Disconnecting prefrontal cortical neurons from the ventral midline thalamus: Loss of specificity due to progressive neural toxicity of an AAV-Cre in the rat thalamus.

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.

Inhibition of the ventral midline thalamus does not alter encoding, short-term holding or retrieval of spatial information in rats performing a water-escape working memory task.

Working memory (WM) is a function operating in three successive phases: encoding (sample trial), holding (delay), and retrieval (test trial) of information. Studies point to a possible implication of the thalamic reuniens nucleus (Re) in spatial WM (SWM). In which of the aforementioned 3 phases the Re has a function is largely unknown. Recently, in a delayed SWM water-escape task, we found that performance during the retrieval trial correlated positively with c-Fos expression in the Re nucleus, suggesting participation in retrieval. Here, we used the same task and muscimol (MUSC) inhibition or DREADD(hM4Di)-mediated inhibition of the Re during information encoding, right thereafter (thereby affecting the holding phase), or during the retrieval trial. A 6-hour delay separated encoding from retrieval. Concerning SWM, MUSC in the Re nucleus did not alter performance, be it during or after encoding, or during evaluation. CNO administered before encoding in DREADD-expressing rats was also ineffective, although CNO-induced inhibition disrupted set shifting performance, as found previously (Quet et al., Brain Struct Function 225, 2020), thereby validating DREADD efficiency. These findings are the first that do not support an implication of the Re nucleus in SWM. As most previous studies used T-maze alternation tasks, which carry high proactive interference risks, an important question to resolve now is whether the Re nucleus is required in (T-maze alternation) tasks using very short information-holding delays (seconds to minutes), and less so in other short-term spatial memory tasks with longer information holding intervals (hours) and therefore reduced interference risks.

Ventral midline thalamus activation is correlated with memory performance in a delayed spatial matching-to-sample task: A c-Fos imaging approach in the rat.

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.

The reuniens and rhomboid nuclei of the thalamus: A crossroads for cognition-relevant information processing?

Over the past twenty years, the reuniens and rhomboid (ReRh) nuclei, which constitute the ventral midline thalamus, have received constantly growing attention. Since our first review article about the functional contributions of ReRh nuclei (Cassel et al., 2013), numerous (>80) important papers have extended anatomical knowledge, including at a developmental level, introduced new and very original electrophysiological insights on ReRh functions, and brought novel results on cognitive and non-cognitive implications of the ReRh. The current review will cover these recent articles, more on Re than on Rh, and their contribution will be approached according to their affiliation with work before 2013. These neuroanatomical, electrophysiological or behavioral findings appear coherent and point to the ReRh nuclei as two major components of a multistructural system supporting numerous cognitive (and non-cognitive) functions. They gate the flow of information, perhaps especially from the medial prefrontal cortex to the hippocampus and back, and coordinate activity and processing across these two (and possibly other) brain regions of major cognitive relevance.

Routes of the thalamus through the history of neuroanatomy.

The most distant roots of neuroanatomy trace back to antiquity, with the first human dissections, but no document which would identify the thalamus as a brain structure has reached us. Claudius Galenus (Galen) gave to the thalamus the name 'thalamus nervorum opticorum', but later on, other names were used (e.g., anchae, or buttocks-like). In 1543, Andreas Vesalius provided the first quality illustrations of the thalamus. During the 19th century, tissue staining techniques and ablative studies contributed to the breakdown of the thalamus into subregions and nuclei. The next step was taken using radiomarkers to identify connections in the absence of lesions. Anterograde and retrograde tracing methods arose in the late 1960s, supporting extension, revision, or confirmation of previously established knowledge. The use of the first viral tracers introduced a new methodological breakthrough in the mid-1970s. Another important step was supported by advances in neuroimaging of the thalamus in the 21th century. The current review follows the history of the thalamus through these technical revolutions from Antiquity to the present day.

The ventral midline thalamus contributes to strategy shifting in a memory task requiring both prefrontal cortical and hippocampal functions.

Electrophysiological and neuroanatomical evidence for reciprocal connections with the medial prefrontal cortex (mPFC) and the hippocampus make the reuniens and rhomboid (ReRh) thalamic nuclei a putatively major functional link for regulations of cortico-hippocampal interactions. In a first experiment using a new water escape device for rodents, the double-H maze, we demonstrated in rats that a bilateral muscimol (MSCI) inactivation (0.70 vs 0.26 and 0 nmol) of the mPFC or dorsal hippocampus (dHip) induces major deficits in a strategy shifting/spatial memory retrieval task. By way of comparison, only dHip inactivation impaired recall in a classical spatial memory task in the Morris water maze. In the second experiment, we showed that ReRh inactivation using 0.70 nmol of MSCI, which reduced performance without obliterating memory retrieval in the water maze, produces an as large strategy shifting/memory retrieval deficit as mPFC or dHip inactivation in the double-H maze. Thus, behavioral adaptations to task contingency modifications requiring a shift toward the use of a memory for place might operate in a distributed circuit encompassing the mPFC (as the potential set-shifting structure), the hippocampus (as the spatial memory substrate), and the ventral midline thalamus, and therein the ReRh (as the coordinator of this processing). The results of the current experiments provide a significant extension of our understanding of the involvement of ventral midline thalamic nuclei in cognitive processes: they point to a role of the ReRh in strategy shifting in a memory task requiring cortical and hippocampal functions and further elucidate the functional system underlying behavioral flexibility.

Whole-body exposure to 2.45 GHz electromagnetic fields does not alter 12-arm radial-maze with reduced access to spatial cues in rats.

Lai et al. [Lai H, Horita A, Guy AW. Microwave irradiation affects radial-arm maze performance in the rat. Bioelectromagnetics 1994;15(2):95-104] reported that exposure of rats to pulsed 2.45 GHz microwaves altered maze performance. Their maze was bordered by 20 cm high opaque walls. Using a maze test based on unrestrained access to spatial cues (no walls), we could not replicate this result [Cassel JC, Cosquer B, Galani R, Kuster N. Whole-body exposure to 2.45 GHz electromagnetic fields does not alter radial-maze performance in rats. Behav Brain Res 2004;155:37-43]. Here, we attempted another replication using a maze apparatus bordered by 30 cm high opaque walls. Performance of exposed rats was normal. These results show that microwave exposure as used herein does not alter spatial working memory, when access to spatial cues is reduced.

Cognitive deficits in aged rats correlate with levels of L-arginine, not with nNOS expression or 3,4-DAP-evoked transmitter release in the frontoparietal cortex.

Aging is associated with altered neurotransmitter function in the brain. In this study, we measured release parameters for acetylcholine (ACh), norepinephrine and serotonin in the frontoparietal cortex of young and aged rats. We also determined cortical amino acid concentrations and nitric oxide (NO) synthase function. Prior to sacrifice, the rats had been tested for Morris water-maze performance. In aged, compared with young rats, we observed a reduction in both uptake of choline and acetylcholine release. Serotonin release and L-arginine concentrations (a precursor of NO) showed an aging-related increase; however, L-citrulline/L-arginine ratios were decreased in aged rats. Moreover, while most age-related changes in transmitter release or neurochemical markers were not related to the learning performance, L-arginine concentrations were positively correlated to cognitive deficits. NO synthase concentrations were not affected by aging. It is suggested that events related to L-arginine-to-L-citrulline/NO metabolism in the frontoparietal cortex may take part in age-related cognitive deficits.

Whole-body exposure to 2.45 GHz electromagnetic fields does not alter anxiety responses in rats: a plus-maze study including test validation.

In a first phase of this investigation, a validation of our elevated plus-maze apparatus was performed in male Sprague-Dawley rats by testing anxiety response at various ambient light intensities (200, 30, 10 and 2.5 lux), as well as the effects of diazepam treatment (0.5 and 1.0 mg/kg, i.p. at 30 lux). Anxiety responses were found to decrease with decreasing light intensity and to be attenuated by diazepam treatment. Subsequently, a separate set of rats was exposed to 2.45 GHz EMFs (2 micros pulse width, 500 pulses per second, whole-body and time averaged of SAR 0.6 W/kg +/-2 dB, brain-averaged SAR of 0.9 W/kg +/-3 dB) for 45 min to assess whether EMF exposure altered anxiety responses in the same apparatus. As we made no a priori hypothesis on whether the effects would be anxiogenic or anxiolytic, part of the rats were tested under an ambient light intensity of 2.5 lux, the other one being tested at 30 lux. The low intensity level set the behavioural baseline for the detection of anxiogenic effects, while the higher one corresponded to the detection of anxiolytic effects. Sham-exposed and naive rats were used as controls. Whatever light intensity was used, EMF exposure failed to induce any significant effect on anxiety responses in the plus maze. The present experiment demonstrates that exposure to EMFs, which was previously found to increase the number of benzodiazepine receptors in the rat cortex [Lai H, Carino MA, Horita A, Guy AW. Single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat. Bioelectromagnetics 1992;13(1):57-66], does not alter anxiety responses assessed in the elevated plus maze.

Whole-body exposure to 2.45 GHz electromagnetic fields does not alter radial-maze performance in rats.

Mobile communication is based on utilization of electromagnetic fields (EMFs) in the frequency range of 0.3-300 GHz. Human and animal studies suggest that EMFs, which are in the 0.1 MHz-300 GHz range, might interfere with cognitive processes. In 1994, a report by Lai et al. [Bioelectromagnetics 15 (1994) 95-104] showed that whole-body exposure of rats to pulsed 2.45 GHz microwaves (2 micros pulse width, 500 pps, and specific absorption rate [SAR] 0.6 W/kg) for 45 min resulted in altered spatial working memory assessed in a 12-arm radial-maze task. Surprisingly, there has been only one attempt to replicate this experiment so far [Bioelectromagnetics 25 (2004) 49-57]; confirmation of the Lai et al. experiment failed. In the present study, rats were tested in a 12-arm radial-maze subsequently to a daily exposure to 2.45 GHz microwaves (2 micros pulse width, 500 pps, and SAR 0.6 W/kg) for 45 min. The performance of exposed rats was comparable to that found in sham-exposed or in naive rats (no contact with the exposure system). Regarding the methodological details provided by Lai et al. on their testing protocol, our results might suggest that the microwave-induced behavioral alterations measured by these authors might have had more to do with factors liable to performance bias than with spatial working memory per se.