DOI 10.1007/s11055-015-0065-0 Neuroscience and Behavioral Physiology, Vol. 45, No. 3, March, 2015
Changes in the Functional Population Response of the Cat Primary Visual Cortex after Short-Term Injections of Propofol on the Background of Continuous Propofol Infusion V. S. Bugrova, R. S. Ivanov, and I. V. Bondar’
Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 99, No. 4, pp. 453–463, April, 2013. Original article submitted September 6, 2012. Revised version received December 3, 2012. The effects of systemic administration of the anesthetic propofol on the amplitude of the optical signal recorded from the neuron population of the primary visual cortex of the cat were studied. The study included development of an anesthesia protocol allowing maintenance of the required depth of anesthesia without introducing artefacts into the experimental data. Optical access to the cerebral cortex was gained at the beginning of the experiment. The effects of propofol on the functional responses of the neuron population were evaluated in the experiments by optical mapping using intrinsic signals. The study addressed the stability of the population response of the brain during the whole experiment and after single doses of propofol. The results showed that prolonged administration of propofol at a constant rate had no effect on the amplitude of the optical signal recorded from the cortical surface. On this background, single concentrated doses induced transient suppression of signal amplitude. Practical recommendations are provided for long-lasting acute neurophysiological experiments using propofol anesthesia. Keywords: propofol, optical recording using intrinsic signals, primary visual cortex, cat, neuron population, functional architecture of the cortex.
Studies of the properties of neurons and the functional architecture of the visual cortical analyzer constitute a difficult task, which has been made easier by the active development of brain mapping methods [2, 5, 6]. Significant advances in our understanding of the organization of working modules in the primary visual cortex have been obtained using optical mapping [3, 11]. This method, of which there are several variants, has high spatial resolution and, using voltage-sensitive dyes, can a be used to record neuron signals with an accuracy of 1 msec. The variant of the method based on the ability of active brain tissue to accumulate deoxyhemoglobin locally has been used widely. Accumulation of the reduced form of hemoglobin is accompanied by changes in the optical properties of the tissue, which can be recorded using highly sensitive CCD cameras.
As the accumulation of metabolites is a slow process (of the order of several seconds), optical mapping using intrinsic signals has low temporal resolution. In addition, the mapping signal can be impaired by noise from global vascular artefacts linked with the cardiac and respiratory cycles [3]. In the classical block scheme for optical mapping experiments, the effects of noise are reduced by synchronizing data recording with physiological cycles. A solution for dealing with a further source of artifacts – slow vasomotor activity [9] – was proposed by Kalatsky and consisted of prolonged signal recording accompanied by cyclic visual stimulation [7]. This approach opened up new potentials for using optical mapping, as it achieved a significant decrease in the time needed for mapping. This allowed testing and comparison of the magnitudes of the response characteristics of the neuron population in different and changing stimulation conditions [1, 7, 10]. However, changes in the animal’s physiological state induced by administration of anesthetics and substances maintaining anesthesia are of critical impor-
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia; e-mail:
[email protected].
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Changes in the Functional Population Response of the Cat Primary Visual Cortex tance in prolonged neurophysiological experiments. Such changes result from the accumulation of active agent in body tissues, leading to degraded execution of brain functions, to the extent of complete suppression. Two different types of anesthetic actions should be discriminated: long-term cumulative and acute, occurring when there is the need for a rapid increase in the depth of anesthesia. Both types of action can have significant effects on the quality of the experimental data. Propofol (2,6-diisopropylphenol) is widely used in non-Russian pharmacological practice, mainly in surgery, though this agent is gradually finding a place in the arsenal of researchers, including neurophysiologists [4]. This is a short-acting substance, used for general anesthesia (induction and maintenance of anesthesia) and sedation in intensive care and surgery. Propofol is not a pain-killer, so it has to be given in combination with analgesics. The literature currently lacks any clear answer to the question of the nature of the effect of propofol on neuron activity in brain mapping, while the duration of use is limited to six hours in experiments on mice [8]. The aim of the present work was to study the effects of propofol on the functional architecture of the primary visual cortex in cats, on the amplitude and intensity of neuron population responses to visual stimulation in long-term optical brain mapping experiments using different methods for substance administration. We needed to identify the optimum concentrations and administration schemes for the anesthetic to provide the required depth of anesthesia without affecting the quality of experimental data. This was addressed by measuring values reflecting the physiological state and depth of anesthesia of the animals during the experiment – heart rate, exhaled air CO2, and respiratory rate; we also analyzed functional maps of the cortical surface at different stages of the experiment. Methods The experimental protocols were approved by the Ethics Committee of the Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences and experiments were performed in compliance with the Directives of the Council of the European Community (86/609/EC) regarding the use of animals for experimental purposes. Acute prolonged experiments were performed on 19 adult cats (mean weight 3.1 kg; age from seven months to four years). Initial anesthesia was induced by administration of ketamine hydrochloride (10 mg/kg) combined with Vetranquil (5–10 mg/kg acepromazine). After analgesia (Arduan, 150–300 μg/kg), cats were intubated and placed on mechanical ventilation. Continuous i.v. infusions of physiological saline containing glucose (0.2%) and propofol (0.4%) at a rate of 1.5–2 ml/h were given to maintain a controlled level of anesthesia in the animals. Functional state was monitored in terms of exhaled air CO2 content (3.8–4.0%), blood oxygen saturation (99.0%), heart rate
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(120–160 bpm), and body temperature (38.5°C). Pupil size was stabilized with atropine (1%), the nictitating membrane was contracted with Neo-Synephrine (10%), and the eyelids were additionally retracted. Corneas were protected from drying using rigid contact lenses; one eye was covered with an opaque patch. Arduan was injected i.v. every 1.5–2.0 h for additional relaxation of the animal. The general anesthetic Butomidor (0.25–0.5 mg/kg) was given at the beginning of the experiment and every 6 h, mixed with atropine (1%); the anti-inflammatory agent dexamethasone (0.5–1.0 mg/kg) was given every 12 h. Continuous propofol infusions were used to maintain a constant level of anesthesia. Nonetheless, it was important for the experimental aims to maintain a level of anesthesia at which a borderline physiological state was produced in the animal, in which the cerebral cortex was maximally functional but the animal maintained the necessary level of anesthesia. In these anesthesia conditions, the animal remained in a stable state for 5–6 h [8], after which transient episodes of decreased (increased) levels of anesthesia could occur, apparent as increases (decreases) in HR and simultaneous increases (decreases) in exhaled air CO2. When this occurred, the level of anesthesia was corrected by temporary cessation of perfusion if the level of unconsciousness had deepened or additional concentrated propofol administration if the level of anesthesia had decreased. At the same time, on the bass of the suggestion that these necessary transient infusions of propofol might affect the functional state of the cortex at some subsequent time point, we also ran an experiment to detect such effects by transient administration of propofol in conditions of a stable level of anesthesia. A total of five cats were used in experiments lasting 1 h. The stimulus was a grid with a spatial frequency of 0.2 cycles/degree and contrast 100%. At 15 min after the beginning of the experiment, a single infusion dose of propofol (2–4 mg/kg) was given. Recording of the optical signal continued for a further 45 min to record any changes in activity over time. The moment of propofol delivery was positioned at the beginning of the 16th stimulation cycle; the experiment included a total of 60 stimulation cycles. Optical access to the cortex was obtained through a trepanned opening (diameter 16 mm) above field 17 of both hemispheres at the following Horsley-Clarke coordinates: center of opening at AP = –3 mm, ML = 0 mm. After removal of the dura mater, respiratory and cardiac pulsatile displacements of the cortex were eliminated by pouring in low melting temperature agarose (3%) and covering this with a cover slip. Scotopic monocular continuous stimulation was used. Visual stimuli were oriented grids (orientations were changed over the range 0–360°) with a rotation period of 60 sec and a spatial frequency of 0.2 cycles/degree. These grids produced optimal activation of fields 17 and 18 in the cat visual cortex. Visual stimuli were presented on a monitor screen positioned 57 cm from the animal’s eyes. The moni-
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Fig. 1. Functional maps (amplitude and phase) from a single cat obtained by stimulation with a grid with a spatial frequency of 0.2 cycles/degree and a contrast of 100%, with differences over 12 h. The dashed and dotted lines outline areas of interest used for further analysis. Lines show areas of interest corresponding to morphological visual fields 17 (dashed line) and 18 (dotted line); small squares of fixed size correspond to areas of interest located in active orientation columns. The size of each map corresponding to a cortical square of 12 × 12 mm. The occipital part of the brain is at the top.
tor was presented at the center of the visual field by projection ophthalmoscopy. A correcting lens was positioned in front of the animal’s eye. The visual stimulus was a rotating and simultaneously moving grid (0.5–0.94 degrees/sec) consisting of black and white bars with a spatial frequency of 1.6 cycles/degree. A CCD camera (Dalsa, USA; light-sensitive CCD matrix of 1024 × 1024 pixels; matrix size 12 × 12 mm) with a “macroscope” was used and an illuminator (630 µm) was placed above the trepanned opening and focused to a depth of about 700 μm from the surface of the cortical surface to avoid artifacts due to movement of blood through large vessels in the pia. The programmable apparatus for recording intrinsic optical signals was developed by VKImaging (USA). This method allows stimulation frequency to be selected, this being different from the frequency of physiological cycles respiratory, cardiac, and vasomotor) which might
influence the quality of functional maps. Fourier analysis was used to process the experimental data for reliable extraction of the mapping signal from the overall set of periodic signals contributing to the overall intensity of the light recorded from a defined tissue volume. Point-by-point analysis of images allowed plotting of phase- and amplitudebased functional maps of the cortex. Depending on the type of stimulation, phase maps of the visual cortex revealed functional modules involved in the analysis of information on stimulus orientation or stimulus movement direction, as well as encoding the position of the stimulus in the animal’s field of vision. Amplitude maps reflect the magnitude of the functional optical signal recorded from the neuron population in response to presentation of the visual stimulus. Figure 1 shows examples of amplitude and phase maps of the cortical surface. Each map was constructed as a result of the accumulation of experimental data over 15 stimula-
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Fig. 2. Changes in major physiological parameters in relation to time from the beginning of the experiment (heart rate (HR), exhaled air CO2 content, and respiratory rate). Arrows show the times of single doses of propofol.
tion cycles (15 min). Discontinuous lines are used to emphasize areas of interest – dashed lines for visual field 17 and dotted lines for visual field 18. Fields were identified in terms of differences in cortical activation by oriented grids with different spatial frequencies. In addition, fixed-size areas of interest corresponding to orientation columns were identified within field 18. Mean neuron response amplitudes were calculated for these areas and were used for subsequent analysis of the experimental data. Results and Discussion The age of the experimental animals was selected such that the visual area of the cortex was completely formed by the time the experiment began and the animals had sufficient visual experience to have developed the normal functional architecture. At the first stage of the experiment, we assessed correlations between physiological parameters on the background of continuous delivery of propofol at constant speed over a prolonged period. Figure 2 shows plots of relationships between physiological parameters, i.e., heart rate, the exhaled air CO2 concentration, and respiratory rate on the one hand and time on the other, from the beginning of the experiment. In this case (experiment c010), the cortical functional map recording duration was 24 h. The initial decrease in HR should be noted (Fig. 2, first plot) from a rate of the order of 200 bpm to 140–150 bpm. As a rule, this effect could be obtained with single doses of propofol
(2.5–4 mg/kg) on the background of constant administration at a rate of 3–7 mg/kg/h. Correction of the exhaled air CO2 level was performed as required by controlling respiratory rate on the mechanical ventilator, which kept the exhaled air CO2 concentration within the normal physiological range (3.8–4.2%). We have now accumulated a significant amount of data on the effects of propofol on physiological parameters during prolonged visual cortex optical mapping experiments. There is no doubt that the state of the animal during experiments depends on the individual characteristics of the body, so accurate measurement of body weight for accurate individual calculation of drug doses is extremely important. Monitoring of physiological parameters using instruments connected at earliest stages of the experiments provides for immediate evaluation of physiological characteristics of an individual animal and detection of the responses of individual cats to administration of propofol. This allows immediate corrections to be made to doses and the rate of delivery of perfusion solution containing the main anesthetic. Figure 3 shows plots of the relationship between HR in different animals and time from the beginning of the experiment. It follows from these plots that correct selection of the dose and rate of administration of propofol provides stable anesthesia and physiological state throughout prolonged experiments. Mean HR in our experiments was 140–150 bpm. However, values in some cases were even lower. Decreases in
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Fig. 3. Plots of changes in HR during entire experiments, four animals. Shaded rectangles in the lower plots show trace segments corresponding to 60-min experiments with acute administration of concentrated propofol solution.
the level of anesthesia were corrected by administration of additional single doses of 2–4 mg/kg/h of propofol. In the two lower plots, the segments during which 60min experiments were performed to study the effects of single supplementary doses of anesthetic on functional maps are highlighted. In one case, the action of a single dose was tested 10 h after the beginning of the experiment; in the other, effects were tested at 7 h. It should be noted that in the second case, the experiment was preceded by transient interruption of constant anesthetic delivery, which was expected to cause a jump in HR. However, single doses of propofol promoted reductions in HR to a level of about 150 bpm. Thus, propofol combined with Butomidor appears to be an effective combination of substances for prolonged neurophysiological experiments. The physiological state of the animal with this combination remains stable in the long term and, in addition, allows for supplementary correction of the level of anesthesia. As noted previously, it was critically important for us to recognize how the structure and amplitude of the response in the visual cortex change during prolonged experiments. This issue was resolved by selecting two maps for each animal, these being constructed at the beginning and end of the experiment in analogous visual stimulation conditions (Fig. 1). We are aware that the cortex is optimally
activated by contrast grids with a spatial frequency of 0.2 cycles/degree and a contrast of 100%. The mean interval between episodes of data collection for the maps being compared was 7–12 h. Detailed comparison of the maps in Fig. 1 shows that the overall structure of the functional maps did not change. On phase maps (Fig. 1, lower row), the structure of the activity pattern remained virtually unaltered, which is evidence that the recorded maps were stable. Similarly, the centers of hypercolumns (orientation “vertices;” the dark points shown by arrows) on amplitude maps occupied the corresponding positions on the two maps. Thus, at 12 h into the experiment, the overall structure of functional maps determined by the positions of orientation columns and the centers of hypercolumns remained unaltered. We also obtained similar results for functional maps from other animals. Thus, this result supplements published data [8] and shows that the functional structure of the visual cortex on the background of continuous perfusion of propofol remains stable in experiments lasting more than 12 h (in our case up to 32 h). Apart from the persistence of structure, the question of changes in the intensity of the cortical response to stimulation over a period of 12 h of continuous perfusion of anesthetic was of particular interest. We performed a special analysis of fixed areas of interest on maps obtained at the begin-
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Fig. 4. Histogram of the distribution of ratios of response intensity in areas of interest on two maps made at the initial and final stages of a prolonged experiment. Data were obtained in experiments on 10 animals and 200 fixed areas of interest.
Fig. 5. Changes in the ratio of cortical response amplitudes in areas of interest plotted for two functional maps obtained at different stages of the experiment addressing the effects of an acute dose of propofol. Drops in cortical activity were seen immediately after propofol administration. Light columns show data for areas of interest in the hemisphere contralateral to the stimulated eye, shaded columns to the ipsilateral hemisphere.
ning and end of the experiment. The mean amplitude of the response to stimulation was calculated for each area of interest. The ratio of the amplitudes of the responses for the same area on different maps at the beginning and at the end of the experiment was determined. Finally, distributions were calculated for these ratios. The distribution histograms in Fig. 4 show that the mean ratio of response amplitudes in these areas of interest on maps at the beginning and end of the experiment was unambiguously greater than unity. Thus, cortical response amplitude in analogous stimulation conditions increased by a factor of about 1.5: the mean value of the distribution was 1.74 and the median was 1.41. This result indicates that continuous and prolonged administration of propofol as anesthetic did not have inhibitory effects on the functional properties of the neuron population in the cat primary visual cortex. Functional maps retained their structure, while the amplitudes of optical signals changed only slightly, tending to increase. We approach interpretation of the increase in the response with caution, as we suggest that the cortex may be subject to the suppressive effect of the initial anesthetic at the beginning of the experiment, this effect disappearing after some period of time. Thus, we found that in conditions of constant-rate infusion of propofol throughout the study, the amplitude of the optical signal and the functional structure of the cortical map did not change. We then described the effects of single doses of concentrated propofol solution on the optical signal on the background of continuous infusion of the agent (which, as stated in the methods section, was often required for maintenance of
the required level of anesthesia). Administration of supplementary doses of propofol decreased cortical activity, which was recorded as an increase in the quantity of light reflected from the cortical surface as a result of a reduction in the deoxyhemoglobin concentration (Fig. 5). This decrease in functionality lasted around 10–15 min and was followed by slow recovery. Particular features of the cortical reaction were of note and may be associated with the individual physiological properties of different animals. Thus, at the qualitative level we found that acute doses of propofol required for maintaining a constant depth of anesthesia induced transient suppression of visual cortex functioning. Further analysis was directed to quantitative assessment of this effect. As in the case of studies of the amplitudes of responses at the beginning and end of the experiment, we used amplitude maps on which areas of interest of fixed size corresponding to orientation sensitivity columns were identified. The programming allowed sequential construction of four functional maps in a 1-h experiment, each of which was averaged for 15 stimulation cycles (Fig. 5). Mean response amplitudes were calculated for each area of interest. The ratio of the mean response amplitude in the map to the mean response amplitude in the first map obtained before acute administration of propofol was then calculated. Thus, the experimental data were normalized in relation to the initial signal level. The plot in Fig. 5 reflects the ratio of the amplitude of the cortical responses before and after administration of acute doses of propofol. At 15 min after administration of the anesthetic, we saw a drop in activity by 25–30% depending on
262 hemisphere. Activity decreased more in the ipsilateral hemisphere. This was followed by onset of a slow recovery of activity which continued 1 h after propofol administration. The time period selected for these experiments was insufficient to detect complete recovery of brain activity. Thus, we observed a critical drop in activity in the visual cortex after acute administration of propofol. Analysis of structural changes did not identify any significant impairments to functional maps. However, response amplitude decreased by 25–30%. This decrease in the cortical response may be critical in using optical mapping based on intrinsic signals and studies of cortical responses to changes in the parameters of the stimulus situation. A very important result was obtained, on the basis of which we can advise investigators using optical mapping based on intrinsic signals to interrupt the collection of experimental data during recovery of normal cortical functionality when supplementary doses of propofol are needed. Conclusions Use of the optical recording method to identify population neuron activity on the basis of intrinsic mapping signals opens new potentials for detecting the fine details of the functional architecture of the cerebral cortex. Thanks to the high spatial resolution and ability to support long-term experiments, this method provides the possibility for new evaluations of data from neurophysiological experiments and to supplement these data. As noted in this study, the method is based on the indirect extraction of data on the activity of the neuron population. An obligate condition for optical mapping experiments is provision of a constant level of anesthesia. As any anesthetic influences the whole body and especially the CNS, it is extremely important to select the optimum dose and administration protocol for appropriate anesthetics in order to obtain the greatest reduction in influences on the quality of the results obtained. The present study investigated the effects of prolonged administration of propofol both on the physiological state of animals during prolonged neurophysiological experiments and on the objectivity of the results obtained. We obtained experimental support for the view that the administration protocol used here provides the required stability
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