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Background: The thalamic reuniens (Re) and rhomboid (Rh) nuclei are bidirectionally connected with the medial prefrontal cortex (mPFC) and the hippocampus (Hip). Fiber-sparing N-methyl-D-aspartate lesions of the ReRh disrupt cognitive functions, including persistence of certain memories. Because such lesions irremediably damage neurons interconnecting the ReRh with the mPFC and the Hip, it is impossible to know if one or both pathways contribute to memory persistence. Addressing such an issue requires selective, pathway-restricted and direction-specific disconnections., New Method: A recent method associates a retrograde adeno-associated virus (AAV) expressing Cre recombinase with an anterograde AAV expressing a Cre-dependent caspase, making such disconnection feasible by caspase-triggered apoptosis when both constructs meet intracellularly. We injected an AAVrg-Cre-GFP into the ReRh and an AAV5-taCasp into the mPFC. As expected, part of mPFC neurons died, but massive neurotoxicity of the AAVrg-Cre-GFP was found in ReRh, contrasting with normal density of DAPI staining. Other stainings demonstrated increasing density of reactive astrocytes and microglia in the neurodegeneration site., Comparison With Existing Methods: Reducing the viral titer (by a 4-fold dilution) and injection volume (to half) attenuated toxicity substantially, still with evidence for partial disconnection between mPFC and ReRh., Conclusions: There is an imperative need to verify potential collateral damage inherent in this type of approach, which is likely to distort interpretation of experimental data. Therefore, controls allowing to distinguish collateral phenotypic effects from those linked to the desired disconnection is essential. It is also crucial to know for how long neurons expressing the Cre-GFP protein remain operational post-infection., Competing Interests: Declaration of Competing Interest none. Conflict of interest The authors have no conflict of interest to declare. Declaration of Generative (AI) and AI-assisted technologies in the Writing Process. No such technologies have been used in the writing process., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The midline thalamus is critical for flexible cognition, memory, and stress regulation in humans and its dysfunction is associated with several neurological and psychiatric disorders, including Alzheimer's disease, schizophrenia, and depression. Despite the pervasive role of the midline thalamus in cognition and disease, there is a limited understanding of its function in humans, likely due to the absence of a rigorous noninvasive neuroimaging methodology to identify its location. Here, we introduce a new method for identifying the midline thalamus in vivo using probabilistic tractography and k-means clustering with diffusion weighted imaging data. This approach clusters thalamic voxels based on data-driven cortical and subcortical connectivity profiles and then segments the midline thalamus according to anatomical connectivity tracer studies in rodents and macaques. Results from two different diffusion weighted imaging sets, including adult data (22-35 years) from the Human Connectome Project (n = 127) and adolescent data (9-14 years) collected at Florida International University (n = 34) showed that this approach reliably classifies midline thalamic clusters. As expected, these clusters were most evident along the dorsal/ventral extent of the third ventricle and were primarily connected to the agranular medial prefrontal cortex (e.g., anterior cingulate cortex), nucleus accumbens, and medial temporal lobe regions. The midline thalamus was then bisected based on a human brain atlas into a dorsal midline thalamic cluster (paraventricular and paratenial nuclei) and a ventral midline thalamic cluster (rhomboid and reuniens nuclei). This anatomical connectivity-based identification of the midline thalamus offers the opportunity for necessary investigation of this region in vivo in the human brain and how it relates to cognitive functions in humans, and to psychiatric and neurological disorders., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Multiple cognitive operations are associated with the emergence of gamma oscillations in the medial prefrontal cortex (mPFC) although little is known about the mechanisms that control this rhythm. Using local field potential recordings from cats, we show that periodic bursts of gamma recur with 1 Hz regularity in the wake mPFC and are locked to the exhalation phase of the respiratory cycle. Respiration organizes long-range coherence in the gamma band between the mPFC and the nucleus reuniens the thalamus (Reu), linking the prefrontal cortex and the hippocampus. In vivo intracellular recordings of the mouse thalamus reveal that respiration timing is propagated by synaptic activity in Reu and likely underlies the emergence of gamma bursts in the prefrontal cortex. Our findings highlight breathing as an important substrate for long-range neuronal synchronization across the prefrontal circuit, a key network for cognitive operations., (© 2023. The Author(s).)

The paraventricular nucleus (PVT) of the midline thalamus is a critical higher-order cortico-thalamo-cortical integration site that plays a critical role in various behaviors including reward seeking, cue saliency, and emotional memory. Anatomical studies have shown that PVT projects to both medial prefrontal cortex (mPFC) and hippocampus (HC). However, dual mPFC-HC projecting neurons which could serve a role in synchronizing mPFC and HC activity during PVT-dependent behaviors, have not been explored. Here we used a dual retrograde adenoassociated virus (AAV) tracing approach to characterize the location and proportion of different projection populations that send collaterals to mPFC and/or ventral hippocampus (vHC) in rats. Additionally, we examined the distribution of calcium binding proteins calretinin (CR) and calbindin (CB) with respect to these projection populations in PVT. We found that PVT contains separate populations of cells that project to mPFC, vHC, and those that innervate both regions. Interestingly, dual mPFC-HC projecting cells expressed neither CR nor CB. Topographically, CB + and CR + containing cells clustered around dual projecting neurons in PVT. These results are consistent with the features of dual mPFC-vHC projecting cells in the nucleus reuniens (RE) and suggestive of a functional mPFC-PVT-vHC system that may support mPFC-vHC interactions in PVT-dependent behaviors., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The reuniens (Re) and rhomboid (Rh) nuclei of the ventral midline thalamus are bi-directionally connected with the hippocampus and the medial prefrontal cortex. They participate in a variety of cognitive functions, including information holding for seconds to minutes in working memory tasks. What about longer delays? To address this question, we used a spatial working memory task in which rats had to reach a platform submerged in water. The platform location was changed every 2-trial session and rats had to use allothetic cues to find it. Control rats received training in a typical response-memory task. We interposed a 6 h interval between instruction (locate platform) and evaluation (return to platform) trials in both tasks. After the last session, rats were killed for c-Fos imaging. A home-cage group was used as additional control of baseline levels of c-Fos expression. C-Fos expression was increased to comparable levels in the Re (not Rh) of both spatial memory and response-memory rats as compared to their home cage counterparts. However, in spatial memory rats, not in their response-memory controls, task performance was correlated with c-Fos expression in the Re: the higher this expression, the better the performance. Furthermore, we noticed an activation of hippocampal region CA1 and of the anteroventral nucleus of the rostral thalamus. This activation was specific to spatial memory. The data point to a possible performance-determinant participation of the Re nucleus in the delayed engagement of spatial information encoded in a temporary memory., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The interactions between the medial prefrontal cortex (mPFC) and the hippocampus (HC) are critical for memory and decision making and have been specifically implicated in several neurological disorders including schizophrenia, epilepsy, frontotemporal dementia, and Alzheimer's disease. The ventral midline thalamus (vmThal), and lateral entorhinal cortex and perirhinal cortex (LEC/PER) constitute major communication pathways that facilitate mPFC-HC interactions in memory. Although vmThal and LEC/PER circuits have been delineated separately we sought to determine whether these two regions share cell-specific inputs that could influence both routes simultaneously. To do this we used a dual fluorescent retrograde tracing approach using cholera toxin subunit-B (CTB-488 and CTB-594) with injections targeting vmThal and the LEC/PER in rats. Retrograde cell body labeling was examined in key regions of interest within the mPFC-HC system including: (1) mPFC, specifically anterior cingulate cortex (ACC), dorsal and ventral prelimbic cortex (dPL, vPL), and infralimbic cortex (IL); (2) medial and lateral septum (MS, LS); (3) subiculum (Sub) along the dorsal-ventral and proximal-distal axes; and (4) LEC and medial entorhinal cortex (MEC). Results showed that dual vmThal-LEC/PER-projecting cell populations are found in MS, vSub, and the shallow layers II/III of LEC and MEC. We did not find any dual projecting cells in mPFC or in the cornu ammonis (CA) subfields of the HC. Thus, mPFC and HC activity is sent to vmThal and LEC/PER via non-overlapping projection cell populations. Importantly, the dual projecting cell populations in MS, vSub, and EC are in a unique position to simultaneously influence both cortical and thalamic mPFC-HC pathways critical to memory. SIGNIFICANCE STATEMENT: The interactions between mPFC and HC are critical for learning and memory, and dysfunction within this circuit is implicated in various neurodegenerative and psychiatric diseases. mPFC-HC interactions are mediated through multiple communication pathways including a thalamic hub through the vmThal and a cortical hub through lateral entorhinal cortex and perirhinal cortex. Our data highlight newly identified dual projecting cell populations in the septum, Sub, and EC of the rat brain. These dual projecting cells may have the ability to modify the information flow within the mPFC-HC circuit through synchronous activity, and thus offer new cell-specific circuit targets for basic and translational studies in memory., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The midline thalamus bidirectionally connects the medial prefrontal cortex (mPFC) and hippocampus (HC) creating a unique cortico-thalamo-cortical circuit fundamental to memory and executive function. While the anatomical connectivity of midline thalamus has been thoroughly investigated, little is known about its cellular organization within each nucleus. Here we used immunohistological techniques to examine cellular distributions in the midline thalamus based on the calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin (CB). We also examined these calcium binding proteins in a population of reuniens cells known to project to both mPFC and HC using a dual fluorescence retrograde adenoassociated virus-based tracing approach. These dual reuniens mPFC-HC projecting cells, in particular, are thought to be important for synchronizing mPFC and HC activity. First, we confirmed the absence of PV + neurons in the midline thalamus. Second, we found a common pattern of CR + and CB + cells throughout midline thalamus with CR + cells running along the nearby third ventricle (3V) and penetrating the midline. CB + cells were consistently more lateral and toward the middle of the dorsal-ventral extent of the midline thalamus. Notably, single-labeled CR + and CB + zones were partially overlapping and included dual-labeled CR + /CB + cells. Within RE, we also observed a CR and CB subzone specific diversity. Interestingly, dual mPFC-HC projecting neurons in RE expressed none of the calcium binding proteins examined, but were contained in nests of CR + and CB + cells. Overall, the midline thalamus was well organized into CR + and CB + rich zones distributed throughout the region, with dual mPFC-HC projecting cells in reuniens representing a unique cell population. These results provide a cytoarchitectural organization in the midline thalamus based on calcium binding protein expression, and set the stage for future cell-type specific interrogations of the functional role of these different cell populations in mPFC-HC interactions., (© 2020 Wiley Periodicals LLC.)

Inhibitory circuits in thalamus and cortex shape the major activity patterns observed by electroencephalogram (EEG) in the adult brain. Their delayed maturation and circuit integration, relative to excitatory neurons, suggest inhibitory neuronal development could be responsible for the onset of mature thalamocortical activity. Indeed, the immature brain lacks many inhibition-dependent activity patterns, such as slow-waves, delta oscillations and sleep-spindles, and instead expresses other unique oscillatory activities in multiple species including humans. Thalamus contributes significantly to the generation of these early oscillations. Compared to the abundance of studies on the development of inhibition in cortex, however, the maturation of thalamic inhibition is poorly understood. Here we review developmental changes in the neuronal and circuit properties of the thalamic relay and its interconnected inhibitory thalamic reticular nucleus (TRN) both in vitro and in vivo, and discuss their potential contribution to early network activity and its maturation. While much is unknown, we argue that weak inhibitory function in the developing thalamus allows for amplification of thalamocortical activity that supports the generation of early oscillations. The available evidence suggests that the developmental acquisition of critical thalamic oscillations such as slow-waves and sleep-spindles is driven by maturation of the TRN. Further studies to elucidate thalamic GABAergic circuit formation in relation to thalamocortical network function would help us better understand normal as well as pathological brain development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

How our internal state is merged with our visual perception of an impending threat to drive an adaptive behavioural response is not known. Mice respond to visual threats by either freezing or seeking shelter. Here we show that nuclei of the ventral midline thalamus (vMT), the xiphoid nucleus (Xi) and nucleus reuniens (Re), represent crucial hubs in the network controlling behavioural responses to visual threats. The Xi projects to the basolateral amygdala to promote saliency-reducing responses to threats, such as freezing, whereas the Re projects to the medial prefrontal cortex (Re→mPFC) to promote saliency-enhancing, even confrontational responses to threats, such as tail rattling. Activation of the Re→mPFC pathway also increases autonomic arousal in a manner that is rewarding. The vMT is therefore important for biasing how internal states are translated into opposing categories of behavioural responses to perceived threats. These findings may have implications for understanding disorders of arousal and adaptive decision-making, such as phobias, post-traumatic stress and addictions.

Spatial navigation requires information about the relationship between current and future positions. The activity of hippocampal neurons appears to reflect such a relationship, representing not only instantaneous position but also the path towards a goal location. However, how the hippocampus obtains information about goal direction is poorly understood. Here we report a prefrontal-thalamic neural circuit that is required for hippocampal representation of routes or trajectories through the environment. Trajectory-dependent firing was observed in medial prefrontal cortex, the nucleus reuniens of the thalamus, and the CA1 region of the hippocampus in rats. Lesioning or optogenetic silencing of the nucleus reuniens substantially reduced trajectory-dependent CA1 firing. Trajectory-dependent activity was almost absent in CA3, which does not receive nucleus reuniens input. The data suggest that projections from medial prefrontal cortex, via the nucleus reuniens, are crucial for representation of the future path during goal-directed behaviour and point to the thalamus as a key node in networks for long-range communication between cortical regions involved in navigation.

Cholinergic interneurons are the only known source of acetylcholine in the rat nucleus accumbens (nAcb); yet there is little anatomical data about their mode of innervation and the origin of their excitatory drive. We characterized the cholinergic and thalamic innervations of nAcb with choline acetyltransferase (ChAT) immunocytochemistry and anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L) from the midline/intralaminar/paraventricular thalamic nuclei. The use of a monoclonal ChAT antiserum against whole rat ChAT protein allowed for an optimal visualization of the small dendritic branches and fine varicose axons of cholinergic interneurons. PHA-L-labeled thalamic afferents were heterogeneously distributed throughout the core and shell regions of nAcb, overlapping regionally with cholinergic somata and dendrites. At the ultrastructural level, several hundred single-section profiles of PHA-L and ChAT-labeled axon terminals were analyzed for morphology, synaptic frequency, and the nature of their synaptic targets. The cholinergic profiles were small and apposed to various neuronal elements, but rarely exhibited a synaptic membrane specialization (5% in single ultrathin sections). Stereological extrapolation indicated that less than 15% of these cholinergic varicosities were synaptic. The PHA-L-labeled profiles were comparatively large and often synaptic (37% in single ultrathin sections), making asymmetrical contacts primarily with dendritic spines (>90%). Stereological extrapolation indicated that all PHA-L-labeled terminals were synaptic. In double-labeled material, some PHA-L-labeled terminals were directly apposed to ChAT-labeled somata or dendrites, but synapses were never seen between the two types of elements. These observations demonstrate that the cholinergic innervation of rat nAcb is largely asynaptic. They confirm that the afferents from midline/intralaminar/paraventricular thalamic nuclei to rat nAcb synapse mostly on dendritic spines, presumably of medium spiny neurons, and suggest that the excitatory drive of nAcb cholinergic interneurons from thalamus is indirect, either via substance P release from recurrent collaterals of medium spiny neurons and/or by extrasynaptic diffusion of glutamate.

Unlike the basal ganglia input from the midline and intralaminar nuclei, the origin and prominence of striatal projections arising in the lateral thalamus varies considerably among mammals being most restricted in the opossum and monkey, most extensive in the rat. To get further insight into the evolution of thalamo-striatal pathways the Madagascar lesser hedgehog tenrec (Afrotheria) was investigated using anterograde and retrograde flow techniques. An extensive medial thalamic region (including presumed equivalents to the paraventricular, parataenial and dorsomedial nuclei as well as the reuniens complex), the rostral (central) and caudal (parafascicular) intralaminar nuclei were shown to give rise to striatal projections. Additional projections originated in the ventral anterolateral nuclear group and regions within and around the medial geniculate complex. Similar to the rat there was also substantial projections from the lateral posterior-pulvinar complex and the ventral posterior nucleus. The fibers terminated extensively across the striatum in a mainly homogeneous fashion. Isolated patches of low-density terminations were found in the caudoputamen. This inhomogeneous labeling pattern appeared similar to one described in the cat with the unlabeled islands showing features of striosomes. The medial and intralaminar nuclei also projected heavily upon the olfactory tubercle. Differential innervation patterns were noted in the polymorphous layer, the deep and the superficial molecular layer.

By utilizing new information from both clinical and experimental (lesion, electrophysiological, and gene-activation) studies with animals, the anatomy underlying anterograde amnesia has been reformulated. The distinction between temporal lobe and diencephalic amnesia is of limited value in that a common feature of anterograde amnesia is damage to part of an "extended hippocampal system" comprising the hippocampus, the fornix, the mamillary bodies, and the anterior thalamic nuclei. This view, which can be traced back to Delay and Brion (1969), differs from other recent models in placing critical importance on the efferents from the hippocampus via the fornix to the diencephalon. These are necessary for the encoding and, hence, the effective subsequent recall of episodic memory. An additional feature of this hippocampal-anterior thalamic axis is the presence of projections back from the diencephalon to the temporal cortex and hippocampus that also support episodic memory. In contrast, this hippocampal system is not required for tests of item recognition that primarily tax familiarity judgements. Familiarity judgements reflect an independent process that depends on a distinct system involving the perirhinal cortex of the temporal lobe and the medial dorsal nucleus of the thalamus. In the large majority of amnesic cases both the hippocampal-anterior thalamic and the perirhinal-medial dorsal thalamic systems are compromised, leading to severe deficits in both recall and recognition.