Iontophoresis, physiology and data acquisition

Transcription

1 Methods: Behavioral training, surgery and electrophysiological recordings were performed as described previously (1) and were in accord with guidelines set by the National Institutes of Health, and approved by the Yale University Institutional Animal Care and Use Committee. Monkeys Oculomotor Delayed Response Task Studies were performed on two adult male rhesus monkeys (Macaca mulatta) trained on the spatial oculomotor delayed response (ODR) task as previously described (1). This task requires the subject to make a memory guided saccade to a remembered visuospatial target (Fig. 1A). After a 3 second inter trial interval (ITI), a central light emitting diode (LED) was illuminated as the fixation target. A trial began when the subject fixated at the central spot for 0.5 seconds (fixation period). After the fixation period, a cue LED was illuminated for 0.5 seconds (cue period) at one of eight peripheral targets located at an eccentricity of 13 0 with respect to the fixation spot. After the cue was extinguished, a second delay period followed. The fixation cue remained lit during the cue and delay periods and the subject was required to maintain central fixation throughout both the cue presentation and the delay period. At the end of the delay, the fixation spot was extinguished which instructed the monkey to make a memory guided saccade to the location where the cue LED had been located. A trial was considered successful if the subject s response was completed within 0.5 seconds of the offset of the fixation LED and was within 2 0 of the correct cue location. Successful responses were rewarded with fruit juice. The position of the cue was randomized over trials. The subject s eye position was monitored with the ISCAN Eye Movement Monitoring System (ISCAN, Burlington, MA), and the ODR task was generated by the TEMPO real time system (Reflective Computing, St. Louis, MO). Recording Locus Prior to recording, Subject H underwent a magnetic resonance image (MRI) scan to obtain exact anatomical co ordinates of brain structures, which guided placement of the chronic recording chambers. MRI compatible materials were used for the implant so that the position of the

2 recording chambers could be confirmed by MRI post implantation. Based on the MRI, the recording chamber for monkey H was positioned over the prefrontal cortex centered 4mm medial to the principal sulcus and 2 mm posterior to its caudal end. An MRI was not performed on Subject M and the recording locus was computed from stereo taxic coordinates calculated from an atlas (2). The locus was subsequently confirmed with histology to be located around the medial bank of caudal Principal Sulcus (Area 46). Fig. S7 shows the position of the recording cylinder for the two subjects and the estimated recording area in PFC. Task related cells obtained in this study were located in a small area ranging from 0 5 mm anterior to the caudal end of the principal sulcus and 1 to 2 mm medial to the principal sulcus. Iontophoresis, physiology and data acquisition SKF 38393, SKF81297, A68930, chelerythrine and SCH23390 (Sigma RBI, St. Louis, MO) were dissolved in triple distilled water (adjusted with HCl or H3PO4 to ph ) to a final concentration of 0.01M and stored in aliquots of 50 µl at 70 0 C. Rp CAMPS (Sigma RBI; Tocris Cookson, Ellisville, MO) was similarly dissolved in water at ph 6 8 (adjusted with NaOH) and stored at 70 0 C. Iontophoretic electrodes were constructed with a 20 µm pitch carbon fiber (ELSI, San Diego, CA) inserted in the central barrel of a seven barrel non filamented capillary glass (Friedrich and Dimmock, Millville, NJ). The assembly was pulled using a custom electrode puller and the tip was beveled to obtain the finished electrode. Finished electrodes had impedances of MΩ. (at 1 khz) and tip sizes of µm. Thus the recording electrode is of very low impedance compared with tungsten electrodes and saline filled glass micro pippete electrodes also used in iontophoretic recordings. The outer barrels of the electrode were then filled with 3 drug solutions (two consecutive barrels each) and the solutions were pushed to the tip of the electrode using compressed air. Typically the impedance of the drug filled outer barrel was in the order of 100MΩ and above. Thus, while the recording electrode is low impedance, the iontophoretic barrels themselves were of very high impedance. A Neurophore BH2 iontophoretic system (Medical Systems Corp., Greenvale, NY) was used to control delivery of the drugs. The various drugs were ejected using the appropriate polarity depending on the charge on the drug

3 molecule at various ejections currents from 5 100nA. The current ejection itself is not expected to have a large effect on the neuronal firing, since these are extra cellular recordings, where the ejection barrel is at some distance from the neurons. Micro stimulation of neurons typically is done with currents in the order of microamperes. Further, different drugs dissolved in water and ejected with the same polarity and the same current magnitude have been shown in previous studies in our laboratory to have dramatically different effects on neuronal firing. If current application alone could cause changes in the physiology of the neuron, then this would not be expected to be the case and any drug applied with the appropriate strength and polarity would cause the same effect. However, in order to further ensure that current effects were not in play in our observed results, we performed saline (Na + and Cl ) ejection controls for positive and negative current ejection respectively. As can be gleaned from Supplementary Fig. S8, current ejections alone with the same polarity and strength do not cause the same effects as the ejection of drugs from iontophoretic electrodes. Sodium ion ejection at positive current polarities did not cause significant suppression like D1 agonist ejections at the same currents at positive polarities. In fact, Na + ejection in a population of 22 units did not cause significant change in neuronal activity. Na + ejection at 25 na (Fig S8A, top panel), 50 na (middle panel) and 100 na did not significantly alter prefrontal activity (t test, P > 0.05) for both preferred and non preferred directions. This is in contrast to a consistent suppression of activity by the D1 agonist. This is readily seen in Fig S8B, where the average change due to Na + ejection at 25 and 100nA is compared with the effects of the various agonists used in this study. Na + ejection at both ejection currents had no significant effects on neuronal activity in both preferred and non preferred directions, in contrast with the D1 agonist, which consistently suppressed PFC neuronal activity. Fig S9 shows a direct comparison of the effects of Na + ejection and D1 agonist ejection on the firing of prefrontal neurons at various current strengths. The traces have been normalized so the effects after drug application can be compared in isolation. Na + ejection (green traces) at +25 na (top panel), + 50 na (middle panel) and na (bottom panel) had non significant effects on neuronal firing (t test, P > 0.05). In contrast, D1 agonist ejections (red traces) at comparable ejection strengths invariably suppressed prefrontal activity. These results reliably indicate that current balancing is not required in the experimental paradigm used in this study. Further, Na + ejection with and without current balancing did not show significant differences (data not shown). It should also be noted, that as the de ionized ph adjusted water which is used to

4 dissolve all drugs in this study has negligible ion content, almost all the current is carried by the drugs themselves. Retention currents of 3 to 8 na were used in a cycled manner (1 sec on, 1 sec off) when not applying drugs. Drug ejection did not create noise in the recording. The retention currents used in this study are similar to those used in many studies from the laboratory, and over the course of multiple studies, we have not encountered systematic non stationarities in neuronal firing when no drug is applied, thus indicating that the drug was not leaking from the barrels at these retention currents. If a neuron was not stable in the control condition, then it was not used for further experiments or analysis. Before the experiments, the impedances of the drug filled barrels were individually determined, and if the impedances were lower than the usual value, higher retention currents were used during the experiments. However, since in these experiments, the determination of adequate retention current magnitude can only be empirical, the strongest evidence to suggest that they were adequate comes from the lack of systematic change in neuronal activity when no drug was applied. Also, since the recording micro electrode was made of carbon fiber and of low impedance compared with saline filled micropipettes, which typically have impedances in the range of several megaohms, current ejection from the drug barrels resulted in very little noise in the recording electrode and spikes recorded from the recording electrode remained stable and isolatable even when high currents such as 100nA were passed in the drug barrels. Occasionally, iontophoretic currents would result in noise, in which case the recording was not pursued. Typically, current balancing is used when the current ejection from the drug barrel can cause noise in the recording electrode. Since, in these experiments, we rarely encountered noise problems, we did not employ current balancing. This altered methodology has been employed in our laboratory and by others previously, and we have several published studies where no current balancing and medium range retention currents were employed. The D1R agonists, antagonist and PKC blocker, chelerythrine used in this study were all ejected with positive current polarity and Rp CAMPS was similarly applied, but the polarity of the ejection and retention currents was reversed due to the opposite polarity of charge on the molecule. The electrode was mounted on a MO 95 micromanipulator (Narishige, East Meadow, NY) in a 25 gauge stainless steel guide tube. The dura was punctured using the guide tube to facilitate access of the electrode to cortex. Extra cellular voltage was amplified using a custom low noise

5 preamplifier (SKYLAB) and band pass filtered (180 Hz 6 khz, 20 db gain, 4 pole Butterworth; Kron Hite, Avon, MA). Signals were digitized (20.83 khz, micro 1401, Cambridge Electronics Design, Cambridge, UK) and acquired using the Spike2 software (CED, Cambridge, UK). Neural activity was analyzed using waveform sorting by a template matching algorithm, which made it possible to isolate more than one unit at the same recording site. Post stimulus time histograms and rastergrams were constructed online to determine the relationship of unit activity to the task. Unit activity was measured in spikes per second. If the rastergrams displayed task related activity, the units were recorded further and pharmacological testing was performed. After unit isolation, a control condition with approximately eight trials at each of eight cue locations was obtained. The control condition was followed by D1 agonist application. Dose dependent effects of the drug were tested in two or more consecutive conditions. Drugs were continuously applied at the relevant current throughout a given condition. Each condition had around 8 (typically 10 or more) trials at each location for statistical analyses of effects. In some reversal experiments, the next condition was started after the effect of the agonist had been established for a few trials. In some experiments, SCH was co applied with the D1 agonist to determine D1R selectivity. In the experiments designed to test the intracellular signaling pathway coupled to the D1 receptor stimulation, Rp camps (camp blocker) or chelerythrine (PKC inhibitor) was either co applied with the D1 agonist at the relevant current (prevention) or co applied with the D1 agonist after D1 agonist application alone (reversal). In other experiments, the D1 agonist application was followed by two conditions, one with co application of the D1 agonist and chelerythrine and the next with the co application of the D1 agonist, chelerythrine and Rp camps, to assess for reversal of D1 agonist effects. Other control experiments were performed to assess the effects of co application of chelerythrine and Rp camps alone. Sodium ions were ejected at positive current polarities and Chloride ions at negative polarities to determine the effects of currents in some experiments. From the various cells recorded in this study, subsets of cells were tested in a particular experiment. Data analysis For purposes of data analysis, each trial in the ODR task was divided into four epochs: Fixation, Cue, Delay and Response (Saccade). The Fixation epoch lasted for 0.5 sec. The

6 Cue epoch lasted for 0.5 sec and corresponds to the stimulus presentation phase of the task. Delay lasted for sec and reflects the mnemonic component of the task. The Response phase started immediately after the Delay epoch and lasted ~1.5 sec. In the present study, we encountered various types of delay related activity. Some cells were activated for the preferred direction during the cue presentation and continued their firing during the delay period, but were only active in the response period for the non preferred direction. Others would exclusively show activity only in the mnemonic period. Delay related activity usually terminated just before the saccade. In this study, cells that showed spatially asymmetric activity in the delay period, irrespective of activations in other epochs were used. These cells were called delay cells (see example in Fig. 1B). Briefly, post stimulus time histograms were constructed for all spatial directions during the trial epoch using 0.1s bin widths. Two way analysis of variance (ANOVA) was used to examine the spatial tuned task related activity with regard to: (1) different periods of the task (cue, delay, and response vs. fixation) and (2) different cue locations. One way ANOVAs were employed to assess the effect of the effect of the drug compared to the control condition. Population histograms were constructed after normalization of neuronal firing rate to the maximal firing rate of the unit during the task. Drug conditions were normalized to the maximal firing rate of the unit during the task in the control condition. Normalized histograms for each unit thus obtained were then averaged across units to get a population histogram. Population spatial curves were obtained by normalizing the neuronal firing in all spatial directions during an epoch to the maximum firing in the preferred direction. This is done, so only effects on tuning can be examined independently of general effects on cell firing. After this normalization step, the maximal activity in the control and drug conditions would be equal. After further normalization across cells (different cells would have different initial control tuning curves and in order to statistically analyse the populaton tuning widths, it was necessary to generate population curves which were equivalent, except for the tuning width) the curves from various cells were then averaged and a population average curve was obtained for all target directions for the drug and control condition. Polar plots were created by taking the individual values of average firing in various directions and plotting them in X Y space in the corresponding angles of the cues that elicited them. These data was normalized, so the maximal firing in the control and drug conditions would be equal, thus delineating effects on size of the plot (the inverse of which corresponds to spatial tuning) alone. In an alternative analysis, the

7 delay period activity was expressed in terms of baseline activity. This was performed to verify that the normalization across conditions and cells was not producing spurious changes. Delay period activity was normalized to fixation period baseline activity in various directions and in different conditions. The fixation period activity occurs before the subject is presented with a cue, and should not be expected to have any spatial information and should be approximately the same in all directions, and thus be cancelled out in subsequent comparisons. In order to ensure that the effects on delay tuning were independent of concomitant effects on spontaneous activity, the population curves were alternatively obtained from the baseline adjusted delay activity and then compared across conditions. As can be seen from comparing Supplementary Fig. S10 and Fig. 2A in the paper, as expected, normalization to the baseline firing did not significantly alter the results obtained here. Tuning analysis We employed various methodologies to assess the spatial tuning of the memory fields of the prefrontal neurons in this study. We first fitted the average curves of firing rates in all directions by using a modified von Mises function, which is the analog of the Gaussian function for circular data. The function that best fitted the data was a linear convolution of two von Mises functions as follows: f=σ n (L n +A n (e bn(cos(θ θn) 1 )) Here n =2 was employed and the parameters L n, A n, B n and θ n were optimized for best fits using non linear least squares fitting with the Levenberg Marquardt algorithm implemented in MATLAB (see The parameter b n determined the width of the curve, L n, the baseline activity and A n, the maximal response in space. After the parameters were fitted, estimates about the width of the tuning curve were made from the fitted parameters. Effects of the drug on tuning width were then analyzed with t tests.

8 We also estimated tuning by loop vector analysis (data not shown). Briefly, loop vectors were constructed from average firing rates in various spatial directions by calculating their contributions to the average vector using the following formulae: X n =r n cosθ Y n =r n sinθ The amplitude of the resultant loop vector was calculated by the formula (X n +Y n ) 0.5. The angle of the resultant vector was calculated by using arctangents. The tuning was estimated by the size of the resultant vector and directly proportional to it. Loop vector analysis resulted in a similar profile for the data, albeit with differences in the significance levels for the various cells. Another method that was used to analyze the data and showed similar results relied on describing the net activation of cells in spatial directions above confidence limits set on the standard deviation of the baseline firing rate. Rodent Pharmacology Subjects Male Sprague Dawley rats (Taconic, NY) weighing g when received (approximately 2 months old) were initially pair housed in filter frame cages. The animals were fed a limited diet of Purina rat chow (16 grams per rat per day) immediately following behavioral testing. Water was available ad libitum. Food rewards during cognitive testing were highly palatable miniature chocolate chips to minimize the need for dietary restriction. Cognitive testing Special care was taken to habituate animals to all stressful procedures (handling, drug injections, etc.). Rats were trained and tested on the delayed alternation task in a T maze, using the methods described in Birnbaum et al. (1). On the first trial, animals were rewarded for entering either arm of the maze. Thereafter, for a total of 10 trials per session, rats were rewarded only if they entered the maze arm that was not previously chosen. Between trials the choice point was wiped with alcohol to remove any olfactory clues. Performance on the delayed alternation task is dependent upon the length of the delay between trials, thus the delay was raised as needed to maintain each individual animals performance at stable baseline performance of approximately

9 70% correct. This baseline allowed for detection of either improvement or impairment in performance following drug administration. Delays employed in this study were typically 5 15s and this is in the range of WM temporal scales. Cannulae Implantation in rats Following behavioral training, rats were anesthetized with a mixture of ketamine (80 mg kg -1, i.p.) and xylazine (10 mg kg -1, i.p.) and implanted with guide cannulae to allow drug infusions into the medial prefrontal cortex (stereotaxic coordinates from bregma and skull surface: anterior mm, lateral ± 0.75 mm, ventral 1.7 mm). The infusion needles projected 2.8 mm below the guide cannula such that the infusion site was 4.5 mm ventral to skull surface. Following surgery rats were individually housed and allowed to recover for at least 1 week. They were treated with Buprenex (0.01 mg kg -1 ) or three days to control pain; a topical antibiotic powder was applied to the wound margin to facilitate healing. Drug treatments were administered only after the animal recovered from surgery and achieved stable performance (60 80% correct) for two consecutive days. Histological analysis at the end of the experiment verified that all animals had correctly placed cannula. Drug administration Details of the infusion procedure can be found in (3). Rats were initially adapted to mock infusions until performance was unaltered by the infusion procedures. SKF81297 (Sigma RBI, Natick, MA) was diluted in sterile saline. Rp camps (Sigma RBI, Natick, MA) was diluted in sterile PBS. SKF81297 (0.1 µg in 0.5µl) and/or Rp camps (42 nmol in 0.5µl) were slowly infused (0.25 µl min -1 ) into the prefrontal cortex. The infusion needles remained in place for 2 minutes following the infusion. Cognitive testing began 15 minutes after the infusion procedure. All animals received 1) vehicle + vehicle, 2) SKF vehicle, 3) Rp camps + vehicle, and 4) SKF Rp camps. There was at least a one week washout period between infusions; animals were required to return to stable baseline performance before additional infusions could be administered. The order of drug treatments for each animal was quasi random to minimize potential order effects. The investigator testing the animal was blind to drug treatment conditions.

10 Data analysis Rodent cognitive data were analyzed using a two way ANOVA with repeated measures with within subjects factors of SKF81297 and Rp camps treatments. User defined contrasts tested paired comparisons, specifically comparing SKF vehicle vs. SKF Rp camps. Statistics were performed using Systat software. REFERENCES (1) SG Birnbaum et al, Science Oct 29; 306(5697): (2) G Paxinos, XF Huang, AW Toga, The rhesus monkey brain in stereo taxic coordinates, Academic Press (3) BP Ramos et al, Neuron Nov 13; 40(4):