Chemogenetic inactivation of the nucleus reuniens and its projections to the orbital cortex produce deficits on discrete measures of behavioral flexibility in the attentional set-shifting task.

The nucleus reuniens (RE) of the ventral midline thalamus is a critical node in the communication between the orbitomedial prefrontal cortex (OFC) and the hippocampus (HF). While RE has been shown to directly participate in memory-associated functions through its connections with the medial prefrontal cortex and HF, less is known regarding the role of RE in executive functioning. Here, we examined the involvement of RE and its projections to the orbital cortex (ORB) in attention and behavioral flexibility in male rats using the attentional set shifting task (AST). Rats expressing the hM4Di DREADD receptor in RE were implanted with indwelling cannulas in either RE or the ventromedial ORB to pharmacologically inhibit RE or its projections to the ORB with intracranial infusions of clozapine-N-oxide hydrochloride (CNO). Chemogenetic-induced suppression of RE resulted in impairments in reversal learning and set-shifting. This supports a vital role for RE in behavioral flexibility - or the ability to adapt behavior to changing reward or rule contingencies. Interestingly, CNO suppression of RE projections to the ventromedial ORB produced impairments in rule abstraction - or dissociable effects elicited with direct RE suppression. In summary, the present findings indicate that RE, mediated in part by actions on the ORB, serves a critical role in the flexible use of rules to drive goal directed behavior. The cognitive deficits of various neurological disorders with impaired communication between the HF and OFC, may be partly attributed to alterations of RE -- as an established intermediary between these cortical structures.

Projections from the five divisions of the orbital cortex to the thalamus in the rat.

The orbital cortex (ORB) of the rat consists of five divisions: the medial (MO), ventral (VO), ventrolateral (VLO), lateral (LO), and dorsolateral (DLO) orbital cortices. No previous report has comprehensively examined and compared projections from each division of the ORB to the thalamus. Using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin, we describe the efferent projections from the five divisions of the ORB to the thalamus in the rat. We demonstrated that, with some overlap, each division of the ORB distributed in a distinct (and unique) manner to nuclei of the thalamus. Overall, ORB projected to a relatively restricted number of sites in the thalamus, and strikingly distributed entirely to structures of the medial/midline thalamus, while completely avoiding lateral regions or principal nuclei of the thalamus. The main termination sites in the thalamus were the paratenial nucleus (PT) and nucleus reuniens (RE) of the midline thalamus, the medial (MDm) and central (MDc) divisions of the mediodorsal nucleus, the intermediodorsal nucleus, the central lateral, paracentral, and central medial nuclei of the rostral intralaminar complex and the submedial nucleus (SM). With some exceptions, medial divisions of the ORB (MO, VO) mainly targeted "limbic-associated" nuclei such as PT, RE, and MDm, whereas lateral division (VLO, LO, DLO) primarily distributed to "sensorimotor-associated" nuclei including MDc, SM, and the rostral intralaminar complex. As discussed herein, the medial/midline thalamus may represent an important link (or bridge) between the orbital cortex and the hippocampus and between the ORB and medial prefrontal cortex. In summary, the present results demonstrate that each division of the orbital cortex projects in a distinct manner to nuclei of the thalamus which suggests unique functions for each division of the orbital cortex.

Structural and functional organization of the midline and intralaminar nuclei of the thalamus.

The midline and intralaminar nuclei of the thalamus form a major part of the "limbic thalamus;" that is, thalamic structures anatomically and functionally linked with the limbic forebrain. The midline nuclei consist of the paraventricular (PV) and paratenial nuclei, dorsally and the rhomboid and nucleus reuniens (RE), ventrally. The rostral intralaminar nuclei (ILt) consist of the central medial (CM), paracentral (PC) and central lateral (CL) nuclei. We presently concentrate on RE, PV, CM and CL nuclei of the thalamus. The nucleus reuniens receives a diverse array of input from limbic-related sites, and predominantly projects to the hippocampus and to "limbic" cortices. The RE participates in various cognitive functions including spatial working memory, executive functions (attention, behavioral flexibility) and affect/fear behavior. The PV receives significant limbic-related afferents, particularly the hypothalamus, and mainly distributes to "affective" structures of the forebrain including the bed nucleus of stria terminalis, nucleus accumbens and the amygdala. Accordingly, PV serves a critical role in "motivated behaviors" such as arousal, feeding/consummatory behavior and drug addiction. The rostral ILt receives both limbic and sensorimotor-related input and distributes widely over limbic and motor regions of the frontal cortex-and throughout the dorsal striatum. The intralaminar thalamus is critical for maintaining consciousness and directly participates in various sensorimotor functions (visuospatial or reaction time tasks) and cognitive tasks involving striatal-cortical interactions. As discussed herein, while each of the midline and intralaminar nuclei are anatomically and functionally distinct, they collectively serve a vital role in several affective, cognitive and executive behaviors - as major components of a brainstem-diencephalic-thalamocortical circuitry.

Discharge characteristics of neurons of nucleus reuniens across sleep-wake states in the behaving rat.

The nucleus reuniens (RE) of the ventral midline thalamus is strongly reciprocally connected with the hippocampus (HF) and medial prefrontal cortex (PFC), serving a critical role in affective and cognitive functioning. While midline thalamic nuclei have been implicated in the modulation of states of arousal and consciousness, few studies have addressed RE's role in behavioral state control. Accordingly, as a first line of investigation, we examined the discharge properties of RE neurons in behaving rats throughout the sleep-wake cycle. We analyzed 153 units in RE which demonstrated heterogeneity in discharge rates and pattern of activity across sleep wake states. Using a rate ratio of activity in wake vs. REM, we found that the majority of cells displayed state-related changes and were classified into distinct cell types, exhibiting their highest discharge rates during active waking (AW), REM sleep, or maintaining equivalent activity across AW/REM. We further distinguished cells as either slow firing (SF = < 10 Hz) or fast firing (FF =>10 Hz) cells. The majority of cells, independent of state-related preference, were SF. FF RE cells were primarily wake active and wake/REM cell types. This diverse set of RE neurons are likely modulated by key brainstem and hypothalamic nuclei, which in turn, drive RE to exert strong effects on its cortical targets during waking and REM sleep. RE may not only act as a node in HF-PFC circuitry, but also as a critical thalamic link in ascending arousal and attentional networks.

Role of the reuniens and rhomboid thalamic nuclei in anxiety-like avoidance behavior in the rat.

The nucleus reuniens (RE) and rhomboid (RH) nuclei of the ventral midline thalamus are reciprocally connected with the prefrontal cortex (PFC) and the hippocampus (HF) and serve as key intermediaries between these structures, regulating cognitive and emotional behaviors. Regarding affective behavior, several recent reports have described the involvement of RE/RH in the acquisition and retention of conditioned fear, but little is known regarding their role (RE/RH) in anxiety-like behaviors. We examined the role of RH/RE on avoidance and defensive behaviors in male Long Evans rats using the elevated plus maze (EPM). We found that the reversible suppression of RE/RH with muscimol increased avoidance behavior to the open arms of the plus maze as shown by: (a) significant reductions in open arm entries; (b) reductions in the mean duration of time spent in the open arms; and (c) significant increases in retreats during open arm exploration. This was coupled with decreases in the number of head dips in the maze. Consistent with these behavioral effects, a single exposure of naïve rats to the plus maze produced significant increases in c-fos expression selectively in RE and RH of midline thalamic nuclei. We posit that RE/RH normally acts to optimize adaptive responses to anxiety-eliciting situations, and disruptions of RE/RH produce severe deficits in coping behaviors-or as shown here increases in avoidance/defensive behaviors. In sum, the present results establish a novel role for RE/RH in anxiety-like avoidance behavior. In addition to its role in attention, working memory, and executive control, RE/RH also regulates adaptative responses to not only fear but also to anxiogenic stimuli. As such, dysfunction of RE/RH may contribute to the amalgamation of symptoms common to many mental health disorders including anxiety, depression, schizophrenia, and PTSD.

No cognitive processing in the unconscious, anesthetic-like, state of sleep.

We review evidence challenging the hypothesis that memories are processed or consolidated in sleep. We argue that the brain is in an unconscious state in sleep, akin to general anesthesia (GA), and hence is incapable of meaningful cognitive processing-the sole purview of waking consciousness. At minimum, the encoding of memories in sleep would require that waking events are faithfully transferred to and reproduced in sleep. Remarkably, however, this has never been demonstrated, as waking experiences are never truly replicated in sleep but rather appear in very altered or distorted forms. General anesthetics (GAs) exert their effects through endogenous sleep-wake control systems and accordingly GA and sleep share several common features: sensory blockade, immobility, amnesia and lack of awareness (unconsciousness). The loss of consciousness in non-REM (NREM) sleep or to GAs is characterized by: (a) delta oscillations throughout the cortex; (b) marked reductions in neural activity (from waking) over widespread regions of the cortex, most pronounced in frontal and parietal cortices; and (c) a significant disruption of the functional connectivity of thalamocortical and corticocortical networks, particularly those involved in "higher order" cognitive functions. Several (experimental) reports in animals and humans have shown that disrupting the activity of the cortex, particularly the orbitofrontal cortex, severely impairs higher order cognitive and executive functions. The profound and widespread deactivation of the cortex in the unconscious states of NREM sleep or GA would be expected to produce an equivalent, or undoubtedly a much greater, disruptive effect on mnemonic and cognitive functions. In conclusion, we contend that the unconscious, severely altered state of the brain in NREM sleep would negate any possibility of cognitive processing in NREM sleep.

Prefrontal Pathways Provide Top-Down Control of Memory for Sequences of Events.

We remember our lives as sequences of events, but it is unclear how these memories are controlled during retrieval. In rats, the medial prefrontal cortex (mPFC) is positioned to influence sequence memory through extensive top-down inputs to regions heavily interconnected with the hippocampus, notably the nucleus reuniens of the thalamus (RE) and perirhinal cortex (PER). Here, we used an hM4Di synaptic-silencing approach to test our hypothesis that specific mPFC→RE and mPFC→PER projections regulate sequence memory retrieval. First, we found non-overlapping populations of mPFC cells project to RE and PER. Second, suppressing mPFC activity impaired sequence memory. Third, inhibiting mPFC→RE and mPFC→PER pathways effectively abolished sequence memory. Finally, a sequential lag analysis showed that the mPFC→RE pathway contributes to a working memory retrieval strategy, whereas the mPFC→PER pathway supports a temporal context memory retrieval strategy. These findings demonstrate that mPFC→RE and mPFC→PER pathways serve as top-down mechanisms that control distinct sequence memory retrieval strategies.

Inactivation of nucleus reuniens impairs spatial working memory and behavioral flexibility in the rat.

The hippocampal formation (HF) and medial prefrontal cortex (mPFC) play critical roles in spatial working memory (SWM). The nucleus reuniens (RE) of the ventral midline thalamus is an important anatomical link between the HF and mPFC, and as such is crucially involved in SWM functions that recruit both structures. Little is known, however, regarding the role of RE in other behaviors mediated by this circuit. In the present study, we examined the role of RE in spatial working memory and executive functioning following reversible inactivation of RE with either muscimol or procaine. Rats were implanted with an indwelling cannula targeting RE and trained in a delayed nonmatch to sample spatial alternation T-maze task. For the task, sample and choice runs were separated by moderate or long delays (30, 60, and 120 s). Following asymptotic performance, rats were tested following infusions of drug or vehicle. Muscimol infused into RE impaired SWM at all delays, whereby procaine only impaired performance at the longest delays. Furthermore, RE inactivation with muscimol produced a failure in win-shift strategy as well as severe spatial perseveration, whereby rats persistently made re-entries into incorrect arms during correction trials, despite the absence of reward. This demonstrated marked changes in behavioral flexibility and response strategy. These results strengthen the role of nucleus reuniens as a pivotal link between hippocampus and prefrontal cortex in cognitive and executive functions and suggest that nucleus reuniens may be a potential target in the treatment of CNS disorders such as schizophrenia, attention deficit hyperactivity disorder, addiction, and obsessive-compulsive disorder, whose symptoms are defined by hippocampal-prefrontal dysfunctions.

Pattern of distribution of serotonergic fibers to the amygdala and extended amygdala in the rat.

As is well recognized, serotonergic (5-HT) fibers distribute widely throughout the forebrain, including the amygdala. Although a few reports have examined the 5-HT innervation of select nuclei of the amygdala in the rat, no previous report has described overall 5-HT projections to the amygdala in the rat. Using immunostaining for the serotonin transporter, SERT, we describe the complete pattern of distribution of 5-HT fibers to the amygdala (proper) and to the extended amygdala in the rat. Based on its ontogenetic origins, the amygdala was subdivided into two major parts, pallial and subpallial components, with the pallial component further divided into superficial and deep nuclei (Olucha-Bordonau et al. 2015). SERT+ fibers were shown to distributed moderately to densely to the deep and cortical pallial nuclei, but, by contrast, lightly to the subpallial nuclei. Specifically, 1) of the deep pallial nuclei, the lateral, basolateral, and basomedial nuclei contained a very dense concentration of 5-HT fibers; 2) of the cortical pallial nuclei, the anterior cortical and amygdala-cortical transition zone rostrally and the posteromedial and posterolateral nuclei caudally contained a moderate concentration of 5-HT fibers; and 3) of the subpallial nuclei, the anterior nuclei and the rostral part of the medial (Me) nuclei contained a moderate concentration of 5-HT fibers, whereas caudal regions of Me as well as the central nuclei and the intercalated nuclei contained a sparse/light concentration of 5-HT fibers. With regard to the extended amygdala (primarily the bed nucleus of stria terminalis; BST), on the whole, the BST contained moderate numbers of 5-HT fibers, spread fairly uniformly throughout BST. The findings are discussed with respect to a critical serotonergic influence on the amygdala, particularly on the basal complex, and on the extended amygdala in the control of states of fear and anxiety. J. Comp. Neurol. 525:116-139, 2017. © 2016 Wiley Periodicals, Inc.

Lesions of the ventral midline thalamus produce deficits in reversal learning and attention on an odor texture set shifting task.

The nucleus reuniens (RE) of the ventral midline thalamus is strongly reciprocally connected with the hippocampus (HF) and the medial prefrontal cortex (mPFC) and has been shown to mediate the transfer of information between these structures. It has become increasingly well established that RE serves a critical role in mnemonic tasks requiring the interaction of the HF and mPFC, but essentially not tasks relying solely on the HF. Very few studies have addressed the independent actions of RE on prefrontal executive functioning. The present report examined the effects of lesions of the ventral midline thalamus, including RE and the dorsally adjacent rhomboid nucleus (RH) in rats on attention and behavioral flexibility using the attentional set shifting task (AST). The task uses odor and tactile stimuli to test for attentional set formation, attentional set shifting, behavioral flexibility and reversal learning. By comparison with sham controls, lesioned rats were significantly impaired on reversal learning and intradimensional (ID) set shifting. Specifically, RE/RH lesioned rats were impaired on the first reversal stage of the task which required a change in response strategy to select a previously non-rewarded stimulus for reward. RE/RH lesioned rats also exhibited deficits in the ability to transfer or generalize rules of the task which requires making the same modality-based choices (e.g., odor vs. tactile) to different sets of stimuli in the ID stage of the task. These results demonstrate that in addition to its role in tasks dependent on HF-mPFC interactions, nucleus reuniens is also critically involved cognitive/executive functions associated with the medial prefrontal cortex. As such, the deficits in the AST task produced by RE/RH lesions suggest the ventral midline thalamus directly contributes to flexible goal directed behavior.

Limbic circuitry of the midline thalamus.

The thalamus was subdivided into three major groups: sensorimotor nuclei (or principal/relay nuclei), limbic nuclei and nuclei bridging these two domains. Limbic nuclei of thalamus (or 'limbic thalamus') consist of the anterior nuclei, midline nuclei, medial division of the mediodorsal nucleus (MDm) and central medial nucleus (CM) of the intralaminar complex. The midline nuclei include the paraventricular (PV) and paratenial (PT) nuclei, dorsally, and the reuniens (RE) and rhomboid (RH) nuclei, ventrally. The 'limbic' thalamic nuclei predominantly connect with limbic-related structures and serve a direct role in limbic-associated functions. Regarding the midline nuclei, RE/RH mainly target limbic cortical structures, particularly the hippocampus and the medial prefrontal cortex. Accordingly, RE/RH participate in functions involving interactions of the HF and mPFC. By contrast, PV/PT mainly project to limbic subcortical structures, particularly the amygdala and nucleus accumbens, and hence are critically involved in affective behaviors such as stress/anxiety, feeding behavior, and drug seeking activities. The anatomical/functional characteristics of MDm and CM are very similar to those of the midline nuclei and hence the collection of nuclei extending dorsoventrally along the midline/paramidline of the thalamus constitute the core of the 'limbic thalamus'.

Pattern of distribution of serotonergic fibers to the orbitomedial and insular cortex in the rat.

As is well recognized, serotonergic (5-HT) fibers distribute widely throughout the brain, including the cerebral cortex. Although some early reports described the 5-HT innervation of the prefrontal cortex (PFC) in rats, the focus was on sensorimotor regions and not on the 'limbic' PFC - or on the medial, orbital and insular cortices. In addition, no reports have described the distribution of 5-HT fibers to PFC in rats using antisera to the serotonin transporter (SERT). Using immunostaining for SERT, we examined the pattern of distribution of 5-HT fibers to the medial, orbital and insular cortices in the rat. We show that 5-HT fibers distribute massively throughout all divisions of the PFC, with distinct laminar variations. Specifically, 5-HT fibers were densely concentrated in superficial (layer 1) and deep (layers 5/6) of the PFC but less heavily so in intermediate layers (layers 2/3). This pattern was most pronounced in the orbital cortex, particularly in the ventral and ventrolateral orbital cortices. With the emergence of granular divisions of the insular cortex, the granular cell layer (layer 4) was readily identifiable by a dense band of labeling confined to it, separating layer 4 from less heavily labeled superficial and deep layers. The pattern of 5-HT innervation of medial, orbital and insular cortices significantly differed from that of sensorimotor regions of the PFC. Serotonergic labeling was much denser overall in limbic compared to non-limbic regions of the PFC, as was striking demonstrated by the generally weaker labeling in layers 1-3 of the primary sensory and motor cortices. The massive serotonergic innervation of the medial, orbital and insular divisions of the PFC likely contributes substantially to well established serotonergic effects on affective and cognitive functions, including a key role in many neurological and psychiatric diseases.

Serotonergic projections and serotonin receptor expression in the reticular nucleus of the thalamus in the rat.

The reticular nucleus (RT) of the thalamus, a thin sheet of GABAergic neurons located between the external medullary lamina and the internal capsule of the thalamus, has functionally distinct afferent and efferent connections with thalamic nuclei, the neocortex, the basal forebrain and the brainstem. RT is critically positioned to rhythmically pace thalamocortical networks leading to the generation of spindle activity during the early phases of sleep and during absence (spike-wave) seizures. Serotonin, acting on 5-HT(1A) receptors on parvalbumin-containing cells of RT, has been implicated in this rhythmicity. However, the precise source(s) of 5-HT afferents to the RT remains to be determined. In the present study, we injected the retrograde tracer, Fluorogold, into dorsal and ventral regions of RT to determine the origins of raphe input to RT. We further characterized the distribution of 5-HT fibers to RT by using immunohistochemistry for 5-HT and for the 5HT transporter (SERT) detection. Finally, we described the presence of the two major postsynaptic 5-HT receptors in RT, 5-HT(1A) and 5-HT(2A) receptors. Our results show that the dorsal raphe nucleus and the supralemniscal nucleus (B9) of the midbrain are the principal sources of raphe projections to RT. In addition, serotonergic fibers (5-HT and SERT positive) were richly distributed throughout RT, and 5-HT(1A) and 5-HT(2A) receptors were highly expressed on RT neurons and dendrites. These findings suggest a significant 5-HT modulatory influence on GABAergic neurons of RT in the control of rhythmical (or spindle) activity in thalamocortical systems directly associated with sleep and possibly with absence seizures.

Pattern of distribution of serotonergic fibers to the thalamus of the rat.

It is well established that serotonergic (5-hydroxytryptamine, 5-HT) fibers, mainly originating from the dorsal and median raphe nuclei of the brainstem, distribute throughout the forebrain, most heavily to 'limbic' forebrain structures. Few reports have examined the distribution of 5-HT fibers to the thalamus and none to our knowledge using immunoprocedures for the detection of the serotonin transporter (SERT)-a very sensitive marker for 5-HT fibers. Using immunohistochemical methods for SERT, we examined the pattern of distribution of 5-HT fibers to the thalamus in the rat. We show that serotonergic fibers are heavily concentrated in midline, intralaminar and association nuclei of the thalamus, and with the exception of the lateral geniculate complex, weakly distributed to principal nuclei of thalamus. Specifically, we demonstrate that 5-HT fibers are densely concentrated in the anteroventral, anteromedial and interanteromedial nuclei of the anterior thalamus, the paraventricular, rhomboid and reuniens nuclei of the midline thalamus, the central medial and central lateral nuclei of the intralaminar thalamus, the intermediodorsal nucleus, the lateral dorsal nucleus, and the dorsal and ventral lateral geniculate nuclei and intergeniculate leaflet of the LGN complex. Less densely innervated sites include the mediodorsal, paracentral, parafascicular, lateral posterior and submedial nuclei of thalamus. Remaining regions of the thalamus, largely consisting of principal nuclei, contained few 5-HT fibers. This pattern of 5-HT innervation indicates that serotonin/ serotonergic fibers mainly affect thalamic nuclei with connections to 'non-principal' or limbic regions of the cortex (or forebrain). This suggests that serotonergic fibers to the thalamus may exert a significant influence on affective and cognitive functions, possibly complementing the actions of 5-HT fibers to other parts of the brain involved in emotional and cognitive behaviors.