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Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex
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RESEARCH ARTICLE R. Cotter et al. / Neuroscience xxx (2020) xxx–xxx
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Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex
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R. Cotter, y S. Winnik y A. Singer and G. Aaron *
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Wesleyan University, Middletown, CT 06459, United States
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Abstract—We measured the sensitivity of cortical circuit activity to small differences in local cortical environments by studying how temperature affects the trajectory of epileptiform events (EEs). EEs evoked via blockade of GABA-A receptors were recorded extracellularly from mouse coronal brain slices containing both hemispheres of anterior cingulate cortex synaptically connected by corpus callosum axons. Preferentially illuminating one hemisphere with the microscope condenser produced temperature differences of 0.1 °C between the hemispheres. The relatively warmer hemisphere typically initiated the EEs that then propagated to the contralateral side, demonstrating temperature directed propagation. Severing the callosum following one hour of EEs showed that the warmer hemisphere possessed a higher rate of EE generation. Further experiments implied that intact callosal circuits were required for the increased EE generation in the warmer hemisphere. We propose a hypothesis whereby callosal circuits can amplify differences in respective hemispheric activity, promoting this directionality in seizure propagation. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.
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Key words: callosotomy, seizure, GABA, GABAB.
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INTRODUCTION
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Using multiple intracranial electrodes, several studies have demonstrated that seizures in the intact human brain follow stereotyped trajectories. That is, the direction of seizure propagation from one seizure to the next is not random, but rather follows a set pattern of trajectories (Jackson et al., 1994; Emerson et al., 1995; McCormick and Contreras, 2001; Schevon et al., 2012). These trajectories vary from patient to patient, just as the phenomenology of seizure patterns can vary between patients. How these patterns become stereotyped remains an open question. Our results show that this stereotypy in seizure propagation can be achieved within an hour between cortical areas in our reduced preparation and can be biased by very small temperature differences. Studying the dynamics of cortical propagation in the brain is difficult as the potential routes of propagation are numerous. This was shown most clearly in a study of seizure propagation between rostral cingulate cortices in a cat, where seizure propagation was maintained between cortices even after the corpus callosum was severed, thus implicating thalamic or brainstem routes
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of alternate propagation (Ralston, 1961). Our callosal preparation is ideal for studying propagation of neuronal activity from one cortical area to another as it retains synaptically coupled neurons as well as two separate cortical areas connected only by a tract of callosal axons that can be cut before or during recorded activity (Walker et al., 2012). The effects of temperature on neuronal circuits have been well documented: neuronal membrane potential, action potential propagation speed, and synaptic activity can all be directly affected (Thompson et al., 1985; Moser et al., 1993; Trevelyan and Jack, 2002; Long and Fee, 2008). In addition, the temperature of brain circuits can alter the frequency of oscillations and rate of activity produced by those circuits (Long and Fee, 2008; Reig et al., 2010; Aronov and Fee, 2012; Stujenske et al., 2015). All of these previous data on temperature effects in neuronal tissue were achieved using temperature perturbations of at least 1 °C, and such temperature differences can occur naturally in the brain between disparate areas due to differences in activity-dependent or, possibly, inflammation-dependent activity (Mrozek et al., 2012). To our knowledge, we are the first group documenting significant effects on neural circuits imposed by a temperature perturbation that is an order of magnitude smaller than that created by any previous study (i.e., 0.1 °C).
*Corresponding author. Address: Biology Department, 52 Lawn Avenue, Wesleyan University, Middletown, CT 06459, United States. E-mail address: [email protected] (G. Aaron). y These authors contributed equally. Abbreviations: ACSF, artificial cerebral spinal fluid; EEs, epileptiform events; IHLs, interhemispheric latencies. https://doi.org/10.1016/j.neuroscience.2019.12.041 0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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EXPERIMENTAL PROCEDURES
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All work involving mice in this study was approved by Wesleyan’s IACUC committee, in accordance with IACUC protocols. The protocol number for this study is 2015_0603_Aaron.
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Preparation of slices
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Slices were prepared as described previously (Walker et al., 2012). Black Swiss mice of both sexes (postnatal days 18–24) were injected intraperitoneally with a mixture of 120 mg/kg ketamine and 10 mg/kg xylazine. The deeply anesthetized mouse was decapitated and the brain was removed and placed in high-sucrose ice-cold artificial cerebral spinal fluid (ACSF) composed of (in mM): 3 magnesium sulfate, 1 calcium chloride, 222 sucrose, 27.1 sodium bicarbonate, 1.5 sodium phosphate, 2.6 potassium chloride, 3 myo-inositol, 2 sodium-pyruvate, 0.4 ascorbic acid, bubbled with carbogen gas (95% O2/5% CO2). The caudal blocking cut was made at a 6 degree angle, using a template blocking device to ensure a consistent blocking angle. 350 lm thick coronal slices were cut with a vibratome (Leica VT1000S). The first slice saved for recording was the first slice to show an intact corpus callosum, and identified as ‘‘slice 1”, the next slice as ‘‘slice 2”, etc., as slices were collected in the rostral to caudal direction. Slices were transferred to warm (34 °C), oxygenated ‘‘1–3 ACSF” immediately after slicing (in mM): 1 calcium chloride, 3 magnesium sulfate, 126 sodium chloride, 26 sodium bicarbonate, 1.1 sodium phosphate, 25 glucose, 3 potassium chloride, 3 myoinositol, 2 sodium-pyruvate, 0.4 ascorbic acid. The slices were left to recover for at least one hour, and during this time they equilibrated to room temperature. During recordings the slices were transferred to a recording chamber (model RC-27, Warner Instruments) and perfused with the same ‘‘1–3 ACSF”. In all recordings the solution was warmed to at least 34 °C prior to perfusion in the chamber. However, we later discovered that the solution cooled significantly during perfusion through the chamber, producing a chamber temperature that was essentially room temperature (between 22 and 27 °C). Epileptiform events (EEs) were evoked pharmacologically by switching to a ‘‘2-1 BIC ACSF” that was the same as ‘‘1–3 ACSF” except for (in mM): 2 calcium chloride, 1 magnesium sulfate, and 0.02 bicuculline.
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In vitro callosotomy
Precut. In a subset of experiments the corpus callosum was severed by hand using a surgical scalpel blade #15 (EMS, Hatfield, PA) while the slice was in the incubation bath, just before it was placed in the recording chamber. The cut was aligned with the central fissure to create a symmetrical cut and avoid any damage to cortex. The slice was examined visually in the incubation chamber and again at 20x magnification to verify a complete bisection.
Midcut. This technique was an improved version of the callosal bisection described in Walker et al., 2012). Following at least an hour of extracellular recordings, the callosum in the slice was bisected by lowering a sapphire blade (WPI, Sarasota, FL) attached to one of the electrode manipulators. In these experiments the slice rested on a vinyl sheet (Plastic Film Corp. of America, 1.5H DPC .010 5400 4200105821) to allow for the sapphire blade to follow through on its cut into the vinyl sheet below. The recordings continued for an additional hour following the cut. The slice was examined after the experiment to ensure that the callosum was fully severed by flipping the slice over and inspecting the underside to ensure a complete cut.
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Light placement. It is important to note how and when light was used in these experiments. When ‘‘light” is discussed, we are referring to the condenser light used to illuminate brain slices for IR-DIC recordings (powered by a 100 W, 12 V halogen bulb in our Olympus BX51 microscope). A 775 nm infrared filter and a polarizing filter were applied to the light in order to create optimal visualization of the slice. Light was either in a central position on the central fissure (Fig. 1 experiments) or lateralized (such as experiments described as LB34, where ‘‘LB” means ‘‘Light bias”). The diameter of the light was between 0.5 and 1.0 mm. In ‘‘moving light” experiments, slices were bathed in bicuculline for 2 h with a lateral light position. After the first hour of the experiment, the light was moved to the contralateral hemisphere.
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Extracellular recordings. We recorded brain slices extracellularly as described previously (Walker et al., 2012). Electrodes were made with borosilicate glass capillary tubes (Sutter instruments) pulled to resistances of 1–3 MX, and electrodes were filled with ACSF. Extracellular signals were amplified 100 using patch clamp amplifier model-2400 (A-M Systems, Inc., Carlsborg, WA, USA), and routed to a personal computer through ITC18 computer interface (Instrutech Corporation, Port Washington, NY, USA). Each electrode was placed in opposite hemispheres. Electrodes were placed equidistant from the interhemispheric tissue in layer 2 and approximately 0.2 mm dorsal to the white matter of the callosum (see Fig. 2 of Walker et al., 2012). Analog signals were digitized at 5 kHz with an Instrutech digitizer and acquired with IGOR software.
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Data analysis of electrophysiology. EEs were detected by combining 2nd derivative analysis of the waveforms in addition to amplitude thresholds, using custom macros written in IGOR (WaveMetrics). Thus, events with strong deflections with regards to slope and amplitude were detected. Detected events were checked by eye against the original recordings to ensure the elimination of false positives and false negatives. The time delays between EEs on either side were calculated (interhemispheric latencies, IHLs). An EE was determined to be ‘‘bilateral” if there was an IHL less than 200 ms. A minimum of 10 bilateral EEs per
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Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Fig. 1. Slices bathed in bicuculline demonstrate directionality with respect to EE propagation. (A) Two examples of 10 min of data collected from hour-long extracellular EE recordings are shown, where a representative example on the left is highly directional, and a rare sample from a recording on the right displays almost no directionality. In each case, the time when each EE occurs is recorded in both hemispheres, and if the latency between those events is relatively short (i.e., less than 200 ms), then it is classified as a bilateral EE that propagated from one hemisphere to the contralateral side. The latency is calculated for each propagating EE and plotted as the interhemispheric latency (IHL) on the y-axis, with the time of EE occurrence on the x-axis. Picking one hemisphere as a reference point, we can then assign values for IHLs, such that a propagating EE that begins in the left hemisphere is denoted as a dot on the L > R side of the y-axis. As shown in this example, there was a strong directionality in that 95% of all propagating EEs began in the left hemisphere. (B) Black dots depicting directionality values, each from 10 different hour long recordings in bicuculline, are ordered left to right from lowest directionality value to highest. Grey dots are from 1000 surrogate data sets based on the same number of experiments (n = 10) and the same number of bilateral EEs from each respective experiment, but each surrogate data set assumes no directionality bias (i.e., every EE has 0.5 probability of originating from the left hemisphere). Each surrogate data set is rank ordered as in the actual data set. (C, left) Directionality measures for all ten slices are shown again as single dots, along with a boxplot and whisker diagram illustrating the range, median, and first and third quartiles of the data. (C, right) While these slices demonstrated significant directionality, there was no bias in that directionality, meaning that neither hemisphere (left or right) was more likely to be the leading hemisphere (p = 0.49 sign rank test for null hypothesis that median = 0.5; p = 1.0 sign test for same hypothesis). For these experiments, the light source was placed between both hemispheres, so that no bias from that source could influence activity. 171 172 173 174 175 176 177 178
hour were required for inclusion in further analysis. In some cases where high rates of EEs occurred (>200/h) a shift predictor was used to determine whether designating bilaterality was justified, as the probability of two EEs from opposite hemispheres occurring within 200 ms increases as the total number of EEs increases. This shift predicting algorithm shifts the time of occurrences for all EEs in one recording by 500 ms,
which should greatly reduce the number of detected bilateral EEs. If such a reduction does not occur, then the hypothesis that IHLs are produced via callosal propagation is undermined and the recording is not eligible for measuring IHLs.
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Surrogate data sets. Directionality is defined as the fraction of bilateral EEs that propagate in the majority
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Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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direction, with the lowest value possible being 0.5, and the highest 1.0. Surrogate data sets were created using IGOR macros with the assumption that an individual EE had equal probability of originating in either hemisphere (i.e., 0.5 probability). These surrogate data sets were based on the actual number of EEs in each of the 10 recordings. Nichrome wire heater. A microheater was designed by running 0.7 A of current through a small loop of nickel chromium wire (Nichrome 30 gauge AWG wire). The length of exposed wire was about 3 mm, and the wire was bent in a narrow u-shape so that the length of exposed heater surface was approximately 1 mm. This heater was powered by a stack of D cell batteries, and current was controlled by a potentiometer. The heater was attached to an electrode manipulator and manually controlled. The slice was placed in the recording chamber, and electrodes placed in each hemisphere. Once electrodes were in place, the heater was lowered down to rest just above the slice and turned on to apply heat to the area. The infrared light was turned off to remove any additional heating variables provided by the light source. Subsequent experiments determined that the temperature difference induced was approximately 1 °C. In vitro slice temperature recordings. In a subset of experiments, very small temperature probes were attached to the recording electrodes using heat shrink tubing and small strips of Parafilm (Physiotemp Instruments, Clifton, NJ. TH-5 thermometer with IT-1E temperature probes). The tips of the temperature probes were thereby positioned within a millimeter of the tips of the extracellular electrodes, resting on the slice surface. The temperature probe on the illuminated side was always within the radius of the light column, allowing temperature difference recordings between the two hemispheres to an accuracy of 0.1 °C.
RESULTS
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We bathed slices in 20 lM bicuculline in order to evoke spontaneous EEs that in most cases propagated from one hemisphere to the other, and we labeled such transcallosally propagating EEs as ‘‘bilateral EEs”. In most of these slices it was the case that these bilateral EEs did not, as expected, arise with equal probability in each hemisphere. Rather, bilateral EEs were highly directional. We define ‘‘directional” numerically as the proportion of bilateral EEs that arise in the hemisphere more often serving as the source of bilateral EEs, with values ranging from 0.5 (equal numbers of bilateral EEs propagating in either direction) to 1.0 (Fig. 1A). Measured against the null hypothesis that either hemisphere has an equal probability of serving as the source of a bilateral EE, there was significantly high directionality observed. We support this statement with the construction of 1000 surrogate data sets. For these data sets we manufactured artificial data where the direction assigned for each IHL was randomly assigned with 0.5 probability of being either from left to right or
right to left. Each data set was composed of 10 experiments, each one representing the same number of IHLs detected in the real data (with the number of IHLs detected per experiment ranging from 13 to 135), and again, the direction of each IHL was determined with a probability of 0.5 (Fig. 1B). These surrogates addressed the null hypothesis that the directionality we observed could arise with a reasonable probability if the individual IHLs were in fact determined by 0.5 probability in either direction. As shown, the highest distribution of directionality values from these 1000 iterations was much less than the distribution of directionality values obtained from the real data. We then investigated whether the identity of the particular hemisphere (left vs. right) was more often the leader in experiments. We thus introduced a measure, ‘‘biased directionality” defined as the proportion of bilateral EEs that originate from a hemisphere determined a priori, in this case, the left hemisphere. There was no evidence for a significant hemispheric bias, as only 5 of 10 experiments showed the left hemisphere as being the leading hemisphere (Fig. 1C). For the above experiments the microscope condenser light was centered between the two hemispheres, so that no intentional temperature bias was introduced experimentally. For all the subsequent experiments described, the condenser light was placed over a single hemisphere, and the hemisphere chosen to be illuminated was usually switched with each new slice recording (i.e., neither favoring the left nor right hemisphere). This bandpass filtered (775 nm) and polarized light was the usual light used to visualize slices for intracellular recordings. We measured a temperature difference of approximately 0.1 °C by placing temperature probes in each hemisphere while illuminating a single hemisphere with the light (Fig. 2A). Repeating the same experiments where we measure bilateral EEs, we found that this temperature difference introduced a significant bias (Fig. 2B, C, ‘‘light bias at 27 °C” or ‘‘LB27”). In order to ensure that the directionality effect was due to temperature and not light, we fabricated a miniature nichrome wire heater, powered by a stack of D batteries and a potentiometer. Placing this nichrome heater just above the recording site of one hemisphere, we were able to increase the temperature of that area by approximately 1 °C during recordings where the condenser light was turned off. This produced an arguably larger effect than that from the light source, as every slice demonstrated a biased directionality greater than 0.5 (Fig. 2C, ‘‘Nich27”). We discovered during the course of measuring temperature in these slices that the slice temperature was almost room temperature despite heating the incoming ACSF (while the incoming ACSF was heated to 34 °C by an in-line heater, the temperature dropped to approximately 27 °C as it flowed across the relatively large chamber). Given the importance of temperature to these experiments, we incorporated a chamber heater to our rig, ensuring that the temperature in the slice was approximately 34 °C. We then repeated experiments
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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and confirmed a temperature difference bias in slices where the average temperature was 34 °C (Fig. 2C, ‘‘LB34”).
Moving light experiments: testing the stability of established bias How stable is the directionality in a slice? We conducted seven 2-h experiments where the light was focused on
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one hemisphere, creating a stable directional propagation from that lighted hemisphere to the dark hemisphere. At the end of 1 h, the light was then switched to the other hemisphere (Fig. 3). The recording with the least directionality was the only one that switched direction with the temperature switch (Fig. 3Aiii); otherwise, all other recordings displayed the same trend, even as one recording appeared perturbed by the switch (Fig. 3Aii).
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Fig. 3. After one hour in bicuculline, directionality is resistant to a reversal of light-induced temperature bias. (A(i)) The IHLs of a recording is shown, where in this case the directionality continued to increase despite the switch in temperature gradient. IHLs >200 ms are not considered propagating events, and thus termed ‘‘unilateral” (uni). (A(ii)) The IHLs of another recording where the temperature switch is coincident with a perturbation of IHLs. (A(iii)) The IHLs of the one and only case (out of 7) where directionality switched in tandem with temperature reversal, showing the appearance of a decisive increase in directionality in line with the new heat bias (directionality for this 2nd hour = 0.94). (B) Summary of seven two hour recordings in BIC, including the cases shown in (A(i–iii)) which are each labeled with (i)–(iii). Temperature directionality bias is shown on the yaxis, where 2 of the 7 recordings displayed a directionality that was against the temperature gradient (i.e., propagating from colder to warmer), and 5 of the 7 recordings displayed a directionality in line with the temperature gradient. After one hour, the light source was moved to the opposite hemisphere, switching the temperature bias. Following the switch, only one recording followed the change in temperature by switching directionality (iii), although that recording was the least directional of all recordings (directionality = 0.55). All of these temperature switch experiments were conducted at about 27 °C, as in LB27 recordings.
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Fig. 2. Microscope condenser light induces a 0.1 °C temperature difference between the hemispheres and a consequent directionality bias. (A) Temperature probes were implanted in each hemisphere at the same respective locations where electrodes would normally be placed to record EEs. The condenser light illuminated one hemisphere only at the 2 min intervals indicated. This produced an average temperature difference of approximately 0.1 °C, with the illuminated hemisphere as the warmer, indicated by red circle and red temperature recording trace. (During EE recordings, the condenser light was on non-stop for the duration of the recording.) (B) Representative example: temperature differences bias the directionality of propagating EEs. Five bilateral EEs are shown, with 4 out of 5 of them originating from the illuminated hemisphere. The bottom inset shows a temporally magnified view of the first of the five bilateral EEs, and a latency of about 50 ms can be seen in that view. (Bi) Several IHLs are shown, including the 5 selected for illustration above (indicated by black line). Propagation proceeds most often from the illuminated to nonilluminated hemisphere, as shown by the predominance of illuminated to non-illuminated IHLs (red dots). (C) Temperature differences significantly bias the directionality of propagating EEs. Three sets of experiments comprising 42 recordings are summarized. The description of each experiment is displayed as a cartoon slice, showing location of the light source, the average temperature difference between slices (D°C) and the average bath temperature (bath°C). Left (LB27): an average of 0.1 °C temperature difference between hemispheres, induced with the condenser light, yields a significant directionality bias during experiments conducted with an approximate bath temperature of 27 °C. Middle (Nich27): directly inducing an average 1.0 °C temperature difference with a miniature nichrome wire heater applied near the cortical surface produces significant directionality bias. Right (LB34): an average of 0.1 °C temperature difference between hemispheres, induced with condenser light, yields a significant directionality bias during experiments conducted with bath temperature of about 34 °C. In all panels, black dots indicate the directionality bias measured from each experiment while box plots show the respective range, median, and first and third quartiles. All biases were rated as having medians significantly greater than 0.5, as measured with a sign test (p < 0.05 for each, n = 16 for LB27, n = 10 for Nich27, n = 17 for LB34). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Midcut vs. precut experiments
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We investigated whether temperature influenced the determination of the leading hemisphere by affecting the rate of EEs. The idea here is that the hemisphere with the higher rate of EEs is more likely to lead the propagation, provided that an EE in one hemisphere produces an EE in the other hemisphere with high reliability, and assuming a refractory period for each EE.
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This is similar to a model of coupled nonlinear oscillators for lamprey swimming (Matsushima and Grillner, 1992), where the spinal segments with the faster rates lead the propagating wave, determining whether the lamprey swims forwards or backwards. While we can’t know the endogenous rate of EEs for both hemispheres while the callosum is intact (especially if the reliability of propagation is nearly 100%, as it is in many slices), we can bisect the callosum after one hour of propagating EEs and then measure the EE rates immediately after bisection (Fig. 4A). As before, we placed the light over one hemisphere and recorded EEs in the presence of 20 mM bicuculline, and in these Midcut experiments, we severed the callosum after this 1 hour, and then continued recording EEs from the separate hemispheres for another hour. Results supported the hypothesis, in that the hemisphere that was the illuminated (warmed) and usually leading hemisphere (in 8/10 experiments) was also the hemisphere that had a higher rate of EEs following callosal bisection (Fig. 4A and 4B). If the recordings are sorted by which hemisphere was leading, then the difference in EE rates after bisection is even more pronounced (Fig. 4B, right). In summary, the leading hemisphere almost always has a higher rate of EEs revealed postbisection (9/10 slices), and the temperature difference can strongly bias which hemisphere becomes the leading hemisphere (in 8/10 slices), resulting in a significant increase in EE rates postbisection for the warmed hemisphere. Regarding this latter result, it seemed hard to believe that such small temperature differences could account for such large differences in EE rates. We therefore performed a related experiment—the Precut experiment—which was identical to the Midcut except in the timing of manipulations, in that the callosum was severed immediately before recordings began (Fig. 4C). Thus, the hemispheres were never connected during the genesis of bicuculline induced EEs in the Precut experiments. In these experiments the recordings lasted
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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2 hours (same as Midcut), and we compared relative rates of EEs in Midcut to Precut during the 2nd hour of EEs. Results showed that the light was not able to produce a significant difference in EE rates if the callosum was bisected prior to EE generation (Fig. 4C), implying that intact callosal circuits amplified the temperature bias and were required to produce these rate differences.
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Potential role of inhibition
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One hypothesis for explaining the difference between Midcut and Precut experiments is that callosallydependent feedforward inhibition might have dampened activity in the relatively cooler side. We wondered whether there was some intact callosal feedforward inhibition that could have reduced rates of EEs in the cooler hemisphere while the callosum was propagating EE activity. In this hypothesis, the 0.1 °C warmer side has only a slightly increased rate of EE generation. However, because of feedforward inhibition, that slight advantage may be relatively augmented, as the cooler hemisphere would receive relatively increased inhibition from the warmed hemisphere, and in turn, would provide less inhibition to the warmed hemisphere. This hypothesis seems less likely in our seizure model using a GABA-A antagonist, but there are other sources of inhibition, such as GABAB receptors. Thus, we repeated the intact bicuculline experiments at 34 °C using bicuculline and a GABAB antagonist (2 lM CGP52432, i.e., ‘‘LB34CGP”). Compared to bicuculline alone, the bicuculline + CGP experiments did not produce significant light-biased directionality (Fig. 5A), providing support for this hypothesis. That BIC + CGP would produce a reduced feedforward inhibition was also indirectly supported by the significant decrease in inter-hemispheric latencies (IHLs) measured in BIC + CGP (Fig. 5B), as well as a significant reduction in the fraction of EEs that failed to propagate across the callosum (fraction unilateral, Fig. 5C). IHLs measure the time delay between EEs measured in opposite hemispheres in the slice, and reducing hindrances to seizure propagation have been shown to increase seizure propagation speeds (Trevelyan et al., 2007; Trevelyan and Schevon, 2013; Paz and Huguenard, 2015). Likewise, the fraction of uni-
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lateral EEs is a measure of unsuccessful seizure propagation, and one would expect that reducing hindrances to seizure propagation should decrease the number of unilateral EEs. Indeed, there is a correlation between the fraction of unilateral EEs and IHLs, as measured from the LB34 data (Fig. 5D).
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DISCUSSION
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Small temperature differences between hemispheres reliably produced a bias in the direction of EE propagation across the callosum in our slice preparation. Whether this phenomenon would also be found in vivo remains to be seen, as the cingulate cortex contains strong connections to subcortical areas that would likely influence ongoing activity. It is likely, however, that such temperature differences could exist between connected areas of the brain: temperature fluctuations in the range of a few tenths of a degree Celsius have been evoked between brain areas during sensory induced increases in brain activity (Delgado and Hanai, 1966; Melzack and Casey, 1967) and during paroxysmal activity (Freund et al., 1989). For cingulate cortex, it is not known whether one hemisphere could maintain significantly higher levels of activity relative to contralateral cortex that could produce temperature differences on the order of an hour time scale, but it seems unlikely. A more plausible scenario might be one in which one hemisphere is damaged to the extent that inflammation and angiogenesis is produced. Several hour-long localized temperature increases on the order of 0.5 °C have been observed in damaged brain tissue in humans (Karaszewski et al., 2009), and it has been shown that cooling cortical tissue can stop seizures in the cooled area and also prevent epileptogenesis (Yang and Rothman, 2001; Yang et al., 2002; Tanaka et al., 2008; Rothman, 2009). Our results suggest that the site of damage where a seizure focus is forming gains more traction in its ability to spread seizures beyond that focus if it remains at even a slightly higher temperature than its neighboring cortical circuits, even if those neighbors are in the opposite hemisphere. Our results also have implications with regard to brain stimulations studies as well as any brain tissue studies that use light. With regards to the former, it has been shown that clinical deep brain stimulation protocols may
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3 Fig. 4. Callosal bisection studies suggest that intact callosal circuits amplify the effects of temperature differences. (A) A representative example of a ‘‘Midcut” experiment is shown, whereby the callosum is severed with a sapphire blade after one hour of being bathed in bicuculline. Top traces are temporally amplified examples taken from the trace below, where the ‘‘CUT” shows when the callosum was bisected (at about 1 h into the recording). Following the cut, EEs from each hemisphere are no longer tightly coupled in time. The IHLs for each EE above are shown below the traces, and following the cut there are only unilateral events (‘‘uni” on the y-axis), indicating that IHLs are greater than 200 ms. Note that the rate of EEs is greater in the illuminated and leading hemisphere following the cut. (B) Midcut experiments: a summary of 10 Midcut experiments, an example of which is shown above in (A). The number of EEs recorded during the 2nd hour (post-bisection) from each hemisphere (light vs. dark) is shown for each recorded slice, with dotted lines connecting the numbers corresponding to each individual slice. The illuminated hemisphere had significantly more EEs than its contralateral dark hemisphere (*p < 0.05, Wilcoxon paired signed rank test). The right panel is the same data as in the left, but slightly reordered according to the leading (‘‘LEAD”) vs. lagging (‘‘LAG”) hemisphere with regard to propagating seizure direction. Note that only two brain slices needed to be reordered (i.e., the leading hemisphere was the illuminated hemisphere in 8/10 slices). Also note that in 9/10 slices that the leading hemisphere had a higher rate of EEs post-bisection (*p < 0.05, Wilcoxon paired signed rank test). (C) The only difference between Midcut and Precut experiments is that in Precut experiments the callosum is bisected immediately before the slice is placed in the chamber to record EEs. Thus, Precut slices never experience EEs with an intact callosum. In contrast to Midcuts, the warmer hemisphere does not have significantly more EEs than its contralateral hemisphere in the dark (p = 0.20, Wilcoxon paired signed rank test). All Midcut and Precut experiments were conducted at about 27 °C (as in LB27 experiments). Box plots show the respective range, median, and first and third quartiles.
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Fig. 5. GABAB inhibition influences callosal propagation and directionality biases. (A) Temperature directionality biases are not significant when GABAB receptors are blocked. Results are compared between bicuculline alone (LB34) and bicuculline with CGP (LBCGP34) during experiments with one hemisphere preferentially illuminated (light biased, LB). Data from LB34 is same as shown in Fig. 2B, and the format of data display is the same as that figure for this panel (*p < 0.05; n.s.: p = 0.58 for LBCGP34, sign test). (B) Blocking GABAB inhibition significantly lowers IHLs, measured from the same experiments with data displayed in (A) (*p < 0.01 rank sum test). (C) Likewise, the fraction of unilateral EEs decreases significantly during GABAB antagonism (*p < 0.01 Wilcoxon rank sum test). Box plots show the respective range, median, and first and third quartiles. (D) Data from LB34 shows that there is a significant correlation between the fraction of EEs that fail to propagate to the other hemisphere (fraction unilateral) and the average IHLs (p < 0.02, r = 0.57, Pearson correlation coefficient). Both measures may reflect levels of inhibition with regards to seizure propagation.
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cause local increases of brain temperature greater than 0.5 °C (Elwassif et al., 2006). Our results directly show that even low levels of condenser light can increase tissue temperature. Furthermore, in vivo studies show that optogenetic fiber optics can cause local temperature increases that are greater than 1.0 °C (Stujenske et al., 2015), and these temperature increases have measurable effects on neuronal activity (Owen et al., 2019). Given the careful studies documenting temperature effects on individual neurons, it is hard to imagine how 0.1 °C differences in temperature would have any effect (Trevelyan and Jack, 2002). Anterior cingulate cortex contain TRPV1 receptors that are sensitive to temperature, but again, whether they would be sensitive to such small temperature differences is uncertain (Steenland et al., 2006). However, it is conceivable that the coopera-
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tive activity of neuronal circuits could amplify indiscernible perturbations of individual neurons, and our findings suggesting that temperature-dependent directionality bias depends on intact callosal circuits supports that interpretation. As shown in Fig. 4, when we cut the callosum prior to recording, producing independent hemispheres, we see no statistically significant effect of temperature differences in these precut slices as compared to the midcut experiments, suggesting that callosal circuits are necessary for the temperature-mediated effects. As described in Fig. 4, the warmer hemisphere became the leader in 8/10 slices, and in 9/10 slices the leading hemisphere demonstrated a higher rate of EEs post-bisection than the lagging hemisphere. These findings are also supportive of the idea that the leading hemisphere is in effect a pacemaker, having a higher endogenous rate of EEs that can be revealed following separation from other brain areas it entrains (Codadu et al., 2019). We also note that once a strong directionality bias is established, it can’t be easily reversed by switching the light source to the opposite hemisphere, implying a consolidation of directionality, possibly via synaptic plasticity mechanisms (Fig. 3). In previous work we documented the steady evolution of bilateral EEs throughout hour long recordings, with decreasing rates of unilateral EEs as well as decreasing IHLs. Those findings which are replicated in part in this study imply changes in network activity that occur well past the expected saturation time for bicuculline to soak into the slice (Walker et al., 2012), supporting a development of network changes that may underlie consolidation of directionality. Further investigation of these consolidation processes may be relevant to the study of how seizure trajectories are stereotyped in vivo (Jackson et al., 1994; Emerson et al., 1995; McCormick and Contreras, 2001; Schevon et al., 2012). The results from GABAB antagonists (Fig. 5) suggest feedforward inhibition as a mechanism for accentuating the differences of activity between hemispheres. Difference augmentation via synaptic inhibition has been showed in brain stem circuits with regards to interaural loudness differences in the superior olive (Tsuchitani, 1977; Moore and Caspary, 1983). Our study may be the first to implicate this kind of mechanism in cortical circuits. This suggestion is further supported by previous work documenting strong feedforward inhibition in these slices (Walker et al., 2012). We propose that callosal GABAB feedforward inhibition can increase the differences in excitability generated by the temperature difference. Bicuculline creates conditions of increased neuronal firing leading to recurring EEs are regular intervals. Bicuculline is known to induce long-term potentiation (LTP) in cortical circuits, which is likely due to the high levels of synchronous activity (Kanter and Haberly, 1993), and there is evidence for multiple forms of LTP in anterior cingulate cortex (Bliss et al., 2016). Furthermore, LTP can have widespread network effects that presumably can alter the dynamics of callosal circuits (Canals et al., 2009). As each hemisphere increases its respective activity rates, we propose that there are consequent LTP-mediated increases in excita-
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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tory synaptic strengths, furthering the general excitation in the hemisphere. As we showed previously, excitation in one hemisphere can result mostly in feedforward inhibition in the contralateral hemisphere (Walker et al., 2012), so that excitation in the one hemisphere could be weakened by excitation in the other. We imagine that one of the two hemispheres is bound to become dominant in this steady development of higher EE rates and mutual inhibition, thus producing strong directionality. Given this is scenario, it is likely that other small perturbations that are consistently applied to one hemisphere would also have outsized effects (different potassium concentrations, for example), such that temperature is just one means of producing a biased directionality in this model system. The hemisphere that becomes dominant seems to be random if no bias is introduced (as shown in Fig. 1C), although small randomly-assigned biases might exist in each slice due to inconsistent slicing, for example. In some cases, those random biases may be stronger than the bias introduced by a very small temperature difference, which could explain why a number of slices did not demonstrate temperature-biased directionality (as in Fig. 2C, especially in the 34 degrees slices with 0.1 temperature difference). Whatever random biases are introduced for each slice, however, it appears that the bias introduced by very small temperature differences can significantly tip the balance of dominance towards the warmer hemisphere.
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FUNDING
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Funding for this project supplied by Wesleyan University, and some of those funds were supplied via the Howard Hughes grant for Summer Research.
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ACKNOWLEDGEMENTS
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Cole Morrissette contributed data to the LB34 group of data. Conflict of interest: none declared.
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REFERENCES
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(Received 5 September 2019, Accepted 23 December 2019) (Available online xxxx)
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RESEARCH ARTICLE R. Cotter et al. / Neuroscience xxx (2020) xxx–xxx
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Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex
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R. Cotter, y S. Winnik y A. Singer and G. Aaron *
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Wesleyan University, Middletown, CT 06459, United States
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Abstract—We measured the sensitivity of cortical circuit activity to small differences in local cortical environments by studying how temperature affects the trajectory of epileptiform events (EEs). EEs evoked via blockade of GABA-A receptors were recorded extracellularly from mouse coronal brain slices containing both hemispheres of anterior cingulate cortex synaptically connected by corpus callosum axons. Preferentially illuminating one hemisphere with the microscope condenser produced temperature differences of 0.1 °C between the hemispheres. The relatively warmer hemisphere typically initiated the EEs that then propagated to the contralateral side, demonstrating temperature directed propagation. Severing the callosum following one hour of EEs showed that the warmer hemisphere possessed a higher rate of EE generation. Further experiments implied that intact callosal circuits were required for the increased EE generation in the warmer hemisphere. We propose a hypothesis whereby callosal circuits can amplify differences in respective hemispheric activity, promoting this directionality in seizure propagation. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.
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Key words: callosotomy, seizure, GABA, GABAB.
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INTRODUCTION
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Using multiple intracranial electrodes, several studies have demonstrated that seizures in the intact human brain follow stereotyped trajectories. That is, the direction of seizure propagation from one seizure to the next is not random, but rather follows a set pattern of trajectories (Jackson et al., 1994; Emerson et al., 1995; McCormick and Contreras, 2001; Schevon et al., 2012). These trajectories vary from patient to patient, just as the phenomenology of seizure patterns can vary between patients. How these patterns become stereotyped remains an open question. Our results show that this stereotypy in seizure propagation can be achieved within an hour between cortical areas in our reduced preparation and can be biased by very small temperature differences. Studying the dynamics of cortical propagation in the brain is difficult as the potential routes of propagation are numerous. This was shown most clearly in a study of seizure propagation between rostral cingulate cortices in a cat, where seizure propagation was maintained between cortices even after the corpus callosum was severed, thus implicating thalamic or brainstem routes
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of alternate propagation (Ralston, 1961). Our callosal preparation is ideal for studying propagation of neuronal activity from one cortical area to another as it retains synaptically coupled neurons as well as two separate cortical areas connected only by a tract of callosal axons that can be cut before or during recorded activity (Walker et al., 2012). The effects of temperature on neuronal circuits have been well documented: neuronal membrane potential, action potential propagation speed, and synaptic activity can all be directly affected (Thompson et al., 1985; Moser et al., 1993; Trevelyan and Jack, 2002; Long and Fee, 2008). In addition, the temperature of brain circuits can alter the frequency of oscillations and rate of activity produced by those circuits (Long and Fee, 2008; Reig et al., 2010; Aronov and Fee, 2012; Stujenske et al., 2015). All of these previous data on temperature effects in neuronal tissue were achieved using temperature perturbations of at least 1 °C, and such temperature differences can occur naturally in the brain between disparate areas due to differences in activity-dependent or, possibly, inflammation-dependent activity (Mrozek et al., 2012). To our knowledge, we are the first group documenting significant effects on neural circuits imposed by a temperature perturbation that is an order of magnitude smaller than that created by any previous study (i.e., 0.1 °C).
*Corresponding author. Address: Biology Department, 52 Lawn Avenue, Wesleyan University, Middletown, CT 06459, United States. E-mail address: [email protected] (G. Aaron). y These authors contributed equally. Abbreviations: ACSF, artificial cerebral spinal fluid; EEs, epileptiform events; IHLs, interhemispheric latencies. https://doi.org/10.1016/j.neuroscience.2019.12.041 0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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EXPERIMENTAL PROCEDURES
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All work involving mice in this study was approved by Wesleyan’s IACUC committee, in accordance with IACUC protocols. The protocol number for this study is 2015_0603_Aaron.
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Preparation of slices
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Slices were prepared as described previously (Walker et al., 2012). Black Swiss mice of both sexes (postnatal days 18–24) were injected intraperitoneally with a mixture of 120 mg/kg ketamine and 10 mg/kg xylazine. The deeply anesthetized mouse was decapitated and the brain was removed and placed in high-sucrose ice-cold artificial cerebral spinal fluid (ACSF) composed of (in mM): 3 magnesium sulfate, 1 calcium chloride, 222 sucrose, 27.1 sodium bicarbonate, 1.5 sodium phosphate, 2.6 potassium chloride, 3 myo-inositol, 2 sodium-pyruvate, 0.4 ascorbic acid, bubbled with carbogen gas (95% O2/5% CO2). The caudal blocking cut was made at a 6 degree angle, using a template blocking device to ensure a consistent blocking angle. 350 lm thick coronal slices were cut with a vibratome (Leica VT1000S). The first slice saved for recording was the first slice to show an intact corpus callosum, and identified as ‘‘slice 1”, the next slice as ‘‘slice 2”, etc., as slices were collected in the rostral to caudal direction. Slices were transferred to warm (34 °C), oxygenated ‘‘1–3 ACSF” immediately after slicing (in mM): 1 calcium chloride, 3 magnesium sulfate, 126 sodium chloride, 26 sodium bicarbonate, 1.1 sodium phosphate, 25 glucose, 3 potassium chloride, 3 myoinositol, 2 sodium-pyruvate, 0.4 ascorbic acid. The slices were left to recover for at least one hour, and during this time they equilibrated to room temperature. During recordings the slices were transferred to a recording chamber (model RC-27, Warner Instruments) and perfused with the same ‘‘1–3 ACSF”. In all recordings the solution was warmed to at least 34 °C prior to perfusion in the chamber. However, we later discovered that the solution cooled significantly during perfusion through the chamber, producing a chamber temperature that was essentially room temperature (between 22 and 27 °C). Epileptiform events (EEs) were evoked pharmacologically by switching to a ‘‘2-1 BIC ACSF” that was the same as ‘‘1–3 ACSF” except for (in mM): 2 calcium chloride, 1 magnesium sulfate, and 0.02 bicuculline.
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In vitro callosotomy
Precut. In a subset of experiments the corpus callosum was severed by hand using a surgical scalpel blade #15 (EMS, Hatfield, PA) while the slice was in the incubation bath, just before it was placed in the recording chamber. The cut was aligned with the central fissure to create a symmetrical cut and avoid any damage to cortex. The slice was examined visually in the incubation chamber and again at 20x magnification to verify a complete bisection.
Midcut. This technique was an improved version of the callosal bisection described in Walker et al., 2012). Following at least an hour of extracellular recordings, the callosum in the slice was bisected by lowering a sapphire blade (WPI, Sarasota, FL) attached to one of the electrode manipulators. In these experiments the slice rested on a vinyl sheet (Plastic Film Corp. of America, 1.5H DPC .010 5400 4200105821) to allow for the sapphire blade to follow through on its cut into the vinyl sheet below. The recordings continued for an additional hour following the cut. The slice was examined after the experiment to ensure that the callosum was fully severed by flipping the slice over and inspecting the underside to ensure a complete cut.
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Light placement. It is important to note how and when light was used in these experiments. When ‘‘light” is discussed, we are referring to the condenser light used to illuminate brain slices for IR-DIC recordings (powered by a 100 W, 12 V halogen bulb in our Olympus BX51 microscope). A 775 nm infrared filter and a polarizing filter were applied to the light in order to create optimal visualization of the slice. Light was either in a central position on the central fissure (Fig. 1 experiments) or lateralized (such as experiments described as LB34, where ‘‘LB” means ‘‘Light bias”). The diameter of the light was between 0.5 and 1.0 mm. In ‘‘moving light” experiments, slices were bathed in bicuculline for 2 h with a lateral light position. After the first hour of the experiment, the light was moved to the contralateral hemisphere.
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Extracellular recordings. We recorded brain slices extracellularly as described previously (Walker et al., 2012). Electrodes were made with borosilicate glass capillary tubes (Sutter instruments) pulled to resistances of 1–3 MX, and electrodes were filled with ACSF. Extracellular signals were amplified 100 using patch clamp amplifier model-2400 (A-M Systems, Inc., Carlsborg, WA, USA), and routed to a personal computer through ITC18 computer interface (Instrutech Corporation, Port Washington, NY, USA). Each electrode was placed in opposite hemispheres. Electrodes were placed equidistant from the interhemispheric tissue in layer 2 and approximately 0.2 mm dorsal to the white matter of the callosum (see Fig. 2 of Walker et al., 2012). Analog signals were digitized at 5 kHz with an Instrutech digitizer and acquired with IGOR software.
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Data analysis of electrophysiology. EEs were detected by combining 2nd derivative analysis of the waveforms in addition to amplitude thresholds, using custom macros written in IGOR (WaveMetrics). Thus, events with strong deflections with regards to slope and amplitude were detected. Detected events were checked by eye against the original recordings to ensure the elimination of false positives and false negatives. The time delays between EEs on either side were calculated (interhemispheric latencies, IHLs). An EE was determined to be ‘‘bilateral” if there was an IHL less than 200 ms. A minimum of 10 bilateral EEs per
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Fig. 1. Slices bathed in bicuculline demonstrate directionality with respect to EE propagation. (A) Two examples of 10 min of data collected from hour-long extracellular EE recordings are shown, where a representative example on the left is highly directional, and a rare sample from a recording on the right displays almost no directionality. In each case, the time when each EE occurs is recorded in both hemispheres, and if the latency between those events is relatively short (i.e., less than 200 ms), then it is classified as a bilateral EE that propagated from one hemisphere to the contralateral side. The latency is calculated for each propagating EE and plotted as the interhemispheric latency (IHL) on the y-axis, with the time of EE occurrence on the x-axis. Picking one hemisphere as a reference point, we can then assign values for IHLs, such that a propagating EE that begins in the left hemisphere is denoted as a dot on the L > R side of the y-axis. As shown in this example, there was a strong directionality in that 95% of all propagating EEs began in the left hemisphere. (B) Black dots depicting directionality values, each from 10 different hour long recordings in bicuculline, are ordered left to right from lowest directionality value to highest. Grey dots are from 1000 surrogate data sets based on the same number of experiments (n = 10) and the same number of bilateral EEs from each respective experiment, but each surrogate data set assumes no directionality bias (i.e., every EE has 0.5 probability of originating from the left hemisphere). Each surrogate data set is rank ordered as in the actual data set. (C, left) Directionality measures for all ten slices are shown again as single dots, along with a boxplot and whisker diagram illustrating the range, median, and first and third quartiles of the data. (C, right) While these slices demonstrated significant directionality, there was no bias in that directionality, meaning that neither hemisphere (left or right) was more likely to be the leading hemisphere (p = 0.49 sign rank test for null hypothesis that median = 0.5; p = 1.0 sign test for same hypothesis). For these experiments, the light source was placed between both hemispheres, so that no bias from that source could influence activity. 171 172 173 174 175 176 177 178
hour were required for inclusion in further analysis. In some cases where high rates of EEs occurred (>200/h) a shift predictor was used to determine whether designating bilaterality was justified, as the probability of two EEs from opposite hemispheres occurring within 200 ms increases as the total number of EEs increases. This shift predicting algorithm shifts the time of occurrences for all EEs in one recording by 500 ms,
which should greatly reduce the number of detected bilateral EEs. If such a reduction does not occur, then the hypothesis that IHLs are produced via callosal propagation is undermined and the recording is not eligible for measuring IHLs.
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Surrogate data sets. Directionality is defined as the fraction of bilateral EEs that propagate in the majority
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direction, with the lowest value possible being 0.5, and the highest 1.0. Surrogate data sets were created using IGOR macros with the assumption that an individual EE had equal probability of originating in either hemisphere (i.e., 0.5 probability). These surrogate data sets were based on the actual number of EEs in each of the 10 recordings. Nichrome wire heater. A microheater was designed by running 0.7 A of current through a small loop of nickel chromium wire (Nichrome 30 gauge AWG wire). The length of exposed wire was about 3 mm, and the wire was bent in a narrow u-shape so that the length of exposed heater surface was approximately 1 mm. This heater was powered by a stack of D cell batteries, and current was controlled by a potentiometer. The heater was attached to an electrode manipulator and manually controlled. The slice was placed in the recording chamber, and electrodes placed in each hemisphere. Once electrodes were in place, the heater was lowered down to rest just above the slice and turned on to apply heat to the area. The infrared light was turned off to remove any additional heating variables provided by the light source. Subsequent experiments determined that the temperature difference induced was approximately 1 °C. In vitro slice temperature recordings. In a subset of experiments, very small temperature probes were attached to the recording electrodes using heat shrink tubing and small strips of Parafilm (Physiotemp Instruments, Clifton, NJ. TH-5 thermometer with IT-1E temperature probes). The tips of the temperature probes were thereby positioned within a millimeter of the tips of the extracellular electrodes, resting on the slice surface. The temperature probe on the illuminated side was always within the radius of the light column, allowing temperature difference recordings between the two hemispheres to an accuracy of 0.1 °C.
RESULTS
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We bathed slices in 20 lM bicuculline in order to evoke spontaneous EEs that in most cases propagated from one hemisphere to the other, and we labeled such transcallosally propagating EEs as ‘‘bilateral EEs”. In most of these slices it was the case that these bilateral EEs did not, as expected, arise with equal probability in each hemisphere. Rather, bilateral EEs were highly directional. We define ‘‘directional” numerically as the proportion of bilateral EEs that arise in the hemisphere more often serving as the source of bilateral EEs, with values ranging from 0.5 (equal numbers of bilateral EEs propagating in either direction) to 1.0 (Fig. 1A). Measured against the null hypothesis that either hemisphere has an equal probability of serving as the source of a bilateral EE, there was significantly high directionality observed. We support this statement with the construction of 1000 surrogate data sets. For these data sets we manufactured artificial data where the direction assigned for each IHL was randomly assigned with 0.5 probability of being either from left to right or
right to left. Each data set was composed of 10 experiments, each one representing the same number of IHLs detected in the real data (with the number of IHLs detected per experiment ranging from 13 to 135), and again, the direction of each IHL was determined with a probability of 0.5 (Fig. 1B). These surrogates addressed the null hypothesis that the directionality we observed could arise with a reasonable probability if the individual IHLs were in fact determined by 0.5 probability in either direction. As shown, the highest distribution of directionality values from these 1000 iterations was much less than the distribution of directionality values obtained from the real data. We then investigated whether the identity of the particular hemisphere (left vs. right) was more often the leader in experiments. We thus introduced a measure, ‘‘biased directionality” defined as the proportion of bilateral EEs that originate from a hemisphere determined a priori, in this case, the left hemisphere. There was no evidence for a significant hemispheric bias, as only 5 of 10 experiments showed the left hemisphere as being the leading hemisphere (Fig. 1C). For the above experiments the microscope condenser light was centered between the two hemispheres, so that no intentional temperature bias was introduced experimentally. For all the subsequent experiments described, the condenser light was placed over a single hemisphere, and the hemisphere chosen to be illuminated was usually switched with each new slice recording (i.e., neither favoring the left nor right hemisphere). This bandpass filtered (775 nm) and polarized light was the usual light used to visualize slices for intracellular recordings. We measured a temperature difference of approximately 0.1 °C by placing temperature probes in each hemisphere while illuminating a single hemisphere with the light (Fig. 2A). Repeating the same experiments where we measure bilateral EEs, we found that this temperature difference introduced a significant bias (Fig. 2B, C, ‘‘light bias at 27 °C” or ‘‘LB27”). In order to ensure that the directionality effect was due to temperature and not light, we fabricated a miniature nichrome wire heater, powered by a stack of D batteries and a potentiometer. Placing this nichrome heater just above the recording site of one hemisphere, we were able to increase the temperature of that area by approximately 1 °C during recordings where the condenser light was turned off. This produced an arguably larger effect than that from the light source, as every slice demonstrated a biased directionality greater than 0.5 (Fig. 2C, ‘‘Nich27”). We discovered during the course of measuring temperature in these slices that the slice temperature was almost room temperature despite heating the incoming ACSF (while the incoming ACSF was heated to 34 °C by an in-line heater, the temperature dropped to approximately 27 °C as it flowed across the relatively large chamber). Given the importance of temperature to these experiments, we incorporated a chamber heater to our rig, ensuring that the temperature in the slice was approximately 34 °C. We then repeated experiments
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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and confirmed a temperature difference bias in slices where the average temperature was 34 °C (Fig. 2C, ‘‘LB34”).
Moving light experiments: testing the stability of established bias How stable is the directionality in a slice? We conducted seven 2-h experiments where the light was focused on
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one hemisphere, creating a stable directional propagation from that lighted hemisphere to the dark hemisphere. At the end of 1 h, the light was then switched to the other hemisphere (Fig. 3). The recording with the least directionality was the only one that switched direction with the temperature switch (Fig. 3Aiii); otherwise, all other recordings displayed the same trend, even as one recording appeared perturbed by the switch (Fig. 3Aii).
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Fig. 3. After one hour in bicuculline, directionality is resistant to a reversal of light-induced temperature bias. (A(i)) The IHLs of a recording is shown, where in this case the directionality continued to increase despite the switch in temperature gradient. IHLs >200 ms are not considered propagating events, and thus termed ‘‘unilateral” (uni). (A(ii)) The IHLs of another recording where the temperature switch is coincident with a perturbation of IHLs. (A(iii)) The IHLs of the one and only case (out of 7) where directionality switched in tandem with temperature reversal, showing the appearance of a decisive increase in directionality in line with the new heat bias (directionality for this 2nd hour = 0.94). (B) Summary of seven two hour recordings in BIC, including the cases shown in (A(i–iii)) which are each labeled with (i)–(iii). Temperature directionality bias is shown on the yaxis, where 2 of the 7 recordings displayed a directionality that was against the temperature gradient (i.e., propagating from colder to warmer), and 5 of the 7 recordings displayed a directionality in line with the temperature gradient. After one hour, the light source was moved to the opposite hemisphere, switching the temperature bias. Following the switch, only one recording followed the change in temperature by switching directionality (iii), although that recording was the least directional of all recordings (directionality = 0.55). All of these temperature switch experiments were conducted at about 27 °C, as in LB27 recordings.
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Fig. 2. Microscope condenser light induces a 0.1 °C temperature difference between the hemispheres and a consequent directionality bias. (A) Temperature probes were implanted in each hemisphere at the same respective locations where electrodes would normally be placed to record EEs. The condenser light illuminated one hemisphere only at the 2 min intervals indicated. This produced an average temperature difference of approximately 0.1 °C, with the illuminated hemisphere as the warmer, indicated by red circle and red temperature recording trace. (During EE recordings, the condenser light was on non-stop for the duration of the recording.) (B) Representative example: temperature differences bias the directionality of propagating EEs. Five bilateral EEs are shown, with 4 out of 5 of them originating from the illuminated hemisphere. The bottom inset shows a temporally magnified view of the first of the five bilateral EEs, and a latency of about 50 ms can be seen in that view. (Bi) Several IHLs are shown, including the 5 selected for illustration above (indicated by black line). Propagation proceeds most often from the illuminated to nonilluminated hemisphere, as shown by the predominance of illuminated to non-illuminated IHLs (red dots). (C) Temperature differences significantly bias the directionality of propagating EEs. Three sets of experiments comprising 42 recordings are summarized. The description of each experiment is displayed as a cartoon slice, showing location of the light source, the average temperature difference between slices (D°C) and the average bath temperature (bath°C). Left (LB27): an average of 0.1 °C temperature difference between hemispheres, induced with the condenser light, yields a significant directionality bias during experiments conducted with an approximate bath temperature of 27 °C. Middle (Nich27): directly inducing an average 1.0 °C temperature difference with a miniature nichrome wire heater applied near the cortical surface produces significant directionality bias. Right (LB34): an average of 0.1 °C temperature difference between hemispheres, induced with condenser light, yields a significant directionality bias during experiments conducted with bath temperature of about 34 °C. In all panels, black dots indicate the directionality bias measured from each experiment while box plots show the respective range, median, and first and third quartiles. All biases were rated as having medians significantly greater than 0.5, as measured with a sign test (p < 0.05 for each, n = 16 for LB27, n = 10 for Nich27, n = 17 for LB34). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Midcut vs. precut experiments
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We investigated whether temperature influenced the determination of the leading hemisphere by affecting the rate of EEs. The idea here is that the hemisphere with the higher rate of EEs is more likely to lead the propagation, provided that an EE in one hemisphere produces an EE in the other hemisphere with high reliability, and assuming a refractory period for each EE.
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This is similar to a model of coupled nonlinear oscillators for lamprey swimming (Matsushima and Grillner, 1992), where the spinal segments with the faster rates lead the propagating wave, determining whether the lamprey swims forwards or backwards. While we can’t know the endogenous rate of EEs for both hemispheres while the callosum is intact (especially if the reliability of propagation is nearly 100%, as it is in many slices), we can bisect the callosum after one hour of propagating EEs and then measure the EE rates immediately after bisection (Fig. 4A). As before, we placed the light over one hemisphere and recorded EEs in the presence of 20 mM bicuculline, and in these Midcut experiments, we severed the callosum after this 1 hour, and then continued recording EEs from the separate hemispheres for another hour. Results supported the hypothesis, in that the hemisphere that was the illuminated (warmed) and usually leading hemisphere (in 8/10 experiments) was also the hemisphere that had a higher rate of EEs following callosal bisection (Fig. 4A and 4B). If the recordings are sorted by which hemisphere was leading, then the difference in EE rates after bisection is even more pronounced (Fig. 4B, right). In summary, the leading hemisphere almost always has a higher rate of EEs revealed postbisection (9/10 slices), and the temperature difference can strongly bias which hemisphere becomes the leading hemisphere (in 8/10 slices), resulting in a significant increase in EE rates postbisection for the warmed hemisphere. Regarding this latter result, it seemed hard to believe that such small temperature differences could account for such large differences in EE rates. We therefore performed a related experiment—the Precut experiment—which was identical to the Midcut except in the timing of manipulations, in that the callosum was severed immediately before recordings began (Fig. 4C). Thus, the hemispheres were never connected during the genesis of bicuculline induced EEs in the Precut experiments. In these experiments the recordings lasted
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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2 hours (same as Midcut), and we compared relative rates of EEs in Midcut to Precut during the 2nd hour of EEs. Results showed that the light was not able to produce a significant difference in EE rates if the callosum was bisected prior to EE generation (Fig. 4C), implying that intact callosal circuits amplified the temperature bias and were required to produce these rate differences.
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Potential role of inhibition
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One hypothesis for explaining the difference between Midcut and Precut experiments is that callosallydependent feedforward inhibition might have dampened activity in the relatively cooler side. We wondered whether there was some intact callosal feedforward inhibition that could have reduced rates of EEs in the cooler hemisphere while the callosum was propagating EE activity. In this hypothesis, the 0.1 °C warmer side has only a slightly increased rate of EE generation. However, because of feedforward inhibition, that slight advantage may be relatively augmented, as the cooler hemisphere would receive relatively increased inhibition from the warmed hemisphere, and in turn, would provide less inhibition to the warmed hemisphere. This hypothesis seems less likely in our seizure model using a GABA-A antagonist, but there are other sources of inhibition, such as GABAB receptors. Thus, we repeated the intact bicuculline experiments at 34 °C using bicuculline and a GABAB antagonist (2 lM CGP52432, i.e., ‘‘LB34CGP”). Compared to bicuculline alone, the bicuculline + CGP experiments did not produce significant light-biased directionality (Fig. 5A), providing support for this hypothesis. That BIC + CGP would produce a reduced feedforward inhibition was also indirectly supported by the significant decrease in inter-hemispheric latencies (IHLs) measured in BIC + CGP (Fig. 5B), as well as a significant reduction in the fraction of EEs that failed to propagate across the callosum (fraction unilateral, Fig. 5C). IHLs measure the time delay between EEs measured in opposite hemispheres in the slice, and reducing hindrances to seizure propagation have been shown to increase seizure propagation speeds (Trevelyan et al., 2007; Trevelyan and Schevon, 2013; Paz and Huguenard, 2015). Likewise, the fraction of uni-
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lateral EEs is a measure of unsuccessful seizure propagation, and one would expect that reducing hindrances to seizure propagation should decrease the number of unilateral EEs. Indeed, there is a correlation between the fraction of unilateral EEs and IHLs, as measured from the LB34 data (Fig. 5D).
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DISCUSSION
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Small temperature differences between hemispheres reliably produced a bias in the direction of EE propagation across the callosum in our slice preparation. Whether this phenomenon would also be found in vivo remains to be seen, as the cingulate cortex contains strong connections to subcortical areas that would likely influence ongoing activity. It is likely, however, that such temperature differences could exist between connected areas of the brain: temperature fluctuations in the range of a few tenths of a degree Celsius have been evoked between brain areas during sensory induced increases in brain activity (Delgado and Hanai, 1966; Melzack and Casey, 1967) and during paroxysmal activity (Freund et al., 1989). For cingulate cortex, it is not known whether one hemisphere could maintain significantly higher levels of activity relative to contralateral cortex that could produce temperature differences on the order of an hour time scale, but it seems unlikely. A more plausible scenario might be one in which one hemisphere is damaged to the extent that inflammation and angiogenesis is produced. Several hour-long localized temperature increases on the order of 0.5 °C have been observed in damaged brain tissue in humans (Karaszewski et al., 2009), and it has been shown that cooling cortical tissue can stop seizures in the cooled area and also prevent epileptogenesis (Yang and Rothman, 2001; Yang et al., 2002; Tanaka et al., 2008; Rothman, 2009). Our results suggest that the site of damage where a seizure focus is forming gains more traction in its ability to spread seizures beyond that focus if it remains at even a slightly higher temperature than its neighboring cortical circuits, even if those neighbors are in the opposite hemisphere. Our results also have implications with regard to brain stimulations studies as well as any brain tissue studies that use light. With regards to the former, it has been shown that clinical deep brain stimulation protocols may
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3 Fig. 4. Callosal bisection studies suggest that intact callosal circuits amplify the effects of temperature differences. (A) A representative example of a ‘‘Midcut” experiment is shown, whereby the callosum is severed with a sapphire blade after one hour of being bathed in bicuculline. Top traces are temporally amplified examples taken from the trace below, where the ‘‘CUT” shows when the callosum was bisected (at about 1 h into the recording). Following the cut, EEs from each hemisphere are no longer tightly coupled in time. The IHLs for each EE above are shown below the traces, and following the cut there are only unilateral events (‘‘uni” on the y-axis), indicating that IHLs are greater than 200 ms. Note that the rate of EEs is greater in the illuminated and leading hemisphere following the cut. (B) Midcut experiments: a summary of 10 Midcut experiments, an example of which is shown above in (A). The number of EEs recorded during the 2nd hour (post-bisection) from each hemisphere (light vs. dark) is shown for each recorded slice, with dotted lines connecting the numbers corresponding to each individual slice. The illuminated hemisphere had significantly more EEs than its contralateral dark hemisphere (*p < 0.05, Wilcoxon paired signed rank test). The right panel is the same data as in the left, but slightly reordered according to the leading (‘‘LEAD”) vs. lagging (‘‘LAG”) hemisphere with regard to propagating seizure direction. Note that only two brain slices needed to be reordered (i.e., the leading hemisphere was the illuminated hemisphere in 8/10 slices). Also note that in 9/10 slices that the leading hemisphere had a higher rate of EEs post-bisection (*p < 0.05, Wilcoxon paired signed rank test). (C) The only difference between Midcut and Precut experiments is that in Precut experiments the callosum is bisected immediately before the slice is placed in the chamber to record EEs. Thus, Precut slices never experience EEs with an intact callosum. In contrast to Midcuts, the warmer hemisphere does not have significantly more EEs than its contralateral hemisphere in the dark (p = 0.20, Wilcoxon paired signed rank test). All Midcut and Precut experiments were conducted at about 27 °C (as in LB27 experiments). Box plots show the respective range, median, and first and third quartiles.
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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Fig. 5. GABAB inhibition influences callosal propagation and directionality biases. (A) Temperature directionality biases are not significant when GABAB receptors are blocked. Results are compared between bicuculline alone (LB34) and bicuculline with CGP (LBCGP34) during experiments with one hemisphere preferentially illuminated (light biased, LB). Data from LB34 is same as shown in Fig. 2B, and the format of data display is the same as that figure for this panel (*p < 0.05; n.s.: p = 0.58 for LBCGP34, sign test). (B) Blocking GABAB inhibition significantly lowers IHLs, measured from the same experiments with data displayed in (A) (*p < 0.01 rank sum test). (C) Likewise, the fraction of unilateral EEs decreases significantly during GABAB antagonism (*p < 0.01 Wilcoxon rank sum test). Box plots show the respective range, median, and first and third quartiles. (D) Data from LB34 shows that there is a significant correlation between the fraction of EEs that fail to propagate to the other hemisphere (fraction unilateral) and the average IHLs (p < 0.02, r = 0.57, Pearson correlation coefficient). Both measures may reflect levels of inhibition with regards to seizure propagation.
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cause local increases of brain temperature greater than 0.5 °C (Elwassif et al., 2006). Our results directly show that even low levels of condenser light can increase tissue temperature. Furthermore, in vivo studies show that optogenetic fiber optics can cause local temperature increases that are greater than 1.0 °C (Stujenske et al., 2015), and these temperature increases have measurable effects on neuronal activity (Owen et al., 2019). Given the careful studies documenting temperature effects on individual neurons, it is hard to imagine how 0.1 °C differences in temperature would have any effect (Trevelyan and Jack, 2002). Anterior cingulate cortex contain TRPV1 receptors that are sensitive to temperature, but again, whether they would be sensitive to such small temperature differences is uncertain (Steenland et al., 2006). However, it is conceivable that the coopera-
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tive activity of neuronal circuits could amplify indiscernible perturbations of individual neurons, and our findings suggesting that temperature-dependent directionality bias depends on intact callosal circuits supports that interpretation. As shown in Fig. 4, when we cut the callosum prior to recording, producing independent hemispheres, we see no statistically significant effect of temperature differences in these precut slices as compared to the midcut experiments, suggesting that callosal circuits are necessary for the temperature-mediated effects. As described in Fig. 4, the warmer hemisphere became the leader in 8/10 slices, and in 9/10 slices the leading hemisphere demonstrated a higher rate of EEs post-bisection than the lagging hemisphere. These findings are also supportive of the idea that the leading hemisphere is in effect a pacemaker, having a higher endogenous rate of EEs that can be revealed following separation from other brain areas it entrains (Codadu et al., 2019). We also note that once a strong directionality bias is established, it can’t be easily reversed by switching the light source to the opposite hemisphere, implying a consolidation of directionality, possibly via synaptic plasticity mechanisms (Fig. 3). In previous work we documented the steady evolution of bilateral EEs throughout hour long recordings, with decreasing rates of unilateral EEs as well as decreasing IHLs. Those findings which are replicated in part in this study imply changes in network activity that occur well past the expected saturation time for bicuculline to soak into the slice (Walker et al., 2012), supporting a development of network changes that may underlie consolidation of directionality. Further investigation of these consolidation processes may be relevant to the study of how seizure trajectories are stereotyped in vivo (Jackson et al., 1994; Emerson et al., 1995; McCormick and Contreras, 2001; Schevon et al., 2012). The results from GABAB antagonists (Fig. 5) suggest feedforward inhibition as a mechanism for accentuating the differences of activity between hemispheres. Difference augmentation via synaptic inhibition has been showed in brain stem circuits with regards to interaural loudness differences in the superior olive (Tsuchitani, 1977; Moore and Caspary, 1983). Our study may be the first to implicate this kind of mechanism in cortical circuits. This suggestion is further supported by previous work documenting strong feedforward inhibition in these slices (Walker et al., 2012). We propose that callosal GABAB feedforward inhibition can increase the differences in excitability generated by the temperature difference. Bicuculline creates conditions of increased neuronal firing leading to recurring EEs are regular intervals. Bicuculline is known to induce long-term potentiation (LTP) in cortical circuits, which is likely due to the high levels of synchronous activity (Kanter and Haberly, 1993), and there is evidence for multiple forms of LTP in anterior cingulate cortex (Bliss et al., 2016). Furthermore, LTP can have widespread network effects that presumably can alter the dynamics of callosal circuits (Canals et al., 2009). As each hemisphere increases its respective activity rates, we propose that there are consequent LTP-mediated increases in excita-
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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tory synaptic strengths, furthering the general excitation in the hemisphere. As we showed previously, excitation in one hemisphere can result mostly in feedforward inhibition in the contralateral hemisphere (Walker et al., 2012), so that excitation in the one hemisphere could be weakened by excitation in the other. We imagine that one of the two hemispheres is bound to become dominant in this steady development of higher EE rates and mutual inhibition, thus producing strong directionality. Given this is scenario, it is likely that other small perturbations that are consistently applied to one hemisphere would also have outsized effects (different potassium concentrations, for example), such that temperature is just one means of producing a biased directionality in this model system. The hemisphere that becomes dominant seems to be random if no bias is introduced (as shown in Fig. 1C), although small randomly-assigned biases might exist in each slice due to inconsistent slicing, for example. In some cases, those random biases may be stronger than the bias introduced by a very small temperature difference, which could explain why a number of slices did not demonstrate temperature-biased directionality (as in Fig. 2C, especially in the 34 degrees slices with 0.1 temperature difference). Whatever random biases are introduced for each slice, however, it appears that the bias introduced by very small temperature differences can significantly tip the balance of dominance towards the warmer hemisphere.
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FUNDING
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Funding for this project supplied by Wesleyan University, and some of those funds were supplied via the Howard Hughes grant for Summer Research.
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ACKNOWLEDGEMENTS
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Cole Morrissette contributed data to the LB34 group of data. Conflict of interest: none declared.
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REFERENCES
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Aronov D, Fee MS (2012) Natural changes in brain temperature underlie variations in song tempo during a mating behavior. PLoS ONE 7 e47856. Bliss TV, Collingridge GL, Kaang BK, Zhuo M (2016) Synaptic plasticity in the anterior cingulate cortex in acute and chronic pain. Nature Reviews Neuroscience 17:485–496. Canals S, Beyerlein M, Merkle H, Logothetis NK (2009) Functional MRI evidence for LTP-induced neural network reorganization. Curr Biol 19:398–403. Codadu NK, Parrish RR, Trevelyan AJ (2019) Region-specific differences and areal interactions underlying transitions in epileptiform activity. J Physiol-London 597:2079–2096. Delgado JM, Hanai T (1966) Intracerebral temperatures in freemoving cats. Am J Physiol 211:755–769. Elwassif MM, Kong Q, Vazquez M, Bikson M (2006) Bio-heat transfer model of deep brain stimulation-induced temperature changes. J Neural Eng 3:306–315. Emerson RG, Turner CA, Pedley TA, Walczak TS, Forgione M (1995) Propagation patterns of temporal spikes. Electroencephalogr Clin Neurophysiol 94:338–348. Freund TF, Buzsaki G, Prohaska OJ, Leon A, Somogyi P (1989) Simultaneous recording of local electrical activity, partial oxygen tension and temperature in the rat hippocampus with a chamber-
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Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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(Received 5 September 2019, Accepted 23 December 2019) (Available online xxxx)
Please cite this article in press as: Cotter R et al. Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.041
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