile d ru g a d m in is­ tra tio n d id seem to w e ak e n the d ire c tio n a l sig n a l c a rrie d by m o st cells, it did not e lim in a te it (as can be seen in the e x am p le C P P c ells p re se n te d in F ig u re 6), a n d in d iv id u a l R a y le ig h a n aly se s p e rfo rm e d on eac h c e ll in d ic a te d th a t all c e lls re ­ m ain e d d ire c tio n a lly tu n e d fo llo w in g d ru g a d m in istratio n . A s in E xperim ent 1, anim als in the C P P condition appeared to show a decrease in m ovem ent follow ing the injection (P reinjec­ tion = 11.8 ± 0.89 crossings/m in, P ostinjection = 7.5 ± 1 . 8

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Saline

Preinjection

CPP

Cell BL35.C2

Preinjection

Cell BL27

Saline

CPP

Figure 4. (A) Sample movement trajectories of the first 2 minutes of Standard session recordings from each condition of Experiment 1. (B) Mean Crossings/Minute for Preinjection and Postinjection Standard sessions in saline and CPP conditions. Error bars represent standard errors of the mean.

crossings/min). Interestingly, animals in the saline condition also showed a decrease in movement following injection (Preinjec­ tion = 14.2 ± 1.4 crossings/min, Postinjection = 7.9 ± 0.6 crossings/min). As a result, the Session X Condition ANOVA showed a significant main effect of Session [F(l, 17) = 29.75, p < .001], but no significant effect of Condition [F(l, 17) = 1.46, p = .243] or Session X Condition interaction [F < 1], Given that both saline and CPP conditions showed decreased movement following injection, this effect is hypothesized to have at least partly occurred because of neophobia to the novel enclosure. Figure 7 displays the preferred direction shifts of individual cells for the relevant session comparisons of this experiment. The

shifts of cells between the Cylinder 1 and the Triangle Learning Session are displayed in Figure 7A. For both Saline and CPP conditions, most cells displayed a preferred direction in the novel triangular environment within 90° of the preferred direction in the familiar cylinder, while a few cells showed larger shifts. Rayleigh analyses of these shifts showed that the shifts of both conditions were significantly nonrandom (ms = 30.0°and 23.6°, rs = 0.5S and .76, ps = .031 and .003 for Saline and CPP conditions, respectively), and the shifts between Saline and CPP conditions did not appear to be remarkably different. It is noted, however, that the relatively small sample sizes may have limited the ability to detect a subtle effect of drug administration. The finding that the

HD CELLS FOLLOWING NMDA BLOCKADE

Day 1

121

Day 2 Triangle Learning

Figure 5.

Overview of the procedure of Experiment 2.

preferred directions between the different environments were fairly similar is assumed to have occurred because the animals were not given disorientation treatment prior to being placed in the novel triangular environment. The critical test for determ ining if the N M DA antagonist interfered w ith the developm ent o f a stable directional reference in the novel environm ent is the com parison o f the tuning functions betw een the T riangle Learning session on D ay 1 and the R otated T riangle Test session on Day 2. A ssum ing the initial exposure o f the anim al to the novel triangular enclosure on D ay 1 resulted in the ability o f the enclosure to control the HD system and also determ ined the preferred direction o f the cell during future exposures to the triangular enclosure, we w ould expect the preferred direction o f the recorded cell to be rotated by 120° in the clockw ise direction in the R otated T ri­ angle T est session on Day 2 relative to the T riangle Learning session. The dashed lines in the exam ple tuning curves in the R otated T riangle T est panels o f Figure 6 show w here the center o f the tuning curves should fall assum ing the tuning functions established on D ay 1 rotated w ith the enclosure. A s indicated in the exam ple tuning curves, cells from anim als in the saline condition typically show ed preferred directions that rotated w ith the triangular enclosure on the R otated T riangle Test sessions; in contrast, cells from the CPP condition appeared to have random preferred directions relative to the enclosure on this critical test. Figure 7B displays these shifts in preferred directions across the total cells sam pled, and further supports these observations. In the case of saline cells, the distribution of shifts between the two sessions appeared centered around 120°, with the Ray­ leigh test indicating that these shift deviations were nonrandomly distributed (m = 110.6°, r = .55, p = .047), and the V-test indicating that the shifts were significantly clustered around the predicted 120° direction (V = 5.39, p = .008). In contrast, shifts from cells in the CPP condition did not appear clustered in the expected 120° position (Figures 6 and 7A) indicating that land­ m ark control o f the HD system was not acquired by the enclosure during the drugged exposure for these animals. This conclusion is supported by the Rayleigh test which showed that the shifts were not significantly nonrandom in this condition (m = 2.60°, r = .316, p > .05) and the V-test, w hich failed to indicate that the shifts were significantly clustered around the expected direction (V = 1.31, p > .05). Lastly, a W ilcoxon-M ann-W hitney compar­

ison o f the absolute angular shifts between these sessions (Batschelet, 1981) found significantly smaller shifts in the Saline condition relative to the enclosure than in the CPP condition [U (10,9) = 17, p < .05], The shift deviations between the Triangle Learning and Stan­ dard Triangle Test sessions (Figure 1C) provided additional as­ sessment o f this issue, as no shift between those sessions would be expected if the initial exposure in the Triangle Learning session resulted in acquisition o f landmark control. As predicted, cells in the saline condition exhibited shift deviations that were signifi­ cantly distributed nonrandomly (m = —3.5°, r = .77, p = .001) and were centered on 0° (V = 7.71, p < .001), while cells in the CPP condition did not show significantly nonrandom shift devia­ tions (m = —71.8°, r = .20, p > .05) and were not centered around the expected 0° for this comparison (V = .60, p > .05). In addition, the absolute shifts observed in the saline condition relative to the enclosure were once again significantly smaller than those o f the CPP condition [U (10,9) = 16, p < .05]. Lastly, shift deviations between the Rotated Triangle Test and Standard Triangle Test sessions (Figure 7D) were analyzed to verify whether cells in the CPP condition that failed to establish cue control during the drugged exposure did so during the R otated T riangle T est session, the first nondrugged exposure to the triangle for those anim als. The dashed line in the Standard T riangle Test panels of exam ple cells show n in Figure 6 indi­ cates w here the preferred direction is predicted to occur if the directional preference from the R otated T riangle T est session rotated w ith the enclosure. The distribution o f shift deviations from this com parison (Figure 7D) indicates that the m ajority of cells from the CPP condition did indeed establish landm ark control on the R otated Triangle T est session, show ing shifts centered around the expected —120° difference betw een the landm arks o f the tw o sessions. R ayleigh tests verified that cells from both conditions dem onstrated significant nonrandom shift deviations betw een these sessions (ms o f -1 1 9 .4 ° and 108.4°, rs o f .78 and .84, for saline and CPP conditions respectively, p s < .01) and the V -tests verified that the shifts w ere centered around the expected - 1 2 0 ° for both conditions (Vs o f 7.84 and 7.36, ps < .001). W hile most of the cells from the saline condition did appear to establish landmark control during the Triangle Learning session (Figures 6 and 7), a few cells failed to do so, similar to the majority

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Example Saline Cells C ylinder 1

Triangle Learning

Rotated Triangle Test

Standard Triangle Test

C ylinder 2

Standard Triangle Test

C ylinder 2

Standard Triangle Test

Cylinder 2

~Z7 270“

Triangle Learning

Rotated Triangle Test 90-

il 270-

Cylinder 1

270*

Triangle Learning

90-

Rotated Triangle Test 90-

Cell GR22

Example CPP Cells Cylinder 1

Triangle Learning

Rotated Triangle Test

a Triangle Learning

0 Rotated Triangle Test

Standard Triangle Test

C ylinder 2

90-

270-

270-

Cylinder 1 90-

Triangle Learning

Rotated Triangle Test

Standard Triangle Test

Cylinder 2

90-

Cell GR41

Figure 6. Representative FR X HD tuning curves from example cells in each condition of Experiment 2. The dashed lines in the Rotated Triangle Test panels indicate the predicted directional preference if the cell stabilized relative to the enclosure during the Triangle Learning session. The dashed lines in the Standard Triangle Test panels indicate the predicted directional preference relative to the Rotated Triangle Test session. The frequency shown in the bottom left of each panel is the peak firing rate of the cell for that session.

HD CELLS FOLLOWING NMDA BLOCKADE

123

Table 2 Effect o f Injection on Basic Directional Characteristics o f HD Cells in a Novel Environment (Experiment 2) Preinjection Saline Peak FR (Hz) Background FR (Hz) DIC Signal to noise Mean vector Length CPP Peak FR (Hz) Background FR (Hz) DIC Signal to noise Mean vector Length

Note.

90.8 2.1 1.38 0.98 0.79

(±9.6) (±0.66) (±0.15) (±0.01) (±.03)

Pre vs. Post

(±12.1) (±1.23) (±0.14) (±.02) (±.04)

r(9) = 0.22, p t(9) = 1.1 l , p r(9) = 1.77, p t(9) = 1.23, p t(9) = 1.08, p

90.9 (±19.8) 14.2 (±4.4) 0.50 (±0.15) 0.80 (±0.08) 0.48 (±.09)

r(8) = 3.13, p t( 8) = 3.04, p r(8) = 2.99, p r(8) = 2.22, p t(8) = 4.62, p

88.5 3.0 1.26 0.96 0.77

122.2 (±12.4) 6.57 (±2.2) 1.12 (±0.33) 0.94 (±0.02) 0.69 (±.05)

= .833 = .292 = .111 = .252 = .309 = .014

= .016 = .017 = .057 = .002

Values are means (±1 SEM). Pre vs. Post = paired two-tailed t test.

of cells in the CPP condition. Given the assumption that landmark control of HD cells in normal animals is established during the initial exposure to a novel environment, these aberrant cells merit additional consideration. Figure 8 presents the HD by FR tuning curves for the three saline cells that failed to develop landmark control by the triangular enclosure on Day 1. The first cell pre­ sented (GR30) showed a pattern of results similar to the majority of the CPP cells, with the preferred direction displayed during the

A

Postinjection

Cylinderl vs Triangle Learning

Rotated Triangle Test unrelated to the orientation of the triangular enclosure on Day 2, suggesting that the initial exposure to the triangle was ineffective at establishing landmark control. For this cell on Day 2, however, the preferred direction displayed in the Rotated Triangle Test session correctly predicted the preferred direction shown in the following Standard Triangle Test, indicat­ ing that the initial session on Day 2 was effective at establishing landmark control. The other two aberrant saline cells shown in

B

Triangle Learning vs Rotated Triangle Test

Figure 7. Scatter diagrams showing the amount of angular shift of directional activity between sessions for cells in each condition of Experiment 2. (A) Although somewhat variable, there was a tendency for cells in both the saline and CPP conditions to show a similar directional preference in the Cylinder 1 and Triangle Learning sessions. (B) While a majority of cells in the saline condition showed a directional preference that rotated with the enclosure in the Rotated Triangle Test session, most cells from the CPP condition showed directional activity unrelated to the landmarks in the test. (C) When comparing directional activity between Triangle Learning and Standard Triangle Test sessions, cells in the CPP condition again showed poor enclosure control of their directional activity. (D) Cells from saline and CPP conditions rotated with the enclosure between Rotated Triangle Test and Standard Triangle Test sessions, indicating that those cells that did not stabilize to the enclosure during the Day 1 Triangle Learning session did so during the first test of Day 2.

BERKOWITZ ET AL.

124

Cylinder 1

Triangle Learning

Rotated Triangle Test

Standard Triangle Test

C ylinder 2

Triangle Learning

Rotated Triangle Test

Standard Triangle Test

C ylinder 2

Rotated Triangle Test

Standard Triangle Test

C ylinder 2

90° C ell G R 30

Cylinder 1

90°

Figure 8. FR X HD tuning curves from the three cells in the saline condition that did not appear to establish landmark control by the triangular enclosure during the Triangular Learning sessions of Experiment 2. The dashed lines in the Rotated Triangle Test panels indicate the predicted directional preference if the cell stabilized relative to the enclosure during the Triangle Learning session. The dashed lines in the Standard Triangle Test panels indicate the predicted directional preference relative to the Rotated Triangle Test session.

Figure 8 (GR3 and GR7) showed a pattern of results suggesting that these cells treated the triangular enclosure as two qualitatively different environments depending on its rotational position. This conclusion is based on the observation that while the preferred direction shifts between the Triangle Learning and Rotated Trian­ gle Test sessions and then again between the Rotated Triangle Test and Standard Triangle Test sessions appeared unrelated to the position of the enclosure landmarks, the preferred directions were the same between the Triangle Learning and Standard Triangle Test sessions. It was as though the cells were treating the triangular enclosure in the standard position as a unique environment (or at least a unique combination of background cues) relative to the triangular enclosure in the rotated position. The validity of our comparison of the preferred directions exhibited over the two days of recording each cell assumes that the identity of the recorded cells did not change over the recording period. As an assessment of this, we compared the preferred directions exhibited in the Cylinder 1 sessions, collected at the start of Day 1, with the preferred directions exhibited in the Cylinder 2 sessions, collected at the end of Day 2. Given that different HD cells typically show different preferred directions even when located close to another (Taube, 1995), a significant change in preferred direction for a given cell would likely indicate that the identity of the cell had changed across the 2 days. Figure 9A provides the directional shifts between those sessions for saline and CPP conditions. As the figure indicates, the preferred directions of recorded cells in the cylinder remained similar across the 2 days, with the Rayleigh tests indicating that shifts from both conditions were significantly nonrandom (ms = 4.9° and

2.4°, rs = .95 and .98, ps < .001 for saline and CPP conditions, respectively). Similarly, the V-tests indicated that the shifts were significantly clustered around 0° for both the saline and CPP condi­ tions (Vs of 9.5 and 8.8, ps < .001, for saline and CPP, respectively). Across both samples of cells, the largest shift observed between the cylinder sessions was 36° with 17 of 19 cells (89%) showing absolute shifts of 24° or less between the two cylinder sessions. The two remaining cells (shifts of 30° and 36°) were both in the saline condition and therefore cannot account for the poor landmark control of the CPP condition. This is consistent with other reports that the preferred directions of HD cells in the same enclosures remain stable even when recorded across multiple days (Taube et al., 1990b). To provide further analysis of this issue, Figure 9B displays the shift relative to the enclosure between the two cylinder sessions and the shift relative to the enclosure between the Triangle Learn­ ing and Rotated Triangle Test sessions for each individual cell. This cell-by-cell analysis indicates that in those instances where there was a large shift in the preferred direction relative to the enclosure in the first two triangle sessions, it was not typically accompanied by a large shift between the cylinder sessions, as would be expected if a loss of cell isolation was responsible for the shifts between the Triangle Learning and Triangle Test sessions.

Summary and Concluding Discussion In order to better define the neurochemical mechanisms respon­ sible for the maintenance and plasticity of the directional signal carried within the HD system, recordings of HD cells were made

HD CELLS FOLLOWING NMDA BLOCKADE

A

Cylinder 1 vs. Cylinder 2

B

O C ylinder 1 vs. C ylinder 2 ▲ Triangle Learning vs. Rot Triangle Test

1601

Saline Cells

CPP Cells

I6O-1

▲

CD