Nucleus Reuniens-Elicited Delta Oscillations Disable the Prefrontal Cortex in Schizophrenia.

Network oscillations are essential for all cognitive functions. Oscillatory deficits are well established in psychiatric diseases and are recapitulated in animal models. They are significantly and specifically affected by pharmacological interventions using psychoactive compounds. Dopamine D4 receptor (D4R) activation was shown to enhance gamma rhythm in freely moving rats and to specifically affect slow delta and theta oscillations in the urethane-anesthetized rat model. The goal of this study was to test the effect of D4R activation on slow network oscillations at delta and theta frequencies during wake states, potentially supporting enhanced functional connectivity during dopamine-induced attention and cognitive processing. Network activity was recorded in the prefrontal cortex (PFC), hippocampus (HC) and nucleus reuniens (RE) in control conditions and after injecting the D4R agonist A-412997 (3 and 5 mg/kg; systemic administration). We found that A-412997 elicited a lasting (~40 min) wake state and drastically enhanced narrow-band delta oscillations in the PFC and RE in a dose-dependent manner. It also preferentially enhanced delta synchrony over theta coupling within the PFC-RE-HC circuit, strongly strengthening PFC-RE coupling. Thus, our findings indicate that the D4R may contribute to cognitive processes, at least in part, through acting on wake delta oscillations and that the RE, providing an essential link between the PFC and HC, plays a prominent role in this mechanism.

BackgroundThe present study used electrophysiological recordings in freely behaving rats to investigate the effects of Dopamine 4 receptor (D4R) activation on slow oscillations during waking states, in which these rhythms play an essential role in interregional communication between prefrontal cortex (PFC) and hippocampus (HC). Network oscillations are essential for all cognitive functions. Oscillatory deficits are well established in psychiatric diseases and are recapitulated in animal models [1-3]. They are significantly and specifically affected by pharmacological interventions using psychoactive compounds [4-8]. D4R activation was shown to enhance gamma rhythm in PFC and HC of freely moving rats [7] and to specifically affect slow delta and theta oscillations in the urethane anesthetized rat model [8].Aims and ObjectivesThe goal of this study was to test the effect of D4R activation on slow network oscillations at delta and theta frequencies during wake states, potentially supporting enhanced functional connectivity during dopamine-induced attention and cognitive processing. The second goal of this study was to test the potential D4R effect on the balance of frequency-tagged bidirectional relationships between the PFC and HC in freely moving rats by recording from the thalamic nucleus reuniens (RE) which provides an anatomical link between the two structures in both directions.MethodNetwork activity was recorded in the PFC, HC, and RE, in control conditions and after injecting the selective D4R agonist A-412997 (3 and 5 mg/kg, systemic administration). Delta (1-4Hz) and theta (5-10Hz) peak power and coherences were analyzed in two post-injection segments, the first immediately after injection with visible alterations (19+/-3 min, range: 5-40 min) and the second lasting until the first sleep period (19+/-2 min; range: 5-36 min) and statistically compared with controls, 20-50 min episodes with motor activity pre-injection and 6.5+/-1.0, 11.3+/-1.2, and 16.3+/-0.6 hours after injection.ResultsWe found that systemic administration of A-412997 elicited lasting (~40 min) wake state and drastically enhanced narrow-band delta oscillations in the PFC and RE. The effect was dose-dependent, being significant after 5 mg/kg but not after 3 mg/kg drug injection. In contrast, moderate increase in theta power did not show region or dose-dependence, but appeared in all structures after both doses. The frequency of theta oscillations decreased in most experiments, being significant over the group at the lower dose. A-412997 also preferentially enhanced delta synchrony over theta coupling within the PFC-RE-HC circuit, strongly strengthening PFC-RE coupling.Discussion & ConclusionThe present data demonstrate involvement of D4R mechanisms in reorganization of the structure of theta-delta coupling in the PFC-RE-HC network. In conjunction with our prior studies [7, 8], this shows that administration of a D4R agonist elicits a lasting awake state, enhanced gamma activity, and preferentially enhanced synchronous delta activity over theta coupling within the PFC-RE-HC circuit. Modulation of fast beta and gamma network oscillations, by slow delta and theta rhythms play a key role in behavior-dependent interregional, cortico-hippocampal coupling. Thus, our findings indicate that the D4R may contribute to cognitive processes, at least in part, through acting on wake delta oscillations.

The nucleus reuniens (RE) of the midline thalamus has been shown to strongly innervate structures of the limbic forebrain, prominently including the hippocampus (HF) and the medial prefrontal cortex (mPFC) and to exert pronounced excitatory effects on HF and mPFC. It was unknown, however, whether RE projections to, and hence actions on, the HF and mPFC originate from a common or largely separate groups of RE neurons. Using fluorescent retrograde tracing techniques, we examined the patterns of distribution of RE cells projecting to HF, to the mPFC or to both sites via axon collaterals. Specifically, injections of the retrograde tracers Fluorogold (FG) or Fluororuby (FR) were made in the mPFC and in various subfields of HF and patterns of single (FG or FR) or double labeled (FG + FR) cells in RE were determined. Pronounced numbers of (single) labeled neurons were present throughout RE with FG or FR injections, and although intermingled in RE, cells projecting to the mPFC were preferentially distributed along the midline or in the perireuniens nucleus (pRE), whereas those projecting to HF occupied a wide mediolateral cross sectional area of RE lying between cells projecting to the mPFC. Approximately, tenfold more labeled cells were present in RE with ventral compared to dorsal CA1 injections. Like single labeled neurons, double labeled cells were found throughout RE, but were most densely concentrated in areas of greatest overlap of FG+ and FR+ neurons or mainly in the lateral one-third of RE, medial to pRE. Depending on specific combinations of injections, double labeled cells ranged from approximately 3-9% of the labeled neurons. The nucleus reuniens has been shown to be a vital link in limbic subcortical-cortical communication and recent evidence indicates a direct RE involvement in hippocampal and medial prefrontal cortical-dependent behaviors. The present findings indicate that RE is critically positioned to influence the HF and mPFC, and their associated behaviors, via separate or collateral projections to these sites.

The nucleus reuniens (RE) is a ventral midline thalamic nucleus that interconnects the medial prefrontal cortex (mPFC) and hippocampus (HPC). Considerable data indicate that HPC-mPFC circuits are involved in contextual and spatial memory; however, it is not clear whether the RE mediates the acquisition or retrieval of these memories. To examine this question, we inactivated the RE with muscimol before either the acquisition or retrieval of pavlovian fear conditioning in rats; freezing served as the index of fear. We found that RE inactivation before conditioning impaired the acquisition of contextual freezing, whereas inactivation of the RE before retrieval testing increased the generalization of freezing to a novel context; inactivation of the RE did not affect either the acquisition or expression of auditory fear conditioning. Interestingly, contextual conditioning impairments were absent when retrieval testing was also conducted after RE inactivation. Contextual memories acquired under RE inactivation were hippocampal independent, insofar as contextual freezing in rats conditioned under RE inactivation was insensitive to intrahippocampal infusions of the NMDA receptor antagonist aminophosphonovalerate. Together, these data reveal that the RE supports hippocampal-dependent encoding of precise contextual memories that allow discrimination of dangerous contexts from safe contexts. When the RE is inactive, however, alternate neural systems acquire an impoverished contextual memory that is expressed only when the RE is off-line.SIGNIFICANCE STATEMENT The midline thalamic nucleus reuniens (RE) coordinates communication between the hippocampus and medial prefrontal cortex, brain areas that are critical for contextual and spatial memory. Here we show that temporary pharmacological inactivation of RE impairs the acquisition and precision of contextual fear memories after pavlovian fear conditioning in rats. However, inactivating the RE before retrieval testing restored contextual memory in rats conditioned after RE inactivation. Critically, we show that imprecise contextual memories acquired under RE inactivation are learned independently of the hippocampus. These data reveal that the RE is required for hippocampal-dependent encoding of precise contextual memories to support the discrimination of safe and dangerous contexts.

ImportanceThe nucleus reuniens (RE) is a crucial component that interconnects the medial prefrontal cortex (mPFC) and hippocampus (HPC), completing the HPC-dependent circuit underlying the regulation of trace fear. We previously demonstrated that RE inactivation during acquisition impaired the encoding of trace fear, while RE inactivation during both the acquisition and retrieval led to heightened trace fear throughout the test session, raising questions about the involvement of HPC-independent circuit in trace fear acquisition without functional RE.ObjectiveTo investigate whether rats without functional RE throughout the entire behavioral sessions can acquire trace fear using an HPC-independent circuit.DesignA balanced factorial design was used to assess the role of the dorsal hippocampus (DH) or ventral hippocampus (VH) in trace fear acquisition with or without functional RE.SettingThe study was conducted in a controlled laboratory environment.ParticipantsAdult male Long-Evans rats were used as experimental subjects.InterventionsConsecutive intracranial micro-infusions of either GABAA receptor agonist “Muscimol” or vehicle targeted the RE during both trace fear acquisition and retrieval. Micro-infusions of the N-methyl-D-aspartate (NMDA) receptor antagonist “DL-2-amino-5-phosphonovaleric acid” or saline targeted the DH or VH during trace fear acquisition.Main OutcomesFear level of respective groups was measured.MeasuresFreezing was quantified as immobility during baseline and conditioned stimulus during trace fear acquisition and retrieval.ResultsControl animals required proper DH or VH NMDA receptor activation for the acquisition of trace fear. Rats without functional RE still acquired trace fear, but independent of DH or VH NMDA receptor activation, suggesting the reliance of an HPC-independent circuit during fear encoding.Conclusions and RelevanceThese findings highlighted potential alternative neural pathways that may support and compensate trace fear acquisition under pathological conditions.Significance StatementImpaired fear regulation resulted in psychiatric disorders like panic disorder and anxiety. Pavlovian trace fear conditioning using male Long-Evans rats as the subjects models human emotional learning. Although hippocampus (HPC)–medial prefrontal cortex (mPFC) interactions facilitate trace fear acquisition, the role of thalamic nucleus reuniens (RE), which connects these regions, is unclear. In this study, we found that in terms of within-session fear expression during acquisition, dorsal hippocampus (DH) N-methyl-D-aspartate (NMDA) receptor blockade impaired fear response only in RE-intact rats, whereas ventral hippocampus (VH) blockade impaired the response regardless of the RE functionality. Critically, rats without functional RE still acquired trace fear memory independent of DH or VH NMDA receptor activation, indicating the recruitment of alternative HPC-independent pathways. These findings highlight possible compensatory pathways engaged following RE dysfunction, providing insight into how the brain adapts under pathological conditions.

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) is the largest of the midline nuclei of the thalamus and the major source of thalamic afferents to the hippocampus and parahippocampal structures. Nucleus reuniens has recently been shown to exert powerful excitatory actions on CA1 of the hippocampus. Few reports on any species have examined afferent projections to nucleus reuniens. By using the retrograde anatomical tracer Fluorogold, we examined patterns of afferent projections to RE in the rat. We showed that RE receives a diverse and widely distributed set of afferents projections. The main sources of input to nucleus reuniens were from the orbitomedial, insular, ectorhinal, perirhinal, and retrosplenial cortices; CA1/subiculum of hippocampus; claustrum, tania tecta, lateral septum, substantia innominata, and medial and lateral preoptic nuclei of the basal forebrain; medial nucleus of amygdala; paraventricular and lateral geniculate nuclei of the thalamus; zona incerta; anterior, ventromedial, lateral, posterior, supramammillary, and dorsal premammillary nuclei of the hypothalamus; and ventral tegmental area, periaqueductal gray, medial and posterior pretectal nuclei, superior colliculus, precommissural/commissural nuclei, nucleus of the posterior commissure, parabrachial nucleus, laterodorsal and pedunculopontine tegmental nuclei, nucleus incertus, and dorsal and median raphe nuclei of the brainstem. The present findings of widespread projections to RE, mainly from limbic/limbic-associated structures, suggest that nucleus reuniens represents a critical relay in the transfer of limbic information (emotional/cognitive) from RE to its major targets, namely, to the hippocampus and orbitomedial prefrontal cortex. RE appears to be a major link in the two-way exchange of information between the hippocampus and the medial prefrontal cortex.

The hippocampus and prefrontal cortex (PFC) are connected in a reciprocal manner: whereas the hippocampus projects directly to the PFC, a polysynaptic pathway that passes through the nucleus reuniens (RE) of the thalamus relays inputs from the PFC to the hippocampus. The present study demonstrates that lesioning and/or inactivation of the RE reduces coherence in the PFC–hippocampal pathway, provokes an antidepressant-like behavioral response in the forced swim test and prevents, but does not ameliorate, anhedonia in the chronic mild stress (CMS) model of depression. Additionally, RE lesioning before CMS abrogates the well-known neuromorphological and endocrine correlates of CMS. In summary, this work highlights the importance of the reciprocal connectivity between the hippocampus and PFC in the establishment of stress-induced brain pathology and suggests a role for the RE in promoting resilience to depressive illness.

Circuit-level communication between disparate brain regions is fundamental for the complexities of the central nervous system operation. Co-ordinated bouts of rhythmic activity between the prefrontal cortex (PFC) and hippocampus (HPC), in particular, are important for mnemonic processes. This is true during awake behavior, as well as during offline states like sleep. We have recently shown that the anatomically interposed thalamic nucleus reuniens (RE) has a role in coordinating slow-wave activity between the PFC and HPC. Here, we took advantage of spontaneous brain state changes occurring during urethane anesthesia in order to assess if PFC-HPC communication was modified during activated (theta) vs. deactivated (slow oscillation: SO) states. These forebrain states are highly similar to those expressed during rapid eye movement (REM) and non-REM stages of natural sleep, respectively. Evoked potentials and excitatory current sinks in the HPC were consistently larger during SO states, regardless of whether PFC or RE afferents were stimulated. Interestingly, PFC stimulation during theta appeared to preferentially use a cortico-cortical pathway, presumably involving the entorhinal cortex as opposed to the more direct RE to HPC conduit. Optogenetic and chemogenetic manipulations of the RE suggested that this state-dependent biasing was mediated by responding in the RE itself. Finally, the phase of both ongoing rhythms also appeared to be an important factor in modulating HPC responses, with maximal field excitatory postsynaptic potentials (EPSPs) occurring during the negative-going phase of both rhythms. Thus, forebrain state plays an important role in how communication takes place across the PFC and HPC, with the RE as a determining factor in how this is shaped. Furthermore, ongoing sleep-like rhythms influence the coordination and perhaps potentiate excitatory processing in this extended episodic memory circuit. Our results have direct implications for activity-dependent processes relevant to sleep-dependent memory consolidation.

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.

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

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

The nucleus reuniens (RE) is the largest of the midline nuclei of the thalamus and exerts strong excitatory actions on the hippocampus and medial prefrontal cortex. Although RE projections to the hippocampus have been well documented, no study using modern tracers has examined the totality of RE projections. With the anterograde anatomical tracer Phaseolus vulgaris leuccoagglutinin, we examined the efferent projections of RE as well as those of the rhomboid nucleus (RH) located dorsal to RE. Control injections were made in the central medial nucleus (CEM) of the thalamus. We showed that the output of RE is almost entirely directed to the hippocampus and "limbic" cortical structures. Specifically, RE projects strongly to the medial frontal polar, anterior piriform, medial and ventral orbital, anterior cingulate, prelimbic, infralimbic, insular, perirhinal, and entorhinal cortices as well as to CA1, dorsal and ventral subiculum, and parasubiculum of the hippocampus. RH distributes more widely than RE, that is, to several RE targets but also significantly to regions of motor, somatosensory, posterior parietal, retrosplenial, temporal, and occipital cortices; to nucleus accumbens; and to the basolateral nucleus of amygdala. The ventral midline thalamus is positioned to exert significant control over fairly widespread regions of the cortex (limbic, sensory, motor), hippocampus, dorsal and ventral striatum, and basal nuclei of the amygdala, possibly to coordinate limbic and sensorimotor functions. We suggest that RE/RH may represent an important conduit in the exchange of information between subcortical-cortical and cortical-cortical limbic structures potentially involved in the selection of appropriate responses to specific and changing sets of environmental conditions.

The present study investigated the short-term and long-term synaptic plasticity of excitatory synapses formed by the nucleus reuniens (RE) and entorhinal cortex (EC) on the distal apical dendrites of CA1 pyramidal cells. RE-CA1 synapses are implicated in memory involving the hippocampus and medial prefrontal cortex. Current source density (CSD) analysis was used to identify excitatory and inhibitory currents following stimulation of RE or medial perforant path (MPP) in urethane-anesthetized mice in vivo. At the distal apical dendrites, RE evoked an initial excitatory sink followed by inhibitory sources at short (~ 30ms) and long (150-200ms) latencies, and often showing gamma (25-40Hz) oscillations. Both RE-evoked and spontaneous gamma-frequency local field potentials displayed the same CSD depth profile. Paired-pulse facilitation (PPF) of the distal excitatory sink at 20-200ms interpulse intervals was observed following RE stimulation, generally higher than that following MPP stimulation. Theta-frequency burst stimulation (TBS) of RE induced input-specific long-term potentiation (LTP) at the distal dendritic CA1 synapses, accompanied by reduction of PPF. After TBS of the MPP, the MPP-CA1 distal dendritic synapse could manifest LTP or long-term depression, but the non-tetanized RE-CA1 synapse was typically potentiated. Heterosynaptic potentiation of the RE to CA1 distal synapses may occur after repeated activity of EC afferents, or spread of MPP stimulus currents to coursing RE afferents. The results indicate a propensity of RE-CA1 distal excitatory synapses to show PPF, LTP and gamma oscillations, all of which may participate in memory processing by RE and EC.

Nucleus reuniens mediates the extinction of contextual fear conditioning