Modulation of medial prefrontal cortical activity using in vivo recordings and optogenetics

The medial prefrontal cortex (mPFC) serves executive functions that are essential for selecting appropriate and inhibiting inappropriate actions. Prefrontal cortex dysfunction has been identified as a key neurobiological correlate of cognitive inflexibility and behavioral disinhibition associated with neuropsychiatric disorders such as drug addiction, obsessive-compulsive disorder, anxiety disorders and schizophrenia[1‐8]. The important role of the mPFC in top-down cognitive control mechanisms is particularly well documented in experimental models of behavioral “extinction” of negative emotions[9‐12].

The infralimbic region of the mPFC inhibits amygdala output to suppress (“extinguish”) aversive behaviors[10, 13‐18]. Increased thickness and activity of the mPFC correlate with successful extinction of negative emotions[19‐22] whereas decreased activity has been implicated in cognitive control deficits in models of extinction[23‐26] and behavioral disinhibition[2]. The concept of behavioral extinction forms the neurobiological basis for certain cognitive behavioral therapies in emotional-affective disorders[11, 27] and chronic pain[28]. Extinction deficits have been proposed as a mechanism of the persistence of pain and its negative affective dimension[29]. Abnormalities in the mPFC are found in human pain patients and in animal pain models[30, 31]. Our studies showed that amygdala-driven abnormal inhibition and decreased output of mPFC pyramidal cells contribute to pain-related impaired decision-making[32]. The underlying mechanism is feedforward inhibition of mPFC pyramidal cell output[32‐34].

Therefore, increasing mPFC output to engage cognitive control systems is a desirable goal in conditions and disorders that are associated with decreased mPFC activity, such as anxiety[35‐37], depression[38, 39] and pain[32]. Differential roles of infralimbic and prelimbic mPFC regions[23], diversity of excitatory and inhibitory neurons[40], and complex pharmacology and neurochemistry[41] present a challenge for the selective control of mPFC activity. Recently developed optogenetic tools allow the activation or inhibition of distinct neuronal cell types within defined brain regions[42, 43] but few studies have analyzed the effects of optogenetic manipulation of mPFC function. Optogenetic activation, but not inhibition, of excitatory mPFC cells inhibited unconditioned social exploratory behavior and fear conditioning[44] and had antidepressant-like effects[38]. Activation of GABAergic mPFC interneurons impaired operant delayed alternation performance[45] but rescued impaired social behavior due to abnormal mPFC excitation[44]. The neuronal effects of optical activation were confirmed using patch-clamp recordings in brain slices[44, 45] and activity markers[38]. Electrophysiological effects of optogenetic manipulations in the mPFC on neuronal activity in vivo were addressed only recently in studies that recorded multiunit activity[44, 46].

The novelty of the present study is the use of single-unit recordings in the intact animal to determine the effect of optical activation of infralimbic pyramidal output on spontaneous and evoked responses of infralimbic pyramidal cells, infralimbic interneurons and prelimbic pyramidal cells. This work is significant because infralimbic output to subcortical brain areas such as the amygdala plays a key role in fear extinction that might be utilized in the treatment of anxiety disorders such as PTSD[17, 23, 47].

The mPFC serves major executive functions and plays an important role in the modulation of emotional processing in subcortical centers such as the amygdala[53‐58]. The present study advances our knowledge about function and manipulation of mPFC neurons in several ways.

First, optogenetic stimulation of ChR2-expressing excitatory neurons in the IL produced not only excitation of IL pyramidal cells but also increased their responsiveness to excitatory inputs driven by peripheral mechanical stimuli. mPFC neurons receive multisensory including somatosensory and nociceptive information[52]. Physiological nociceptive signals that normally activate mPFC output cells[32] likely serve protective functions such as attention, awareness and appraisal[59‐61]. mPFC responses are believed to be related to the affective value of the stimulus which is consistent with the close reciprocal connections between mPFC and limbic forebrain structures such as the amygdala that provide emotion- and value-based information[32, 33, 51, 52]. Our data suggest that optical stimulation provides a tool to increase pyramidal output not only through direct excitation but also by facilitating afferent input to drive pyramidal output.

Second, our results directly demonstrate an inverse interaction between infra- and prelimbic mPFC regions. Activation of IL output inhibited PL pyramidal cells, which likely involved feedforward inhibition[62]. Differential roles of infralimbic and prelimbic mPFC regions have been proposed with regard to their modulation of emotional processing associated with conditioned fear[15, 23]. Specifically, IL plays a critical role in fear extinction likely through direct excitatory projections to a cluster of inhibitory neurons (intercalated cells) interposed between input and output regions of the amygdala[16]. Stimulation of IL facilitates extinction[63] and causes inhibition of amygdala output neurons[64]. Increased IL activity correlates with successful fear extinction[19, 22] and decreased IL activity with extinction deficits[23‐26]. In contrast, PL is involved in expression and renewal of fear[15, 23]. Stimulation of the PL results in freezing behavior (Vidal-Gonzalez et al., 2006) and increased activation of amygdala input neurons[14]. Evoked responses of PL neurons correlate with fear conditioning and persistent activity with extinction deficits[65] whereas PL inactivation impairs fear expression[23]. Our data show that IL activation can inhibit PL output, suggesting that IL-mediated extinction mechanisms may not only involve direct interactions with the amygdala but also control of PL-driven facilitatory influences on fear expression.

The results of this study are significant because impaired mPFC function is associated with several neuropsychiatric disorders[1‐8]. Modulating mPFC output may be utilized in the treatment of anxiety disorders such as PTSD[17, 23, 47] and optogenetic strategies to increase excitatory or inhibitory processes in the mPFC have been suggested as novel treatment strategies in neuropsychiatric disorders such as depression[66] and schizophrenia[67].

In a previous study we used electrical stimulation of labeled afferent fibers from the amygdala in the IL to show feedforward inhibition of PL pyramidal cells[32‐34]. While these data are not fully comparable with the results of optical stimulation in the IL in the present study, they do agree on the presence of a circuit involving the IL that leads to inhibition of PL pyramidal cells. There are likely different cortical and extracortical sources that can engage feedforward inhibition of PL neurons (and IL neurons for that matter); these cannot easily be distinguished using electrical stimulation which may activate interneurons, pyramidal cells or fibers of passage. In contrast, optogenetics-based stimulation allows the activation of a defined population of neurons (IL pyramidal cells in the present study), which is one of the key advantages of this technology that we used here to show IL-induced inhibition of PL pyramidal cells.

As a technical consideration, the differential effects of optical stimulation on different neuronal populations and the lack of effect in animals injected with control virus argue against nonspecific confounding factors, including heating as the result of high light power, toxicity at high expression levels or long-term expression, and transient changes in ion balance[43]. The stimulus intensities used in this study (1, 5 and 10 mW) are well within the suitable range for optogenetic control despite the stimulation of a small volume of tissue[38]. We used the channelrhodopsin variant ChR2(H134R) that is widely used and can drive precise low-frequency spike trains[50].

The electrophysiological in vivo results directly demonstrate the optogenetic modulation of mPFC activity in a region- and cell type-specific manner, which is significant because optogenetic tools may be useful in neuropsychiatric disorders associated with impaired mPFC function[43, 68]. The selective, controlled, cell type-specific intervention can provide important insights into neurobiological mechanisms of complex brain functions and disorders.

Our results show an inverse relationship between IL and PL, suggesting that some IL functions may not only involve direct projections to subcortical limbic structures but also engage inhibitory control over PL output. The effects on background and evoked activity suggest that optical stimulation provides a tool to increase pyramidal output not only through direct excitation but also by facilitating the effectiveness of afferent drives.