Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

In Vivo Characterization of Neurophysiological Diversity in the Lateral Supramammillary Nucleus during Hippocampal Sharp-wave Ripples of Adult Rats.

The extent of the networks that control the genesis and modulation of hippocampal sharp-wave ripples (SPW-Rs), which are involved in memory consolidation, remains incompletely understood. Here, we performed a detailed in vivo analysis of single cell firing in the lateral supramammillary nucleus (lSuM) during theta and slow oscillations, including SPW-Rs, in anesthetized rats. We classified neurons as SPW-R-active and SPW-R-unchanged according to whether or not they increased their firing during SPW-Rs. We show that lSuM SPW-R-active neurons increase their firing prior to SPW-Rs peak power and prior to hippocampal excitatory cell activation. Moreover, lSuM SPW-R-active neurons show increased firing activity during theta and slow oscillations as compared to unchanged neurons. These results suggest that a sub-population of lSuM neurons can interact with the hippocampus during SPW-Rs, raising the possibility that the lSuM may modulate memory consolidation.

Computing hubs in the hippocampus and cortex.

Neural computation occurs within large neuron networks in the dynamic context of varying brain states. Whether functions are performed by specific subsets of neurons and whether they occur in specific dynamical regimes remain poorly understood. Using high-density recordings in the hippocampus, medial entorhinal, and medial prefrontal cortex of the rat, we identify computing substates where specific computing hub neurons perform well-defined storage and sharing operations in a brain state-dependent manner. We retrieve distinct computing substates within each global brain state, such as REM and nonREM sleep. Half of recorded neurons act as computing hubs in at least one substate, suggesting that functional roles are not hardwired but reassigned at the second time scale. We identify sequences of substates whose temporal organization is dynamic and stands between order and disorder. We propose that global brain states constrain the language of neuronal computations by regulating the syntactic complexity of substate sequences.

The Nucleus Reuniens Controls Long-Range Hippocampo-Prefrontal Gamma Synchronization during Slow Oscillations.

Gamma oscillations are involved in long-range coupling of distant regions that support various cognitive operations. Here we show in adult male rats that synchronized bursts of gamma oscillations bind the hippocampus (HPC) and prefrontal cortex (mPFC) during slow oscillations and slow-wave sleep, a brain state that is central for consolidation of memory traces. These gamma bursts entrained the firing of the local HPC and mPFC neuronal populations. Neurons of the nucleus reuniens (NR), which is a structural and functional hub between HPC and mPFC, demonstrated a specific increase in their firing before gamma burst onset, suggesting their involvement in HPC-mPFC binding. Chemical inactivation of NR disrupted the temporal pattern of gamma bursts and their synchronization, as well as mPFC neuronal firing. We propose that the NR drives long-range hippocampo-prefrontal coupling via gamma bursts providing temporal windows for information exchange between the HPC and mPFC during slow-wave sleep.SIGNIFICANCE STATEMENT Long-range coupling between hippocampus (HPC) and prefrontal cortex (mPFC) is believed to support numerous cognitive functions, including memory consolidation occurring during sleep. Gamma-band synchronization is a fundamental process in many neuronal operations and is instrumental in long-range coupling. Recent evidence highlights the role of nucleus reuniens (NR) in consolidation; however, how it influences hippocampo-prefrontal coupling is unknown. In this study, we show that HPC and mPFC are synchronized by gamma bursts during slow oscillations in anesthesia and natural sleep. By manipulating and recording the NR-HPC-mPFC network, we provide evidence that the NR actively promotes this long-range gamma coupling. This coupling provides the hippocampo-prefrontal circuit with a novel mechanism to exchange information during slow-wave sleep.

Visual Perception in Anterior Temporal Lobectomy.

PURPOSE: To study visual perception in patients with anterior temporal lobectomy. MATERIALS AND METHODS: We explored some aspects of visual perception and compared the results obtained from 14 control subjects and 14 patients with unilateral anterior temporal lobectomy. Each group included 7 men and 7 women and the same age distribution (patients and controls: age range 27-48 years; mean 37 years). All subjects underwent a conventional ophthalmic examination and were tested for color perception, stereopsis, texture perception, face recognition, and visual illusions. To quantify color, stereoscopic, and texture perception they performed a visuomotor task that required a rapid response to a visual stimulus. Reaction times were measured under several conditions. RESULTS: Mild visual field defects involving the superior quadrant contralateral to the lobectomy were found in five patients; two other patients presented more severe defects. Lobectomized patients showed a lower number of correct trials than normal subjects when performing tasks involving color and texture perception. These patients also had longer reaction times for color, stereoscopic, and texture stimulus detection. Face recognition and perception of illusory images were preserved after unilateral anterior temporal lobectomy. CONCLUSION: Our data indicate that patients with anterior temporal lobectomy show moderate deficits in color, stereo, and texture perception, with no impairment in complex visual stimuli perception.

Neural activity in monkey amygdala during performance of a multisensory operant task.

In this paper, we study the potential involvement of monkey amygdala in the evaluation of value encoding of visual and auditive stimuli associated with reward or no reward. We recorded the activity of 93 extracellular neurons from the monkey right amygdala, while performing a multisensory operant task. The activity of 78 task-related neurons was studied. Of these, 13 neurons (16%) responded to the value of visual stimuli, 22 neurons (28%) responded after the presentation of visual stimuli, 22 neurons (28%) showed an inhibition around the lever-pressing and were classified as action related neurons and 22 neurons (28%) responded after reward delivery. These findings suggest that neurons in the amygdala play a role in encoding value and processing visual information, participate in motor regulation and are sensitive to reward. The activity of these neurons did not change in the evaluation of auditive stimuli. These data support the hypothesis that amygdala neurons are specific to each sensory modality and that different groups of amygdala neurons process visual and auditive information.

Putamen neurons process both sensory and motor information during a complex task.

The putamen has classically been considered to be primarily a motor structure. It is involved in a broad range of roles and its neurons have been postulated to function as pattern classifiers of behaviourally significant events. However, its specific role in motor and sensory processing is still unclear. For the purpose of better categorizing putamen neurons, we trained two rhesus monkeys to perform multisensory operant tasks by using complex stimuli such as short videoclips. Trials involved image or soundtrack or both. Some stimuli required a motor response associated to reward, whereas others did not require response and produced no reward. We found that neurons in the putamen showed pure visual responses, action-related activity, and reward responses. Insofar as action-related activity, preparation of movement, movement execution, and withholding of movement involved three different putamen neuron populations. Moreover, our data suggest an involvement of putamen neurons in processing primary rewards and visual events in a complex task, which may contribute to reinforcement learning through stimulus-reward association.

Time course of cold cataract development in anesthetized mice.

PURPOSE: The aim of the present study was to study the time-course development of cold cataract in mice under general anesthesia. METHODS: We anesthetized five groups of 10 mice (12 weeks old) with 400 mg/Kg intraperitoneal injections of chloral hydrate and exposed them to 0, 7, 15, 23, and 37°C for 1 hr. Cataract development was assessed and graded as no cataract, mild, medium, or severe at 10, 20, 30, 45, and 60 min after the exposure started. For quantification purposes, a value from 0 to 3 was assigned to each cataract grade, and the median value was calculated for each group and time point (cataract index, CI). RESULTS: The CI for each temperature fitted a negative exponential equation. We found that four mice of the 37°C group, nine of the 23°C group, and all animals of the 15, 7, and 0°C groups developed cataract. The cataract started at 10 min after exposure to 0°C and at 20 min when exposed to 7, 15, and 23°C. The speed of development and CI significantly increased with lower temperatures. Similar results were observed when the procedure was repeated 48 hr later in the 15, 23, and 37°C groups. In all instances the cataract was reversible. CONCLUSION: Our findings show that cold cataract development is temperature dependent and that cataract formation starts between 10 and 20 min after exposure to low temperature. This finding is relevant for those experimental settings in which clear ocular media are required.

Orientation preference and horizontal disparity sensitivity in the monkey visual cortex.

PURPOSE: Disparity sensitivity may be explained by interocular positional differences of the receptive fields (RF) of visual cortical cells or by interocular shifts of the On and Off RF subregions. Since this latter model assumes shifts are orthogonal to the orientation of the RF, cells with disparity sensitivity should be oriented. The objective of the present study is to test this assumption. METHODS: Single unit recordings were performed in areas V1 and V2 of two Macaca mulatta. For assessing disparity sensitivity, we generated dynamic random dot stereograms. A stereofigure was flashed over the cell RF with different horizontal disparities. To assess orientation sensitivity we used a flashing bar with eight orientations, in several positions over the cell RF in a pseudorandom manner. RESULTS: We found no relationship between sensitivity to horizontal disparity and orientation preference in V1 and V2 cells. CONCLUSIONS: Our data indicate that horizontal disparity sensitivity and orientation preference are unrelated properties. This favors the notion that sensitivity to horizontal disparity is mostly based on RF interocular horizontal positional differences.

Spatial frequency components influence cell activity in the inferotemporal cortex.

We studied the correlation between the spatial frequency of complex stimuli and neuronal activity in the monkey inferotemporal (IT) cortex while performing a task that required visual recognition. Single-cell activity was recorded from the right IT cortex. The frequency components of the images used as stimuli were analyzed by using a fast Fourier transform, and a modulus was obtained for 40 spatial frequency ranges from 0.3 to 11.1 cycles/deg. We recorded 82 cells showing statistically significant responses (analysis of variance, P < 0.05) to at least one of the images used as a stimulus. Seventy-eight percent of these cells (n = 64) showed significant responses to at least three images, and in two thirds of them (n = 42), we found a statistically significant correlation (P < 0.05) between cell response and the modulus amplitude of at least one frequency range present in the images. Our results suggest that information about spatial frequency of the visual images is present in the IT cortex.

Chloral hydrate anesthesia and lens opacification in mice.

PURPOSE: To study the effect of chloral hydrate in the eye lens in mice. METHODS: We instilled tropicamide in the eyes of two groups of eight 129 strain wild-type mice and injected chloral hydrate intraperitoneally (400 mg/kg). A drop of hypromellosum solution was additionally instilled in the eye of the second group to reduce corneal dehydration. The lens was inspected at several time intervals with a standard slit lamp biomicroscope after the injection. RESULTS: No lens opacification was found in any animal after the topical administration of tropicamide. On the contrary, the intraperitoneal administration of chloral hydrate produced lens opacification in both groups of animals. CONCLUSIONS: We show that chloral hydrate, when used intraperitoneally, induces acute and reversible lens opacification in all animals. In addition, we found that instillation of hypromellose solution does not prevent the lens opacification caused by chloral hydrate injection. We hypothesized some plausible explanations for these findings.

Activity of neurons in the caudate and putamen during a visuomotor task.

Evidence supporting a role of the caudate and putamen nuclei in associative learning is present. We recorded the activity of 21 caudate and 26 putamen cells in one macaque monkey while performing a visuomotor task, which involved a visual stimulus and the execution of a motor response. Ninety-one percent of caudate cells and 65% of putamen cells showed changes in activity while the monkey was performing the task. Approximately half of the caudate cells and one third of the putamen cells showed changes in activity without a motor response. Our results show that caudate and putamen cells are activated regardless of the presence or absence of a motor action. These findings are consistent with the idea that these nuclei may play a role in associative learning.