Effects of chronic intermittent ethanol exposure on orbitofrontal and medial prefrontal cortex-dependent behaviors in mice

In humans, stroke or trauma-induced damage to the orbitofrontal cortex (OFC) or medial prefrontal cortex (mPFC) results in impaired cognitive flexibility. Alcoholics also exhibit similar deficits in cognitive flexibility, suggesting that the OFC and mPFC are susceptible to alcohol-induced dysfunction. The present experiments investigated this issue using an attention set-shifting assay in ethanol dependent adult male C57BL/6J mice. Ethanol dependence was induced by exposing mice to repeated cycles of chronic intermittent ethanol (CIE) vapor inhalation. Behavioral testing was conducted 72 hours or 10 days following CIE exposure to determine whether ethanol-induced changes in OFC-dependent (reversal learning) and mPFC-dependent (set-shifting) behaviors are long lasting. During early ethanol abstinence (72 hrs), CIE mice showed reduced reversal learning performance as compared to controls. Reversal learning deficits were revealed as greater number of trials to criterion, more errors made, and a greater difficulty in performing a reversal learning task relative to baseline performance. Furthermore, the magnitude of the impairment was greater during reversal of a simple discrimination rather than reversal of an intra-dimensional shift. Reversal learning deficits were no longer present when mice were tested 10 days after CIE exposure, suggesting that ethanol-induced changes in OFC function can recover. Unexpectedly, performance on the set-shifting task was not impaired during abstinence from ethanol. These data suggest reversal learning, but not attention set-shifting, is transiently disrupted during short-term abstinence from CIE. Given that reversal learning requires an intact OFC, these findings support the idea that the OFC may be vulnerable to the cognitive impairing actions of ethanol.     

Dissociations between medial prefrontal cortical subregions in the modulation of learning and action

The medial prefrontal cortex (mPFC) has been implicated in various attentional functions. This experiment examined the involvement of mPFC subregions in the allocation of attention in learning and action as a function of the predictive accuracy of cues. Rats with dorsal (encompassing anterior cingulate, prelimbic, and infralimbic cortices) or ventral (encompassing mainly infralimbic and dorsopeduncular cortices and tenia tecta) mPFC lesions were trained in a multiple-choice discrimination task in which operant nosepoke responses to some visual cues were consistently (100%) reinforced (CRF) with food, whereas responses to other visual cues were partially (50%) reinforced (PRF). In challenge tests designed to assess attention in the control of action, responding was directed more to CRF cues than to PRF cues in sham and dorsal mPFC-lesioned rats, but ventral mPFC-lesioned rats showed similar levels of responding to both CRF and PRF cues. Nevertheless, when given a choice between simultaneously presented CRF and PRF cues in a cue competition test, all groups responded more to CRF cues. In a subsequent Pavlovian overshadowing phase designed to assess attention in the acquisition of new learning, previously trained CRF cues overshadowed conditioning to novel auditory cues more than did PRF cues in dorsal mPFC-lesioned rats, whereas the opposite pattern was observed in sham and ventral mPFC-lesioned rats. These results suggest a dissociation within the mPFC in the use of reinforcement prediction information to allocate attention for new learning and for the control of action.     

Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat

The medial prefrontal cortex (mPFC) has been associated with diverse functions including attentional processes, visceromotor activity, decision making, goal directed behavior, and working memory. Using retrograde tracing techniques, we examined, compared, and contrasted afferent projections to the four divisions of the mPFC in the rat: the medial (frontal) agranular (AGm), anterior cingulate (AC), prelimbic (PL), and infralimbic (IL) cortices. Each division of the mPFC receives a unique set of afferent projections. There is a shift dorsoventrally along the mPFC from predominantly sensorimotor input to the dorsal mPFC (AGm and dorsal AC) to primarily ‘limbic’ input to the ventral mPFC (PL and IL). The AGm and dorsal AC receive afferent projections from widespread areas of the cortex (and associated thalamic nuclei) representing all sensory modalities. This information is presumably integrated at, and utilized by, the dorsal mPFC in goal directed actions. In contrast with the dorsal mPFC, the ventral mPFC receives significantly less cortical input overall and afferents from limbic as opposed to sensorimotor regions of cortex. The main sources of afferent projections to PL/IL are from the orbitomedial prefrontal, agranular insular, perirhinal and entorhinal cortices, the hippocampus, the claustrum, the medial basal forebrain, the basal nuclei of amygdala, the midline thalamus and monoaminergic nuclei of the brainstem. With a few exceptions, there are few projections from the hypothalamus to the dorsal or ventral mPFC. Accordingly, subcortical limbic information mainly reaches the mPFC via the midline thalamus and basal nuclei of amygdala. As discussed herein, based on patterns of afferent (as well as efferent) projections, PL is positioned to serve a direct role in cognitive functions homologous to dorsolateral PFC of primates, whereas IL appears to represent a visceromotor center homologous to the orbitomedial PFC of primates.     

Naltrexone reverses age-induced cognitive deficits in rats

We evaluated young (3-4 months) and aged (22-24 months) male Sprague-Dawley rats in an attentional set-shifting procedure that assessed reversal, intradimensional shift (IDS), and extradimensional shift (EDS) discrimination learning tasks within one test session. These aspects of discrimination learning are sensitive to damage to distinct regions of frontal cortex. Compared to young animals, aged rats were significantly impaired on the EDS task and did not demonstrate significant impairment on the reversal or IDS tasks. The opioid antagonist naltrexone (2mg/kg, ip) was administered to young and aged rats prior to testing to assess possible improvements in aged-related cognitive impairments. Naltrexone (2mg/kg) attenuated the impairments in cognitive function in the EDS task for aged animals, but did not alter any task performance in the younger group. These results suggest that normal aging in rats is associated with impaired medial frontal cortex function as assessed by this attentional set-shifting procedure and opioid mediated mechanisms may represent a therapeutic target for drugs to improve cognitive deficits associated with aging.     

Differential laminar effects of amphetamine on prefrontal parvalbumin interneurons

The increase in excitatory outflow from the medial prefrontal cortex is critical to the development of sensitization to amphetamine. There is evidence that psychostimulant-induced changes in dopamine-GABA interactions are key to understanding the behaviorally sensitized response. The objective of this study was to characterize the effects of different amphetamine paradigms on the Fos activation of GABAergic interneurons that contain parvalbumin in the medial prefrontal cortex. Although a sensitizing, repeated regimen of amphetamine induced Fos in all cortical layers, only layer V parvalbumin-immunolabeled cells were activated in the infralimbic and prelimbic cortices. Repeated amphetamine treatment was also associated with a loss of parvalbumin immunoreactivity in layer V, but only in the prelimbic cortex. An acute amphetamine injection to naive rats was associated with an increase in Fos, but in parvalbumin-positive neurons of the prelimbic cortex, where it was preferentially induced in layer III. These data indicate that distinct substrates mediate the response to repeated or acute amphetamine treatment. They also suggest that a sensitizing amphetamine regimen directs medial prefrontal cortex (mPFC) outflow, via changes in inhibitory neuron activation, toward subcortical centers important in reward.     

The effect of catecholaminergic depletion within the prelimbic and infralimbic medial prefrontal cortex on recognition memory for recency, location, and objects

There is good evidence that the medial prefrontal cortex (mPFC) is involved in different aspects of recognition memory. However, the mPFC is a heterogeneous structure, and the contribution of the prelimbic (PL) and infralimbic (IL) cortices to recognition memory has not been investigated. Similarly, the role of different neuromodulators within the mPFC in these processes is poorly understood. To this end, we tested animals with 6-hydroxydopamine (6-OHDA) lesions of the PL and IL mPFC on three tests of object recognition memory that required judgments about recency, object location, and object identity. In the recency task, lesions to both PL and IL severely impaired animals' ability to differentiate between old (earlier presented) and recently presented familiar objects. Relative to sham and PL animals, the IL lesion also disrupted performance on the object location task. However, both lesions left novel object recognition intact. These data confirm previous reports that the mPFC is not required for discriminations based on the relative familiarity of individual objects. However, these results demonstrate that catecholamines within the PL cortex are crucial for relative recency judgments and suggest a possible role for neural processing within the IL in the integration of information about object location.     

Noradrenergic modulation of cognitive function in rat medial prefrontal cortex as measured by attentional set shifting capability

The brain noradrenergic system is thought to facilitate neuronal processes that promote behavioral activation, alertness, and attention. One region in which norepinephrine may exert such effects is the medial prefrontal cortex, which has been implicated in many cognitive functions including arousal, attention, motivation, working memory, response inhibition, and behavioral flexibility. The present study addressed the modulatory influence of noradrenergic neurotransmission in medial prefrontal cortex on cognitive function in rats, as measured by performance in an attentional set shifting task. In experiment 1, we tested effects of increasing and decreasing brain noradrenergic neurotransmission by systemic administration of the alpha2-adrenergic autoreceptor antagonist and agonist drugs, atipamezole and clonidine, respectively. Atipamezole pretreatment significantly improved performance on the stages of the attentional task requiring an extradimensional shift in attention, and those involving stimulus reversals, whereas clonidine had no effect at any stage. In experiment 2, we then tested effects of microinjecting alpha1- or beta-adrenergic receptor antagonists into medial prefrontal cortex on the enhancement of performance on the extradimensional task produced by atipamezole. The atipamezole-induced enhancement of performance on the extradimensional set shifting task was blocked by alpha1-, but not beta-adrenergic receptor antagonists in medial prefrontal cortex. Neither antagonist alone had any effect on extradimensional set shift performance in the absence of atipamezole-induced enhancement. These results indicate that elevating noradrenergic activity at alpha1-receptors in medial prefrontal cortex facilitates cognitive performance of rats in an attentional set-shifting task, which may contribute to the role of norepinephrine in behavioral state changes such as arousal, or to the beneficial cognitive effects of psychotherapeutic drugs that target noradrenergic neurotransmission.     

Glutamate receptors in the rat medial prefrontal cortex regulate set-shifting ability

The authors examined set-shifting abilities in rats injected with antagonists of N-methyl-D-aspartate (NMDA) receptors (MK801) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (LY293558) into the medial prefrontal cortex (mPFC). Set-shifting was assessed with a maze-based task requiring a switch between brightness and texture discrimination strategies. Intra-mPFC injection of MK801 prior to training on the 2nd discrimination impaired discrimination strategy acquisition. The MK801-induced deficit was due to increased perseverative responding. AMPA receptor blockade also impaired acquisition of the 2nd discrimination; these impairments were due to more general cognitive deficits. Results suggest that, within the mPFC, both AMPA and NMDA receptors are necessary for set-shifting, and that NMDA receptor hypofunction impairs the capacity to modify existing knowledge or to inhibit responses that are no longer appropriate.     

Catecholaminergic depletion within the prelimbic medial prefrontal cortex enhances latent inhibition

Latent inhibition (LI) refers to the reduction in conditioning to a stimulus that has received repeated non-reinforced pre-exposure. Investigations into the neural substrates of LI have focused on the nucleus accumbens (NAc) and its inputs from the hippocampal formation and adjacent cortical areas. Previous work has suggested that lesions to the medial prefrontal cortex (mPFC), another major source of input to the NAc, do not disrupt LI. However, a failure to observe disrupted LI does not preclude the possibility that a particular brain region is involved in the expression of LI. Moreover, the mPFC is a heterogeneous structure and there has been no investigation of a possible role of different regions within the mPFC in regulating LI under conditions that prevent LI in controls. Here, we tested whether 6-hydroxydopamine (6-OHDA)-induced lesions of dopamine (DA) terminals within the prelimbic (PL) and infralimbic (IL) mPFC would lead to the emergence of LI under conditions that do produce LI in controls (weak pre-exposure). LI was measured in a thirst motivated conditioned emotional response procedure with 10 pre-exposures to a noise conditioned stimulus (CS) and two conditioning trials. Sham-operated and IL-lesioned animals did not show LI and conditioned to the pre-exposed CS at comparable levels to the non-pre-exposed controls. 6-OHDA lesions to the PL, however, produced potentiation of LI. These results provide the first demonstration that the PL mPFC is a component of the neural circuitry underpinning LI.     

Cue familiarity is represented in monkey medial prefrontal cortex during visuomotor association learning

To examine functional roles of the medial prefrontal cortex (mPFC) in visuomotor association learning, neuronal activity in the mPFC of a behaving monkey was recorded during this learning. The monkey was presented a cueing visual stimulus, and required to push, pull or turn a manipulator according to the cue following a delay period. Under the control condition, three cues (circle, triangle and square) instructed the monkey to the three responses in a block of trials. After 2 months of training the animal was familiar with these cue-response associations. Under the learning condition, two of the three familiar cues and one novel cue were presented in a block. The monkey initially did not know what the novel cue instructed at first and learned a new cue-response association by trial and error. Neurons in the mPFC showed marked responses to cue presentation, and cue responses changed depending on whether cues were familiar or novel. A group of mPFC neurons responded to novel cues, but not to familiar cues. Another group of neurons responded to familiar cues, but not to novel cues. In a subgroup of these familiar cue-selective neurons, cue response was increased under the learning condition compared to the control condition. These results suggest that mPFC neurons differentiate between familiar and novel instructions, and that the neurons responsive to familiar stimuli enhance their modulations when both familiar and novel instructions have to be processed during task performance.     

Prefrontal cortical mechanisms underlying delayed alternation in mice

The prefrontal cortex (PFC) has been implicated in the maintenance of task-relevant information during goal-directed behavior. Using a combination of lesions, local inactivation, and optogenetics, we investigated the functional role of the medial prefrontal cortex (mPFC) in mice with a novel operant delayed alternation task. Task difficulty was manipulated by changing the duration of the delay between two sequential actions. In experiment 1, we showed that excitotoxic lesions of the mPFC impaired acquisition of delayed alternation with long delays (16 s), whereas lesions of the dorsal hippocampus and ventral striatum, areas connected with the PFC, did not produce any deficits. Lesions of dorsal hippocampus, however, significantly impaired reversal learning when the rule was changed from alternation to repetition. In experiment 2, we showed that local infusions of muscimol (an agonist of the GABA(A) receptor) into mPFC impaired performance even when the animal was well trained, suggesting that the mPFC is critical not only for acquisition but also for successful performance. In experiment 3, to examine the mechanisms underlying the role of GABAergic inhibition, we used Cre-inducible Channelrhodopsin-2 to activate parvalbumin (PV)-expressing GABAergic interneurons in the mPFC of PV-Cre transgenic mice as they performed the task. Using whole cell patch-clamp recording, we demonstrated that activation of PV-expressing interneurons in vitro with blue light in brain slices reliably produced spiking and inhibited nearby pyramidal projection neurons. With similar stimulation parameters, in vivo stimulation significantly impaired delayed alternation performance. Together these results demonstrate a critical role for the mPFC in the acquisition and performance of the delayed alternation task.     

Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat

The medial prefrontal cortex (mPFC) participates in several higher order functions including selective attention, visceromotor control, decision making and goal-directed behaviors. We discuss the role of the infralimbic cortex (IL) in visceromotor control and the prelimbic cortex (PL) in cognition and their interactions in goal-directed behaviors in the rat. The PL strongly interconnects with a relatively small group of structures that, like PL, subserve cognition, and together have been designated the 'PL circuit.' These structures primarily include the hippocampus, insular cortex, nucleus accumbens, basolateral nucleus of the amygdala, the mediodorsal and reuniens nuclei of the thalamus and the ventral tegmental area of the midbrain. Lesions of each of these structures, like those of PL, produce deficits in delayed response tasks and memory. The PL (and ventral anterior cingulate cortex) (AC) of rats is ideally positioned to integrate current and past information, including its affective qualities, and act on it through its projections to the ventral striatum/ventral pallidum. We further discuss the role of nucleus reuniens of thalamus as a major interface between the mPFC and the hippocampus, and as a prominent source of afferent limbic information to the mPFC and hippocampus. We suggest that the IL of rats is functionally homologous to the orbitomedial cortex of primates and the prelimbic (and ventral AC) cortex to the lateral/dorsolateral cortex of primates, and that the IL/PL complex of rats exerts significant control over emotional and cognitive aspects of goal-directed behavior.     

Efferent connections of the medial prefrontal cortex in the rabbit

The different cytoarchitectonic regions of the medial prefrontal cortex (mPFC) have recently been shown to play divergent roles in associative learning in rabbits. To determine if these subareas of the mPFC, including areas 24 (anterior cingulate cortex), 25 (infralimbic cortex), and 32 (prelimbic cortex) have differential efferent connections with other cortical and subcortical areas in the rabbit, anterograde and retrograde tracing experiments were performed using thePhaseolus vulgaris leukoagglutinin (PHA-L), and horseradish peroxidase (HRP) techniques. All three areas showed local dorsal-ventral projections into each of the other areas, and a contralateral projection to the homologous area on the other side of the brain. All three also revealed a trajectory through the striatum, resulting in heavy innervation of the caudate nucleus, the claustrum, and a lighter projection to the agranular insular cortex. The thalamic projections of areas 24 and 32 were similar, but not identical, with projections to the mediodorsal nucleus (MD) and all of the midline nuclei. However, the primary thalamic projections from area 25 were to the intralaminar and midline nuclei. All three areas also projected to the ventromedial and to a lesser extent to the ventral posterior thalamic nuclei. Projections were also observed in the lateral hypothalamus, in an area just lateral to the descending limb of the fornix. Amygdala projections from areas 32 and 24 were primarily to the lateral, basolateral and basomedial nuclei, but area 25 also projected to the central nucleus. All three areas also showed projections to the midbrain periaqueductal central gray, median raphe nucleus, ventral tegmental area, substantia nigra, locus coeruleus and pontine nuclei. However, only areas 24 and the more dorsal portions of area 32 projected to the superior colliculus. Area 25 and the ventral portions of area 32 also showed a bilateral projection to the parabrachial nuclei and dorsal and ventral medulla. The dorsal portions of area 32, and all of area 24 were, however, devoid of these projections. It is suggested that these differential projections are responsible for the diverse roles that the cytoarchitectonic subfields of the mPFC have been demonstrated to play in associative learning.     

Delayed alternation in adolescent and adult male and female rats

The prefrontal cortex continues to develop throughout adolescence in several species, and our laboratory has demonstrated that during adolescence there is a decrease in neurons in the rat medial prefrontal cortex (mPFC). A PFC-dependent task, the delayed alternation task, was used in the present study to examine the function of the mPFC while it is still maturing in rats of both sexes. A deficit was found in adolescents when compared to adults during 15- and 60-s delays but not at other delays (5, 10, 30, and 90 s). Furthermore, adolescents committed more perseverative errors. No significant sex differences occurred at any delay for either age group; however, in the no delay training sessions, adolescent males reached criterion faster than females. These results indicate that performance on a mPFC-dependent task improves between adolescence and adulthood.     

Fos expression in the brains of rats performing an attentional set-shifting task

Impairments in executive function and cognitive control are a common feature of neuropsychiatric and neurodegenerative disorders. A promising behavioral paradigm for elucidating the neural mechanisms of executive function is extradimensional/intradimensional (ED/ID) shifting, which places demands on executive function by requiring the adjustment of behavioral responses based on affective or attentional information. To augment the understanding of the brain systems required for these aspects of executive function, we examined the induction of Fos protein in rats tested in the ED/ID paradigm. We found increased Fos-like immunoreactivity (Fos-LI) in several cortical areas, including medial and orbital frontal cortex (OFC), in rats performing affective or attentional shifts relative to rats performing control discriminations. However, increased Fos-LI was also present in rats that performed a yoked number of additional control discrimination trials, without affective or attentional shifting. These observations suggest that cortical networks required for affective and attentional shifting are also activated during comparable discrimination tasks that do not require shifting, consistent with a role for these networks in monitoring ongoing behavior even in situations in which adaptation to changing behavioral demands is not required.     

Effects of excitotoxic lesions in the ventral striatopallidal–thalamocortical pathway on odor reversal learning: inability to extinguish an incorrect response

The role of the ventral striatopallidal pathway and related cortical areas in stimulus-reward association reversal behavior was studied by inducing bilateral lesions with the excitotoxin, N-methyl-D-aspartate (NMDA) at restricted sites in the system. The areas lesioned were the ventral pallidum (VP), the ventral striatum (VS), the medial prefrontal cortex (mPFC) [i.e., the prelimbic (PL) and infralimbic (IL) cortexes], and the orbital cortex [i.e., the dorsolateral orbital (DLO), ventral lateral orbital (VLO), and lateral orbital (LO) cortexes]. Rats with lesions of the dorsal caudate nucleus and putamen (CPu) served as a positive control in this study. Water-deprived rats were trained on a go, no-go two-odor olfactory discrimination task to respond to one odor (S+) with water as a reward and to suppress responding to the other odor (S-). The rats were then tested for their ability to reverse the associated stimuli. The number of errors made before successfully learning the stimulus-reward association were measured in relation to a sham lesion control group which did not receive injections of NMDA. In experimental rats, the lesions did not affect their ability to learn stimulus-reward associations for novel odors, but did result in an increase in the number of false alarms after the significance of the associated stimuli were reversed. That is, the lesioned animals persisted in responding to the formerly rewarded but now unrewarded stimulus. Rats with damage to the CPu did not show a significant effect when compared with the controls during reversal problems. The results support the hypothesis that the ventral striatopallidal system, together with related thalamic and frontal cortical structures, functions in reversal learning by suppressing inappropriate responses to stimuli that are no longer rewarded.     

Effects of rat medial prefrontal cortex lesions on olfactory serial reversal and delayed alternation tasks

When reward reinforcement in a two-choice discrimination task is regularly changed from one stimulus to another immediately after one learning acquisition session, the learning efficiency of a rat increases as if the rat has come to recognize this regularity of reversal. To investigate how the rat medial prefrontal cortex (mPFC) is involved in such improvement, we examined the performance of mPFC-lesioned rats in a serial reversal task of olfactory discrimination. The performance of other mPFC-lesioned rats in a delayed alternation task was also analyzed using the same apparatus to evaluate the contribution of the mPFC to working memory. The mPFC-lesioned rats demonstrated selective difficulty in the second reversal session in the serial reversal task and also showed performance impairment in the delayed alternation task. These results suggest that the rat mPFC mediating working memory is involved in early progress in learning efficiency during experiences of multiple reversals, which may be relevant to cognitive operations in reversal learning beyond a one-time reversal of stimulus response associations.     

Distinct manifestations of executive dysfunction in aged rats

Different components of executive function such as working memory, attention, and cognitive flexibility can be dissociated behaviorally and mechanistically; however, the within-subject influences of normal aging on different aspects of executive function remain ill-defined. To better define these relationships, young adult and aged male F344 rats were cross-characterized on an attentional set-shifting task that assesses cognitive flexibility and a delayed response task that assesses working memory. Across tasks, aged rats were impaired relative to young; however, there was significant variability in individual performance within the aged cohort. Notably, performance on the set-shifting task and performance at long delays on the delayed response task were inversely related among aged rats. Additional experiments showed no relationship between aged rats' performance on the set-shifting task and performance on a hippocampal-dependent spatial reference memory task. These data indicate that normal aging can produce distinct manifestations of executive dysfunction, and support the need to better understand the unique mechanisms contributing to different forms of prefrontal cortical-supported executive decline across the lifespan.     

The role of medial prefrontal cortex in context-specific inhibition during reversal learning of a visual discrimination

Rats with medial prefrontal cortex or sham lesions were trained on a visual discrimination task designed for the eight-arm radial maze. After reaching asymptotic performance on this task, both groups were divided into sub-groups that would experience reversal learning in the same or different context from original training. The results showed that both groups reversed in the different context had accelerated learning compared to the groups reversed in the same context. Reversal learning in rats with medial prefrontal cortex damage was faster than sham animals in the same context. These and other results from a transfer test suggest that the medial prefrontal cortex participates in the behavioral effects of a context-specific inhibitory association acquired during visual discrimination learning.