Encapsulation of the growth factor neurotrophin-3 in heparinised poloxamer hydrogel stabilises bioactivity and provides sustained release.

Poloxamer-based hydrogels show promise to stabilise and sustain the delivery of growth factors in tissue engineering applications, such as following spinal cord injury. Typically, growth factors such as neurotrophin-3 (NT-3) degrade rapidly in solution. Similarly, poloxamer hydrogels also degrade readily and are, therefore, only capable of sustaining the release of a payload over a small number of days. In this study, we focused on optimising a hydrogel formulation, incorporating both poloxamer 188 and 407, for the sustained delivery of bioactive NT-3. Hyaluronic acid blended into the hydrogels significantly reduced the degradation of the gel. We identified an optimal hydrogel composition consisting of 20 % w/w poloxamer 407, 5 % w/w poloxamer 188, 0.6 % w/w NaCl, and 1.5 % w/w hyaluronic acid. Heparin was chemically bound to the poloxamer chains to enhance interactions between the hydrogel and the growth factor. The unmodified and heparin-modified hydrogels exhibited sustained release of NT-3 for 28 days while preserving the bioactivity of NT-3. Moreover, these hydrogels demonstrated excellent cytocompatibility and had properties suitable for injection into the intrathecal space, underscoring their suitability as a growth factor delivery system. The findings presented here contribute valuable insights to the development of effective delivery strategies for therapeutic growth factors for tissue engineering approaches, including the treatment of spinal cord injury.

Translating ultrasound-mediated drug delivery technologies for CNS applications.

Ultrasound enhances drug delivery into the central nervous system (CNS) by opening barriers between the blood and CNS and by triggering release of drugs from carriers. A key challenge in translating setups from in vitro to in vivo settings is achieving equivalent acoustic energy delivery. Multiple devices have now been demonstrated to focus ultrasound to the brain, with concepts emerging to also target the spinal cord. Clinical trials to date have used ultrasound to facilitate the opening of the blood-brain barrier. While most have focused on feasibility and safety considerations, therapeutic benefits are beginning to emerge. To advance translation of these technologies for CNS applications, researchers should standardise exposure protocol and fine-tune ultrasound parameters. Computational modelling should be increasingly used as a core component to develop both in vitro and in vivo setups for delivering accurate and reproducible ultrasound to the CNS. This field holds promise for transformative advancements in the management and pharmacological treatment of complex and challenging CNS disorders.

Generation of direct current electrical fields as regenerative therapy for spinal cord injury: A review.

Electrical stimulation (ES) shows promise as a therapy to promote recovery and regeneration after spinal cord injury. ES therapy establishes beneficial electric fields (EFs) and has been investigated in numerous studies, which date back nearly a century. In this review, we discuss the various engineering approaches available to generate regenerative EFs through direct current electrical stimulation and very low frequency electrical stimulation. We highlight the electrode-tissue interface, which is important for the appropriate choice of electrode material and stimulator circuitry. We discuss how to best estimate and control the generated field, which is an important measure for comparability of studies. Finally, we assess the methods used in these studies to measure functional recovery after the injury and treatment. This work reviews studies in the field of ES therapy with the goal of supporting decisions regarding best stimulation strategy and recovery assessment for future work.

Neuron arbor geometry is sensitive to the limited-range fractal properties of their dendrites.

Fractal geometry is a well-known model for capturing the multi-scaled complexity of many natural objects. By analyzing three-dimensional images of pyramidal neurons in the rat hippocampus CA1 region, we examine how the individual dendrites within the neuron arbor relate to the fractal properties of the arbor as a whole. We find that the dendrites reveal unexpectedly mild fractal characteristics quantified by a low fractal dimension. This is confirmed by comparing two fractal methods-a traditional "coastline" method and a novel method that examines the dendrites' tortuosity across multiple scales. This comparison also allows the dendrites' fractal geometry to be related to more traditional measures of their complexity. In contrast, the arbor's fractal characteristics are quantified by a much higher fractal dimension. Employing distorted neuron models that modify the dendritic patterns, deviations from natural dendrite behavior are found to induce large systematic changes in the arbor's structure and its connectivity within a neural network. We discuss how this sensitivity to dendrite fractality impacts neuron functionality in terms of balancing neuron connectivity with its operating costs. We also consider implications for applications focusing on deviations from natural behavior, including pathological conditions and investigations of neuron interactions with artificial surfaces in human implants.

Investigating the influence of ultrasound parameters on ibuprofen drug release from hydrogels.

Hydrogels are promising ultrasound-responsive drug delivery systems. In this study, we investigated how different ultrasound parameters affected drug release and structural integrity of self-healing hydrogels composed of alginate or poloxamers. The effects of amplitude and duty cycle at low frequency (24 kHz) ultrasound stimulation were first investigated using alginate hydrogels at 2% w/v and 2.5% w/v. Increasing ultrasound amplitude increased drug release from these gels, although high amplitudes caused large variations in release and damaged the gel structure. Increasing duty cycle also increased drug release, although a threshold was observed with the lower pulsed 50% duty cycle achieving similar levels of drug release to a continuous 100% duty cycle. Poloxamer-based hydrogels were also responsive to the optimised parameters at low frequency (24 kHz, 20% amplitude, 50% duty cycle for 30 s) and showed similar drug release results to a 2.5% w/v alginate hydrogel. Weight loss studies demonstrated that the 2% w/v alginate hydrogel underwent significant erosion following ultrasound application, whereas the 2.5% w/v alginate and the poloxamer gels were unaffected by application of the same parameters (24 kHz, 20% amplitude, 50% duty cycle for 30 s). The rheological properties of the hydrogels were also unaffected and the FTIR spectra remained unchanged after low frequency ultrasound stimulation (24 kHz, 20% amplitude, 50% duty cycle for 30 s). Finally, high-frequency ultrasound stimulation (1 MHz, 3 W.cm-2, 50% duty cycle) was also trialled; the alginate gels were less responsive to this frequency, while no statistically significant impact on drug release was observed from the poloxamer gels. This study demonstrates the importance of ultrasound parameters and polymer selection in designing ultrasound-responsive hydrogels.

Investigating Fractal Analysis as a Diagnostic Tool That Probes the Connectivity of Hippocampal Neurons.

Many of nature's fractal objects benefit from the favorable functionality that results from their pattern repetition at multiple scales. Our recent research focused on the importance of fractal scaling in establishing connectivity between neurons. Fractal dimension D A of the neuron arbors was shown to relate to the optimization of competing functional constraints-the ability of dendrites to connect to other neurons versus the costs associated with building the dendrites. Here, we consider whether pathological states of neurons might affect this fractal optimization and if changes in D A might therefore be used as a diagnostic tool in parallel with traditional measures like Sholl analyses. We use confocal microscopy to obtain images of CA1 pyramidal neurons in the coronal plane of the dorsal rat hippocampus and construct 3-dimensional models of the dendritic arbors using Neurolucida software. We examine six rodent groups which vary in brain condition (whether they had lesions in the anterior thalamic nuclei, ATN) and experience (their housing environment and experience in a spatial task). Previously, we showed ATN lesions reduced spine density in hippocampal CA1 neurons, whereas enriched housing increased spine density in both ATN lesion and sham rats. Here, we investigate whether ATN lesions and experience also effect the complexity and connectivity of CA1 dendritic arbors. We show that sham rats exposed to enriched housing and spatial memory training exhibited higher complexity (as measured by D A ) and connectivity compared to other groups. When we categorize the rodent groups into those with or without lesions, we find that both categories achieve an optimal balance of connectivity with respect to material cost. However, the D A value used to achieve this optimization does not change between these two categories, suggesting any morphological differences induced by the lesions are too small to influence the optimization process. Accordingly, we highlight considerations associated with applying our technique to publicly accessible repositories of neuron images with a broader range of pathological conditions.

A Subdural Bioelectronic Implant to Record Electrical Activity from the Spinal Cord in Freely Moving Rats.

Bioelectronic devices have found use at the interface with neural tissue to investigate and treat nervous system disorders. Here, the development and characterization of a very thin flexible bioelectronic implant inserted along the thoracic spinal cord in rats directly in contact with and conformable to the dorsal surface of the spinal cord are presented. There is no negative impact on hind-limb functionality nor any change in the volume or shape of the spinal cord. The bioelectronic implant is maintained in rats for a period of 12 weeks. The first subdural recordings of spinal cord activity in freely moving animals are presented; rats are plugged in via a recording cable and allowed to freely behave and move around on a raised platform. Recordings contained multiple distinct voltage waveforms spatially localize to individual electrodes. This device has great potential to monitor electrical signaling in the spinal cord after an injury and in the future, this implant will facilitate the identification of biomarkers in spinal cord injury and recovery, while enabling the delivery of localized electroceutical and chemical treatments.

Dorsal CA1 hippocampal place cells form a multi-scale representation of megaspace.

Spatially firing "place cells" within the hippocampal CA1 region form internal maps of the environment necessary for navigation and memory. In rodents, these neurons have been almost exclusively studied in small environments (<4 m2). It remains unclear how place cells encode a very large open 2D environment that is commensurate with the natural environments experienced by rodents and other mammals. Such an ethologically realistic environment would require a complex spatial representation, capable of simultaneously representing space at multiple overlapping fine-to-coarse informational scales. Here, we show that in a "megaspace" (18.6 m2), the majority of dorsal CA1 place cells exhibited multiple place subfields of different sizes, akin to those observed along the septo-temporal axis. Furthermore, the total area covered by the subfields of each cell was not correlated with the number of subfields, and increased with the scale of the environment. The multiple different-sized subfields exhibited by place cells in the megaspace suggest that the ensemble population of subfields form a multi-scale representation of space within the dorsal hippocampus. Our findings point to a new dorsal hippocampus ensemble coding scheme that simultaneously supports navigational processes at both fine- and coarse-grained resolutions. VIDEO ABSTRACT.

Enriched Environment Procedures for Rodents: Creating a Standardized Protocol for Diverse Enrichment to Improve Consistency across Research Studies.

Exposure to environmental enrichment has beneficial effects on learning and memory, diverse neurobiological effects, and promotes recovery of function after brain injury. The effect of enrichment is produced by a combination of increased social interaction, physical activity, spatial complexity, and novelty. Procedures in the literature have, however, been idiosyncratic with poor consistency in the manner or extent to which protocols provide consistent enrichment. We provide an environmental enrichment protocol that can be easily replicated with minor details determined locally so that animals across cohorts and cages all experience a comparable level of enrichment. Procedures are outlined to generate and use a daily pool of suitably varied objects using a standardized format, with objects systematically varied up to a 40-day continuous period. Together with using a large group of rats in a suitably-sized cage, and regular shifting of the position of food and water and cage location, these procedures have produced robust effects in different laboratories and rat strain, thereby improving comparisons within and across laboratories. Non-enriched comparisons can vary, but typically would include grouped animals in standard laboratory housing without objects and with stable food and water locations. Enrichment is a safe non-pharamacological tool to examine behavioral and neurobiological processes in animal models of the lifespan, brain dysfunction and injury.

Reservoir computing model of prefrontal cortex creates novel combinations of previous navigation sequences from hippocampal place-cell replay with spatial reward propagation.

As rats learn to search for multiple sources of food or water in a complex environment, they generate increasingly efficient trajectories between reward sites. Such spatial navigation capacity involves the replay of hippocampal place-cells during awake states, generating small sequences of spatially related place-cell activity that we call "snippets". These snippets occur primarily during sharp-wave-ripples (SWRs). Here we focus on the role of such replay events, as the animal is learning a traveling salesperson task (TSP) across multiple trials. We hypothesize that snippet replay generates synthetic data that can substantially expand and restructure the experience available and make learning more optimal. We developed a model of snippet generation that is modulated by reward, propagated in the forward and reverse directions. This implements a form of spatial credit assignment for reinforcement learning. We use a biologically motivated computational framework known as 'reservoir computing' to model prefrontal cortex (PFC) in sequence learning, in which large pools of prewired neural elements process information dynamically through reverberations. This PFC model consolidates snippets into larger spatial sequences that may be later recalled by subsets of the original sequences. Our simulation experiments provide neurophysiological explanations for two pertinent observations related to navigation. Reward modulation allows the system to reject non-optimal segments of experienced trajectories, and reverse replay allows the system to "learn" trajectories that it has not physically experienced, both of which significantly contribute to the TSP behavior.

Interoceptive Insular Cortex Mediates Both Innate Fear and Contextual Threat Conditioning to Predator Odor.

The insular cortex (IC), among other brain regions, becomes active when humans experience fear or anxiety. However, few experimental studies in rats have implicated the IC in threat responses. We have recently reported that inactivation of the primary interoceptive cortex (pIC) during pre-training, or the intra-pIC blockade of protein synthesis immediately after training, impaired the consolidation of auditory fear conditioning. The present study was designed to investigate the role of the pIC in innate and learned defensive responses to predator odor. Freezing behavior was elicited by single or repetitive exposures to a collar that had been worn by a domestic cat. Sessions were video-recorded and later scored by video observation. We found that muscimol inactivation of the pIC reduced the expression of freezing reaction in response to a single or repeated exposure to cat odor. We also found that pIC inactivation with muscimol impaired conditioning of fear to the context in which rats were exposed to cat odor. Furthermore, neosaxitoxin inactivation of the pIC resulted in a prolonged and robust reduction in freezing response in subsequent re-exposures to cat odor. In addition, freezing behavior significantly correlated with the neural activity of the IC. The present results suggest that the IC is involved in the expression of both innate and learned fear responses to predator odor.

A new rat-compatible robotic framework for spatial navigation behavioral experiments.

BACKGROUND: Understanding the neural substrate of information encoding and processing requires a precise control of the animal's behavior. Most of what has been learned from the rodent navigational system results from relatively simple tasks in which the movements of the animal is controlled by corridors or walkways, passive movements, treadmills or virtual reality environments. While a lot has been and continues to be learned from these types of experiments, recent evidence has shown that such artificial constraints may have significant consequences on the functioning of the neural circuits of spatial navigation. NEW METHODS: We present a novel and alternative approach for effectively controlling the precise direction and speed of movement of the animal in an ethologically realistic environment, using a small robot (Sphero). RESULTS: We describe the robotic framework and demonstrate its use in replicating pre-programmed or rat-recorded paths. We show that the robot can control the movement of a rat in order to produce specific trajectories and speeds. We demonstrate that the robot can be used to aid the rat in learning a spatial memory task in a large and complex environment. We show that dorsal hippocampal CA1 place cells do not remap when the rat is following the robot. Comparison with existing method(s): Our framework only involves positive motivation and has been tested together with wireless electrophysiology in large and complex environments. CONCLUSIONS: Our robotic framework can be used to design novel tasks and experiments in which electrophysiological recordings would be largely devoid of maze or task-dependent artifacts.

Lesions of the Head Direction Cell System Increase Hippocampal Place Field Repetition.

A central tenet of systems neuroscience is that the mammalian hippocampus provides a cognitive map of the environment. This view is supported by the finding of place cells, neurons whose firing is tuned to specific locations in an animal's environment, within this brain region. Recent work, however, has shown that these cells repeat their firing fields across visually identical maze compartments [1, 2]. This repetition is not observed if these compartments face different directions, suggesting that place cells use a directional input to differentiate otherwise similar local environments [3, 4]. A clear candidate for this input is the head direction cell system. To test this, we disrupted the head direction cell system by lesioning the lateral mammillary nuclei and then recorded place cells as rats explored multiple, connected compartments, oriented in the same or in different directions. As shown previously, we found that place cells in control animals exhibited repeated fields in compartments arranged in parallel, but not in compartments facing different directions. In contrast, the place cells of animals with lesions of the head direction cell system exhibited repeating fields in both conditions. Thus, directional information provided by the head direction cell system appears essential for the angular disambiguation by place cells of visually identical compartments.

Place field repetition and spatial learning in a multicompartment environment.

Recent studies have shown that place cells in the hippocampus possess firing fields that repeat in physically similar, parallel environments. These results imply that it should be difficult for animals to distinguish parallel environments at a behavioral level. To test this, we trained rats on a novel odor-location task in an environment with four parallel compartments which had previously been shown to yield place field repetition. A second group of animals was trained on the same task, but with the compartments arranged in different directions, an arrangement we hypothesised would yield less place field repetition. Learning of the odor-location task in the parallel compartments was significantly impaired relative to learning in the radially arranged compartments. Fewer animals acquired the full discrimination in the parallel compartments compared to those trained in the radial compartments, and the former also required many more sessions to reach criterion compared to the latter. To confirm that the arrangement of compartments yielded differences in place cell repetition, in a separate group of animals we recorded from CA1 place cells in both environments. We found that CA1 place cells exhibited repeated fields across four parallel local compartments, but did not do so when the same compartments were arranged radially. To confirm that the differences in place field repetition across the parallel and radial compartments depended on their angular arrangement, and not incidental differences in access to an extra-maze visual landmark, we repeated the recordings in a second set of rats in the absence of the orientation landmark. We found, once again, that place fields showed repetition in parallel compartments, and did not do so in radially arranged compartments. Thus place field repetition, or lack thereof, in these compartments was not dependent on extra-maze cues. Together, these results imply that place field repetition constrains spatial learning.

The head direction cell system and behavior: The effects of lesions to the lateral mammillary bodies on spatial memory in a novel landmark task and in the water maze.

The head direction system is composed of neurons found in a number of connected brain areas that fire in a sharply tuned, directional way. The function of this system, however, has not been fully established. To assess this, we devised a novel spatial landmark task, comparable to the paradigms in which stimulus control has been assessed for spatially tuned neurons. The task took place in a large cylinder and required rats to dig in a specific sand cup, from among 16 alternatives, to obtain a food reward. The reinforced cup was in a fixed location relative to a salient landmark, and probe sessions confirmed that the landmark exerted stimulus control over the rats' cup choices. To assess the contribution of the head direction cell system to this memory task, half of the animals received ibotenic acid infusions into the lateral mammillary nuclei (LMN), an essential node in the head direction network, while the other received sham lesions. No differences were observed in performance of this task between the 2 groups. Animals with LMN lesions were impaired, however, in reversal learning on a water maze task. These results suggest that the LMN, and potentially the head direction cell system, are not essential for the use of visual landmarks to guide spatial behavior.

Anterior thalamic nuclei lesions and recovery of function: Relevance to cognitive thalamus.

Injury to the anterior thalamic nuclei (ATN) and their neural connections is the most consistent neuropathology associated with diencephalic amnesia. ATN lesions in rats produce memory impairments that support a key role for this region within an extended hippocampal system of complex overlapping neural connections. Environmental enrichment is a therapeutic tool that produces substantial, although incomplete, recovery of memory function after ATN lesions, even after the lesion-induced deficit has become established. Similarly, the neurotrophic agent cerebrolysin, also counters the negative effects of ATN lesions. ATN lesions substantially reduce c-Fos expression and spine density in the retrosplenial cortex, and reduce spine density on CA1 neurons; only the latter is reversed by enrichment. We discuss the implications of this evidence for the cognitive thalamus, with a proposal that there are genuine interactions among different but allied thalamo-cortical systems that go beyond a simple summation of their separate effects.

Anterior thalamic lesions reduce spine density in both hippocampal CA1 and retrosplenial cortex, but enrichment rescues CA1 spines only.

Injury to the anterior thalamic nuclei (ATN) may affect both hippocampus and retrosplenial cortex thus explaining some parallels between diencephalic and medial temporal lobe amnesias. We found that standard-housed rats with ATN lesions, compared with standard-housed controls, showed reduced spine density in hippocampal CA1 neurons (basal dendrites, -11.2%; apical dendrites, -9.6%) and in retrospenial granular b cortex (Rgb) neurons (apical dendrites, -20.1%) together with spatial memory deficits on cross maze and radial-arm maze tasks. Additional rats with ATN lesions were also shown to display a severe deficit on spatial working memory in the cross-maze, but subsequent enriched housing ameliorated their performance on both this task and the radial-arm maze. These enriched rats with ATN lesions also showed recovery of both basal and apical CA1 spine density to levels comparable to that of the standard-housed controls, but no recovery of Rgb spine density. Inspection of spine types in the CA1 neurons showed that ATN lesions reduced the density of thin spines and mushroom spines, but not stubby spines; while enrichment promoted recovery of thin spines. Comparison with enriched rats that received pseudo-training, which provided comparable task-related experience, but no explicit spatial memory training, suggested that basal CA1 spine density in particular was associated with spatial learning and memory performance. Distal pathology in terms of reduced integrity of hippocampal and retrosplenial microstructure provides clear support for the influence of the ATN lesions on the extended hippocampal system. The reversal by postoperative enrichment of this deficit in the hippocampus but not the retrosplenial cortex may indicate region-specific mechanisms of recovery after ATN injury.