cortex during object localization

Transcription

1 1 The role of parvalbumin- positive interneurons in rodent primary somatosensory cortex during object localization NEUR 494 Honors Thesis Jason Strawbridge University of Southern California Faculty Advisor: Andrew Hires May 13 th, 2015

2 2 Abstract Our internal representation of the external world is based on patterns of neural activity in our cortex. These patterns are profoundly influenced by inhibitory neurons. The most numerous class of inhibitory neurons in the cortex express parvalbumin (PV+). The function of these neurons during active sensation is unknown. We sought to determine the role PV+ interneurons play in the cortical processing of the sense of touch in the mouse whisker system. We addressed how PV+ neuron activity shapes the activity patterns in primary somatosensory cortex (S1) associated with whisker- object contact and whisker motion during whisker- mediated object localization. We recorded whisker motion and object contact with 1ms precision, calculated sensory input, and correlated these variables spiking patterns in S1 neurons. We then optogentically silenced PV+ interneuron activity in the whisker representation of S1 during object localization and compared how the encoding of sensory and motor variables differed between these two states. Finally, we linked these changes in neural activity to shifts in perception of object location. This revealed underlying computational mechanisms by which object location is perceptually constructed from neural activity in S1. Introduction Sensation, or the initial stage in detection of stimulus properties, is a relatively well- understood physiological process. Humans, along with many other species, are able to sense the external world through a number of biochemical pathways activated by stimulus- driven receptors within the cells of sensory organs. The human finger, for example, contains multiple classes of mechanoreceptors that respond to varying degrees of touch, pressure, and vibration 1. Action potentials produced by these receptors are sent to the brain via the central nervous system, where they are processed in the somatosensory cortex in order to create a recognizable percept. Yet the actual process of perception, unlike sensation, is not clearly defined. Decoding the nature of conscious perception is especially difficult in somatosensory regions, where perceptions of sensory signals are built from neural activity patterns within complex parallel and hierarchical circuits. Some fundamental characteristics of processing within primary somatosensory cortex (S1) have been identified; previous research within S1 has revealed the neuronal connections underlying S1 circuits 2,3, and has established a morphological and physiological understanding of the neurons that make up these connections 4. However, the functional organization of circuits underlying perceptual processes in S1 has yet to be determined. One particularly significant obstacle in the way of understanding this functional organization is a lack of knowledge regarding the effects of interneuron influence within layers of somatosensory cortex. The construction of neural activity patterns is highly regulated by feed- forward inhibition from GABAergic interneurons. Previous work has identified parvalbumin- positive (PV+) interneurons as the primary source of this inhibition 5,6, and has revealed the mechanisms by which PV+ interneurons regulate excitatory spiking rates 7. Although these findings provide

3 3 valuable information about the synaptic activity of PV+ interneurons, a quantitative understanding of how these interneurons influence conscious perception has yet to be developed. We sought to shed light on the nature of touch perception by investigating the role that PV+ interneurons play in shaping touch- evoked neural activity patterns. We conducted our investigation of PV+ interneurons by performing electrophysiological and behavioral analysis in mice during optogenetic manipulation. Mice are ideal experimental subjects for the exploration of sensory processing mechanisms because of their cortical similarities to human models; many aspects of cortical circuits that process touch are conserved across both humans and mice 2. Additionally, the nature of whisker- based tactile mechanisms allows precise measurement of sensory input. Mice sweep their whiskers back- and- forth to detect, locate and identify objects 8,9, much like humans use their fingers. Precise measurement of touch input can be performed by tracking whisker deformations with high- speed video recording 10. Furthermore, the head- fixed procedures 11,12 and optogenetic techniques previously established in mice models mice offer a degree of experimental control necessary for the manipulation of specific behaviors and neuron subtypes. In mice, whiskers perform the duty of active touch sensation; signal transduction is mediated by mechanoreceptors within the whisker follicle 17. Inputs from sensory receptors within the whiskers ascend through the principal trigeminal nucleus to the ventral posterior medial (VPM) thalamic nucleus, where they are then relayed to the primary somatosensory cortex (S1) 18,19. Neuronal input from each individual whisker is processed topographically in a single barrel within S1 20. Intrinsic signal imaging of animals trimmed to a single whisker permits the identification of barrel circuitry based on whisker input and allows us to pinpoint the location of neural processing within S1 for individual whiskers 21. Furthermore, this mapping enables precise targeting during optogenetic manipulation, so that optogenetically- tagged cell types are only inhibited or activated within a specific cortical area 22. Previous research within rodent primary somatosensory cortex has revealed important information regarding the mechanisms underlying general tactile processing within barrel circuitry. The parallel and hierarchical processing streams that form the neural basis of touch perception operate between multiple layers of cortex within S1 2. Sensory input from whisker mechanoreceptors first arrives in layers 4 (L4) and 5B after ascending through the VPM of the thalamus 18,23. These signals ascend to L3 and then descend to the pyramidal neurons of L5B, which serve as the major output centers from S While the precise functions of neural activity patterns within L3 and 5B have yet to be determined conclusively, functional circuitry within L4 has been characterized in greater detail. In one experiment, optogenetic stimulation of L4 excitatory neurons in S1 using touch- like patterns evoked illusory perception of object location 22. Follow- up research has indicated that excitatory cells within L4 of S1 are actually able to represent the time of touch onset, strength, and direction with high precision 27 ; these results suggest

4 4 that L4 extracts a when and what touch signal from whisker input. Furthermore, these studies have found evidence supporting the idea that local feedfoward inhibition from PV+ interneurons cancels excitatory signals from the VPM generated by self- motion; suggesting that PV+ interneuron activity allows cells in L4 to discriminate the temporal and spatial nature of a stimulus 27. The implication that PV+ interneuron inhibition might regulate object localization 23 was the basis of our current investigation. While the results of previous experiments targeting excitatory cells and PV+ cells within L4 provided a clearer look at the mechanisms underlying tactile perception, the conclusions drawn from these results had notable limitations. We hoped to expand upon the results from earlier experiments while avoiding these limitations. A key distinguishing feature of our current research is the utilization of a conditional discrimination paradigm, an adaptation of the tactile discrimination paradigm used in previous experiments 18. In the experimental paradigm used during earlier research, mice were required to determine the spatial location of an object relative to their current position 18. However, sample object stimuli in these experiments were presented in only two positions (go and no- go); mice may have been able to solve the task by simply limiting their whisking range and subsequently identifying the presence of a pole within that range. As such, there is a possibility that these mice were not discriminating precise pole location and were performing the task without referencing an internal motor model. In comparison, mice performing a continuous discrimination task based on variable pole position (multiple go and no- go positions) are more likely to use an internal motor model that reflects the precise spatial location of the pole. In a continuous discrimination task, the degree of motor- referenced discrimination can be quantified by the difference in lick probability between go trials and no- go trials when at least one touch occurs during the trial. An important goal of our research was to quantify the mechanism by which PV+ interneuron inhibition influenced object perception. Conclusions from previous investigations of excitatory spiking in S1 have suggested that object location might be encoded by either excitatory spike count or excitatory spike pattern 18. An encoding method based on spike count implies that a greater number of excitatory spikes correspond to the perception of closer object location (due to a greater number of object touches), and that less spikes corresponds to a further location (less touches) 18. In contrast, an encoding method based on spike pattern implies that specific patterns of neural activity correspond to specific object locations in space 18. Our hypothesis was that mice would encode object location during the continuous discrimination task by counting the number of touch- evoked spikes in S1. In order to test this hypothesis, we altered both timing precision and number of touch- evoked spikes in S1 by inhibiting local PV+ inhibitory interneurons; we believed that silencing these cells would increase overall excitatory activity within S1, leading to a greater number of spikes, and shifting the animal s perception of object location to more proximal positions.

5 5 Results PV+ interneurons within somatosensory cortex fire in response to object contact and whisker movement Mice were trimmed to a single whisker (C2) in order to maintain precise stimulus control, and to ensure that sensory input during head- fixed behavior was introduced at distinct and quantifiable locations. Electrophysiological recordings were targeted to the C2 whisker representation within the barrel field via intrinsic signal imaging. Cell- attached pipette recordings were made within the C2 barrel in an effort to identify PV+ interneurons. PV+ interneurons were distinguished by their fast spike- rate and distinct waveforms. Figure 1. Left: microcircuit diagram of feed- forward inhibition from PV+ interneurons onto excitatory pyramidal cells within L4 of primary somatosensory cortex; recording electrodes were targeted to PV+ interneurons. Right: spike waveform used to distinguish whether cell recorded from was in fact a PV+ interneuron. Electrophysiological recordings of spiking activity during behavior from identified PV+ interneurons were paired with high- speed whisker tracking video in order to determine the relationship between PV+ cell activity and sensorimotor input. An increase in PV+ interneuron spike rate was observed during whisking behavior and was found to correlate with whisking strength. Furthermore, an increase in PV+ interneuron spike rate immediately following the onset of touch indicated a touch- aligned response from the cell. Touches with greater force demonstrated an enhanced spiking response compared to those with lesser force. Figure 2. Bottom (left): PV+ cell activity immediately followed object- touch. Bottom (right): Spike rate of PV+ cell increased with greater whisking strength. Top: PV+ cell spiking was greater when whiskers hit objects with higher impact force.

6 6 Optogenetic inhibition of PV+ interneurons increases the number of spikes from excitatory neurons within primary somatosensory cortex Cell- attached pipette recordings were made from excitatory cells within the C2 barrel of S1. Excitatory cells were identified by a short- latency spiking response to object touch and by their recognizable waveform. Initial recordings, in the absence of PV+ photoinhibition, served as a baseline for excitatory cell activity. Recordings were then made from excitatory cells during PV+ photoinhibition at varying light intensities of 5mW, 10mW, and 20mW. Selective transgenic expression of halorhodopsin, a light- gated chloride ion channel, permitted the optogenetic inhibition of PV+ interneurons. Recordings from excitatory neurons during photoinhibition of PV+ cells revealed an increase in excitatory spike rate during the illumination period. Furthermore, this increase in excitatory spiking that positively correlated with photoinhibition intensity; a slight increase in spike rate compared to baseline and controls (0mW) was observed at 5mW (p<0.002), and a large increase in spike rate was observed at 20mw (p<1e- 29). Figure 3. Left: Raster plot of excitatory spiking patterns in S1 during photoinhibition of PV+ cells at various intensities and during control trials (no photoinhibition). Right: Mean spike rate of excitatory cells increases during photoinhibition of PV+ cell activity. Higher spike rates correlate with higher photoinhibition intensities. Silencing the activity of PV+ interneurons shifts perception of pole location in the proximal direction during tactile discrimination Mice used as experimental subjects underwent roughly 2 weeks of behavioral training in order to learn the continuous discrimination task. In this task, mice were required to accurately determine which side of a virtual boundary a pole was presented on. Pole positions anterior to the virtual boundary represented no- go trials, during which the mice learned to refrain from licking. Conversely, pole positions posterior to the virtual boundary represented go trials, during which mice learned to lick, in order to obtain a water reward. Go and no- go trials were

7 7 presented with random but equal probability (50%), and pole position within both go and no- go ranges varied randomly along the anterior- posterior axis. Accurate hits during go trials led to water rewards, and false alarms during no- go trials led to time- outs (~3 seconds). Correct rejections during no- go trials were not rewarded (no water given), and misses during go trials were not punished (no timeout). Figure 4. Object localization task performed during behavioral experiments. Left: during go- trials, a pole is presented in locations closer to the animal s head; a licking response indicates perception of closer pole location and leads to a water reward. Right: during no- go trials, the pole is presented in farther locations; licking responses will lead to false alarms and time- outs. The absence of a licking response indicates perception of farther object location. Once subjects achieved a consistent, high level of accuracy during their training sessions (~75% accuracy or higher), we manipulated the activity of PV+ interneurons using photoinhibition of the targeted S1 area mapped during intrinsic signal imaging. Photoinhibition was delivered during roughly 20% of all trials within each behavioral session, and occurred pseudorandomly among trials. Performance curves for photoinhibition- mediated trials were plotted separately from the baseline curves in order to quantify the effects of PV+ inhibition on tactile discrimination ability. The intensity of the photoinihibiting pulse train remained fixed for the duration of a single behavioral session but varied between sessions; laser power alternated between 5mW, 10mW, and 20mW. Separate performance curves were plotted for each mw value in order to measure the relative behavioral differences between the varying intensities. Grand mean psychometric performance curves plotted for control trials (no photoinhibition) demonstrated that the subject was adept at performing the discrimination task. For trials in which the pole was in the most proximal positions, the probability of a lick was relatively high (70%). Lick probability decreased as the pole moved toward the virtual boundary, and remained relatively low for all trials in which the pole was in the no- go ranges (30%). Grand mean psychometric performance curves plotted during photoinhibition trials, however, exhibited a significantly different behavioral result. During photoinhibition trials, the probability of a lick was as high as 90% in the most proximal locations. This increase in lick probability was seen throughout the continuum, in both go and no- go ranges; during no- go trials, the subject s probability of licking rose to around 60-70%.

8 8 Figure 5. Lick probability during trials with various pole locations. During control trials (no photoinhibition, black dots), lick probability was relatively high in proximal pole locations and relatively low for distal pole locations. During PV+ cell photoinhibition trials (orange), lick probability was higher relative to control trials for all pole positions. Discussion The results obtained from electrophysiological recordings of PV+ interneurons within the C2 representation of the somatosensory cortex demonstrate the involvement of PV+ cell activity in whisker- mediated touch sensation. Furthermore, the recordings from excitatory neurons during PV+ cell inhibition reveal the importance of PV+ cell activity in regulating the neural firing patterns of excitatory neurons in S1. These results show that PV+ cells have the appropriate activity to regulate both the pattern and number of touch- evoked excitatory spikes in S1. The results of PV+ inhibition during tactile discrimination behavior reveal that the silencing of PV+ cell activity, and the increase in excitatory spike rate that follows, shifts the animal s perception of object location to more proximal positions. This suggests that the animal is encoding object location based on a spike- count mechanism, not a spike- pattern mechanism. If object location were encoded by the recognition of specific spike- patterns, then an increase in overall spike rate (as a result of PV+ cell inhibition) would disrupt the spike pattern and impair the animal s ability to discriminate pole location. In this scenario, we would expect to see a flattening of the psychometric performance curve; discriminatory ability would be impaired and performance would trend toward chance levels, regardless of pole location. However, performance curves plotted for photoinhibition trials showed a markedly different effect: photoinhibition increased lick probability among all trials, in both go and no- go ranges. These results, instead, support our original hypothesis that object location is encoded with spike count; an increase in spike rate during PV+ cell inhibition leads to an increase in lick probability because the animal is counting the number of S1 spikes in order to determine the precise spatial location of the pole.

9 9 Figure 6. Psychometric performance curves generated from PV+ cell photoinhibition during object localization behavior (right) more closely support a spatial encoding mechanism based on spike count in S1 (pink curve, right). Although our results provide evidence for a spike- count hypothesis, these results have a few significant limitations. First and foremost, the behavioral data gathered in our research comes from only a single mouse over the course of 5 photoinhibition sessions. In order to eliminate the possibility of subject- to- subject variability and provide more conclusive evidence for a spike- count hypothesis, we need to focus our efforts on obtaining similar data from additional trials across a larger number of mice. Additionally, future experiments should aim to quantify the manner in which spike patterns are changed during photoinhibition of PV+ interneurons; such quantification is necessary in order to completely exclude the possibility that mice might be using a pattern- based spike model for object location encoding. Finally, while these results do provide evidence for a hypothesized function of PV+ interneurons within primary somatosensory cortex, they are limited in the sense that they do not reveal information regarding PV+ interneuron function within specific layers of the cortex. Previous studies have suggested that excitatory neurons within somatosensory regions play layer- specific roles in sensory processing; future efforts to investigate the details of PV+ interneuron function should target individual layers in order to quantify potential differences between PV+ interneuron functions throughout the processing circuit. Methods Surgery for head- fixation Mice (~2-6 months old) were deeply anesthetized with 2% (volume in oxygen) isoflurane and then mounted into a stereotaxic apparatus on top of a heated blanket. After mice were fixed into the apparatus, isoflurane levels were lowered to 1.5%. The eyes were covered with petroleum jelly, and the scalp was cleaned with ethanol

10 10 and betadine. Topical anesthesia was achieved via the injection of a 0.5% marcaine solution under the scalp. Ketofen was injected subcutaneously to prevent inflammation and buprenorphine (opioid analgesic) was injected into the intraperitoneal cavity. A flap of skin (approximately 1cm) was removed from the dorsal skull, and any leftover periostium was removed. After the skull was sterilized with cotton swabs, bone was scraped with a scalpel to ensure proper bonding of glue. A thin layer of cyanoacrylic glue was placed onto the skull, and the head plate was placed directly on top of the glue. Dental acrylic was used to cover any exposed glue and to cement the head plate in the correct position. Intrinsic signal imaging In order to precisely target the primary somatosensory cortex during optogenetic stimulation, intrinsic signal optical imaging was performed on each mouse prior to behavioral training. These images, obtained by capturing small activity- based changes from the illuminated brain, enabled accurate morphological mapping of the dorsal cortex. Imaging was performed through the skull in animals anesthetized with isoflurane (1.5% volume in oxygen). Figure 7. Left: illustration of intrinsic signal imaging technique used to target the C2 whisker representation within primary somatosensory cortex. Middle: changes in oxygen content of hemoglobin are revealed during illumination with 640nm (red) light. Right: changes in blood volume allow imaging of arterial structure during illumination with 525nm (green) light. Water restriction In order to produce the necessary motivation required for behavioral training, mice were put on water restriction; this produced thirst- mediated motivation necessary for learning the task. Water restriction, as opposed to food restriction, was used as a motivator due to the significant number of health problems that have been documented in mice placed on food restriction in previous research. Water restriction typically began at least three days after surgery, allowing the mice

11 11 enough time to recover from the operation. During water restriction, mice were given exactly 1mL of water each day, which was dispensed into bowls inside the cages. Dry food was available to the mice in unlimited quantity. Each day, while mice were given water, each mouse was also scored on a health assessment sheet. Health scores were determined by monitoring 4 different aspects of mouse behavior and appearance. First, movement was observed and monitored for abnormal gait. Second, posture and grooming were assessed by taking note of any hunched stances or ruffled fur. Third, signs of eating and drinking were determined by observing the number of feces and urine traces in the cage. Fourth, signs of dehydration were monitored by pinching the fur of the mouse and looking for prolonged tenting of the fur upon its retraction. Finally, each mouse was weighed in order to determine whether or not the rate of weight loss was at an unhealthy level. Each health assessment category was given a number score, with higher numbers indicating unhealthy appearances or behavior. Mice that received a health score of 4 or greater were given an additional 0.5 ml of water for the day. Mice that received a health score of 6 or greater were removed from water restriction and given access to unlimited water. Mice that obtained water rewards via behavioral training were allowed to continue their training until they wanted to stop on their own volition. Mice that received less than 0.6 ml of water during behavioral training sessions were given supplemental water up to 0.6 ml. Head- fixation and behavioral set- up Mice perform tactile discrimination via whisker- mediated sensation; head- fixation of the mice ensured that the resting position of any given whisker was fixed at a single value, enabling the quantification of whisker movement. Prior to head- fixation, mice were placed into an acrylic tube so that movement of the body was limited. Then, in order to achieve head- fixation, the head plate was inserted into stainless steel holders inside the behavioral apparatus. The head plate was secured to the holders by the tightening of thumbscrews, which clamped the head plate into place. The acrylic tube containing the mouse was coated in aluminum foil and linked to a grounding wire, in order to provide an electrical connection between the mouse and a lickport placed roughly 1mm away from the mouth. Extension of the tongue and contact with the lickport effectively completed the electrical circuit and provided a signal to the behavioral software, indicating that a lick was made. The MATLAB- based BControl software was used for behavioral control and measurement. High- frame video acquisition of whisker movement was obtained using StreamPix software via a Basler 504k camera under 940nm illumination.

12 12 Precision tracking of whisker motion was achieved using the Janelia Whisker Tracker program. Figure 8. Head- fixed behavioral set- up for tactile discrimination paradigm. Behavioral facilitation, whisker imaging, photoinhibition, and electrophysiological recording can all be performed simultaneously when using head- fixed experimental paradigms. Optogenetic photoinhibition Optogenetic tagging of specific cell types permitted neural manipulation during behavior. PV- Cre knock- in mice were crossed to AI- 39 mice that expressed halorhodopsin in Cre, permitting the optogenetic manipulation of PV+ interneurons. Photoinhibition was remotely controlled along with behavior through the use of BControl software on a single computer. A separate computer was used to run Ephus software, which triggered high- speed video recording for each trial, and aligned video recording with photoinhibition pulses. Light pulses were delivered to target regions within S1 of the exposed cortex by a 473nm laser under the control of an acousto- optical modulator and a real- time Linux system. In order to prevent mice from responding to the sound of the laser shutter opening and not to the targeted photoinhibition itself, a sham pulse of roughly 0.1mW was delivered during non- inhibition trials onto the head plate of the mouse. Figure 9. Selective illumination of mouse cortex during delivery of a 4 second 10mW laser pulse. Intrinsic signal images allowed identification of C2 whisker representation in primary somatosensory cortex; photoinhibition was targeted to the identified representation.

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