Imaging lipophilic regions in rodent brain tissue with halogenated BODIPY probes.

The effect of halogen substitution in fluorescent BODIPY species was evaluated in the context of staining lipids in situ within brain tissue sections. Herein we demonstrate that the halogenated species maintain their known in vitro affinity when applied to detect lipids in situ in brain tissue sections. Interestingly, the chlorine substituted compound revealed the highest specificify for white matter lipids. Furthermore, the halogen substituted compounds rapidly detected lipid enriched cells, in situ, associated with a case of brain pathology and neuroinflammation.

Characterization of Ionic and Lipid Gradients within Corpus Callosum White Matter after Diffuse Traumatic Brain Injury in the Rat.

There is increased recognition of the effects of diffuse traumatic brain injury (dTBI), which can initiate yet unknown biochemical cascades, resulting in delayed secondary brain degeneration and long-term neurological sequela. There is limited availability of therapies that minimize the effect of secondary brain damage on the quality of life of people who have suffered TBI, many of which were otherwise healthy adults. Understanding the cascade of biochemical events initiated in specific brain regions in the acute phase of dTBI and how this spreads into adjacent brain structures may provide the necessary insight into drive development of improved therapies. In this study, we have used direct biochemical imaging techniques (Fourier transform infrared spectroscopic imaging) and elemental mapping (X-ray fluorescence microscopy) to characterize biochemical and elemental alterations that occur in corpus callosum white matter in the acute phase of dTBI. The results provide direct visualization of differential biochemical and ionic changes that occur in the highly vulnerable medial corpus callosum white matter relative to the less vulnerable lateral regions of the corpus callosum. Specifically, the results suggest that altered ionic gradients manifest within mechanically damaged medial corpus callosum, potentially spreading to and inducing lipid alterations to white matter structures in lateral brain regions.

Laminar-specific encoding of texture elements in rat barrel cortex.

KEY POINTS: For rats texture discrimination is signalled by the large face whiskers by stick-slip events. Neural encoding of repetitive stick-slip events will be influenced by intrinsic properties of adaptation. We show that texture coding in the barrel cortex is laminar specific and follows a power function. Our results also show layer 2 codes for novel feature elements via robust firing rates and temporal fidelity. We conclude that texture coding relies on a subtle neural ensemble to provide important object information. ABSTRACT: Texture discrimination by rats is exquisitely guided by fine-grain mechanical stick-slip motions of the face whiskers as they encounter, stick to and slip past successive texture-defining surface features such as bumps and grooves. Neural encoding of successive stick-slip texture events will be shaped by adaptation, common to all sensory systems, whereby receptor and neural responses to a stimulus are affected by responses to preceding stimuli, allowing resetting to signal novel information. Additionally, when a whisker is actively moved to contact and brush over surfaces, that motion itself generates neural responses that could cause adaptation of responses to subsequent stick-slip events. Nothing is known about encoding in the rat whisker system of stick-slip events defining textures of different grain or the influence of adaptation from whisker protraction or successive texture-defining stick-slip events. Here we recorded responses from halothane-anaesthetized rats in response to texture-defining stimuli applied to passive whiskers. We demonstrate that: across the columnar network of the whisker-recipient barrel cortex, adaptation in response to repetitive stick-slip events is strongest in uppermost layers and equally lower thereafter; neither whisker protraction speed nor stick-slip frequency impede encoding of stick-slip events at rates up to 34.08 Hz; and layer 2 normalizes responses to whisker protraction to resist effects on texture signalling. Thus, within laminar-specific response patterns, barrel cortex reliably encodes texture-defining elements even to high frequencies.

Orientation selectivity in rat primary visual cortex emerges earlier with low-contrast and high-luminance stimuli.

In natural vision, rapid and sustained variations in luminance and contrast change the reliability of information available about a visual scene, and markedly affect both neuronal and behavioural responses. The hallmark property of neurons in primary visual cortex (V1), orientation selectivity, is unaffected by changes in stimulus contrast, but it remains unclear how sustained differences in mean luminance and contrast affect the time-course of orientation selectivity, and the amount of information that neurons carry about orientation. We used reverse correlation with characterize the temporal dynamics of orientation selectivity in rat V1 neurons under four luminance-contrast conditions. We show that orientation selectivity and mutual information between neuronal responses and stimulus orientation are invariant to contrast or mean luminance. Critically, the time-course of the emergence of orientation selectivity was affected by both factors; response latencies were longer for low- than high-luminance gratings, and surprisingly, response latencies were also longer for high- than low-contrast gratings. Modelling suggests that luminance-modulated changes in feedforward gain, in combination with hyperpolarization caused by high contrasts can account for our physiological data. The hyperpolarization at high contrasts may increase signal-to-noise ratios, whereas a more depolarized membrane may lead to greater sensitivity to weak stimuli.

Masking reduces orientation selectivity in rat visual cortex.

In visual masking the perception of a target stimulus is impaired by a preceding (forward) or succeeding (backward) mask stimulus. The illusion is of interest because it allows uncoupling of the physical stimulus, its neuronal representation, and its perception. To understand the neuronal correlates of masking, we examined how masks affected the neuronal responses to oriented target stimuli in the primary visual cortex (V1) of anesthetized rats (n = 37). Target stimuli were circular gratings with 12 orientations; mask stimuli were plaids created as a binarized sum of all possible target orientations. Spatially, masks were presented either overlapping or surrounding the target. Temporally, targets and masks were presented for 33 ms, but the stimulus onset asynchrony (SOA) of their relative appearance was varied. For the first time, we examine how spatially overlapping and center-surround masking affect orientation discriminability (rather than visibility) in V1. Regardless of the spatial or temporal arrangement of stimuli, the greatest reductions in firing rate and orientation selectivity occurred for the shortest SOAs. Interestingly, analyses conducted separately for transient and sustained target response components showed that changes in orientation selectivity do not always coincide with changes in firing rate. Given the near-instantaneous reductions observed in orientation selectivity even when target and mask do not spatially overlap, we suggest that monotonic visual masking is explained by a combination of neural integration and lateral inhibition.

Traumatic Brain Injury and Neuronal Functionality Changes in Sensory Cortex.

Traumatic brain injury (TBI), caused by direct blows to the head or inertial forces during relative head-brain movement, can result in long-lasting cognitive and motor deficits which can be particularly consequential when they occur in young people with a long life ahead. Much is known of the molecular and anatomical changes produced in TBI but much less is known of the consequences of these changes to neuronal functionality, especially in the cortex. Given that much of our interior and exterior lives are dependent on responsiveness to information from and about the world around us, we have hypothesized that a significant contributor to the cognitive and motor deficits seen after TBI could be changes in sensory processing. To explore this hypothesis, and to develop a model test system of the changes in neuronal functionality caused by TBI, we have examined neuronal encoding of simple and complex sensory input in the rat's exploratory and discriminative tactile system, the large face macrovibrissae, which feeds to the so-called "barrel cortex" of somatosensory cortex. In this review we describe the short-term and long-term changes in the barrel cortex encoding of whisker motion modeling naturalistic whisker movement undertaken by rats engaged in a variety of tasks. We demonstrate that the most common form of TBI results in persistent neuronal hyperexcitation specifically in the upper cortical layers, likely due to changes in inhibition. We describe the types of cortical inhibitory neurons and their roles and how selective effects on some of these could produce the particular forms of neuronal encoding changes described in TBI, and then generalize to compare the effects on inhibition seen in other forms of brain injury. From these findings we make specific predictions as to how non-invasive extra-cranial electrophysiology can be used to provide the high-precision information needed to monitor and understand the temporal evolution of changes in neuronal functionality in humans suffering TBI. Such detailed understanding of the specific changes in an individual patient's cortex can allow for treatment to be tailored to the neuronal changes in that particular patient's brain in TBI, a precision that is currently unavailable with any technique.

Differential susceptibility of cortical and subcortical inhibitory neurons and astrocytes in the long term following diffuse traumatic brain injury.

Long-term diffuse traumatic brain injury (dTBI) causes neuronal hyperexcitation in supragranular layers in sensory cortex, likely through reduced inhibition. Other forms of TBI affect inhibitory interneurons in subcortical areas but it is unknown if this occurs in cortex, or in any brain area in dTBI. We investigated dTBI effects on inhibitory neurons and astrocytes in somatosensory and motor cortex, and hippocampus, 8 weeks post-TBI. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY), and somatostatin (SOM) and glial fibrillary acidic protein (GFAP), a marker for astrogliosis during neurodegeneration. Despite persistent behavioral deficits in rotarod performance up to the time of brain extraction (TBI = 73.13 ± 5.23% mean ± SEM, Sham = 92.29 ± 5.56%, P < 0.01), motor cortex showed only a significant increase, in NPY neurons in supragranular layers (mean cells/mm2 ± SEM, Sham = 16 ± 0.971, TBI = 25 ± 1.51, P = 0.001). In somatosensory cortex, only CR+ neurons showed changes, being decreased in supragranular (TBI = 19 ± 1.18, Sham = 25 ± 1.10, P < 0.01) and increased in infragranular (TBI = 28 ± 1.35, Sham = 24 ± 1.07, P < 0.05) layers. Heterogeneous changes were seen in hippocampal staining: CB+ decreased in dentate gyrus (TBI = 2 ± 0.382, Sham = 4 ± 0.383, P < 0.01), PV+ increased in CA1 (TBI = 39 ± 1.26, Sham = 33 ± 1.69, P < 0.05) and CA2/3 (TBI = 26 ± 2.10, Sham = 20 ± 1.49, P < 0.05), and CR+ decreased in CA1 (TBI = 10 ± 1.02, Sham = 14 ± 1.14, P < 0.05). Astrogliosis significantly increased in corpus callosum (TBI = 6.7 ± 0.69, Sham = 2.5 ± 0.38; P = 0.007). While dTBI effects on inhibitory neurons appear region- and type-specific, a common feature in all cases of decrease was that changes occurred in dendrite targeting interneurons involved in neuronal integration. J. Comp. Neurol. 524:3530-3560, 2016. © 2016 Wiley Periodicals, Inc.

Environmental Enrichment Attenuates Traumatic Brain Injury: Induced Neuronal Hyperexcitability in Supragranular Layers of Sensory Cortex.

We have previously demonstrated that traumatic brain injury (TBI) induces significant long-term neuronal hyperexcitability in supragranular layers of sensory cortex, coupled with persistent sensory deficits. Hence, we aimed to investigate whether brain plasticity induced by environmental enrichment (EE) could attenuate abnormal neuronal and sensory function post-TBI. TBI (n = 22) and sham control (n = 21) animals were randomly assigned housing in either single or enriched conditions for 7-9 weeks. Then, in terminal experiments, extracellular recordings were obtained from barrel cortex neurons in response to whisker motion, including those mimicking motion in awake animals undertaking different tasks. Long-term EE exposure (6 weeks) attenuated TBI-induced hyperexcitability in layers 2-3, such that neuronal activity in TBI animals exposed to EE was restored to control levels. Little to no EE-induced changes in population neuronal responses occurred in input layer 4 and output layer 5. However, single-cell responses demonstrated EE-induced hypoexcitation in L4 post-TBI. EE was also able to fully ameliorate sensory hypersensitivity post-TBI, although it was not found to improve motor function. Long-term enrichment post-TBI induces changes at both the population and single-cell level in the sensory cortex, where EE may act to restore the excitation/inhibition balance in supragranular cortical layers.

Environmental enrichment and the sensory brain: the role of enrichment in remediating brain injury.

The brain's life-long capacity for experience-dependent plasticity allows adaptation to new environments or to changes in the environment, and to changes in internal brain states such as occurs in brain damage. Since the initial discovery by Hebb (1947) that environmental enrichment (EE) was able to confer improvements in cognitive behavior, EE has been investigated as a powerful form of experience-dependent plasticity. Animal studies have shown that exposure to EE results in a number of molecular and morphological alterations, which are thought to underpin changes in neuronal function and ultimately, behavior. These consequences of EE make it ideally suited for investigation into its use as a potential therapy after neurological disorders, such as traumatic brain injury (TBI). In this review, we aim to first briefly discuss the effects of EE on behavior and neuronal function, followed by a review of the underlying molecular and structural changes that account for EE-dependent plasticity in the normal (uninjured) adult brain. We then extend this review to specifically address the role of EE in the treatment of experimental TBI, where we will discuss the demonstrated sensorimotor and cognitive benefits associated with exposure to EE, and their possible mechanisms. Finally, we will explore the use of EE-based rehabilitation in the treatment of human TBI patients, highlighting the remaining questions regarding the effects of EE.

Erythropoietin improves motor and cognitive deficit, axonal pathology, and neuroinflammation in a combined model of diffuse traumatic brain injury and hypoxia, in association with upregulation of the erythropoietin receptor.

BACKGROUND: Diffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia. METHODS: Sprague-Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30 minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-α (5000 IU/kg) or saline at 1 and 24 hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests; 2) axonal pathology (NF-200); 3) callosal degradation (hematoxylin and eosin stain); 3) dendritic loss (MAP2); 4) expression and localisation of the EPO receptor (EpoR); 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1ß. RESULTS: EPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI + Hx). A single dose of EPO at 1 hour reduced axonal damage in the white matter of TAI + Hx rats at 1 day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI + Hx rats; however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1 day after TAI + Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1ß to sham levels 2 hours after TAI + Hx, concomitant to a decrease in CD68 positive cells at 7 and 14 days. CONCLUSIONS: When administered EPO, TAI + Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO's neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO's neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.

Environmental enrichment causes a global potentiation of neuronal responses across stimulus complexity and lamina of sensory cortex.

Enriched social and physical housing produces many molecular, anatomical, electrophysiological and behavior benefits even in adult animals. Much less is known of its effects on cortical electrophysiology, especially in how sensory cortex encodes the altered environment, and extant studies have generally been restricted to neurons in input laminae in sensory cortex. To extend the understanding of how an enriched environment alters the way in which cortex views the world, we investigated enrichment-induced changes in neuronal encoding of sensory stimuli across all laminae of the rat barrel cortex receiving input from the face whisker tactile system. Animals were housed in Enriched (n = 13) or Isolated housing (n = 13) conditions for 8 weeks before extracellular recordings were obtained from barrel cortex in response to simple whisker deflections and whisker motions modeling movements seen in awake animals undertaking a variety of different tasks. Enrichment resulted in increases in neuronal responses to all stimuli, ranging from those modeling exploratory behavior through to discrimination behaviors. These increases were seen throughout the cortex from supragranular layers through to input Layer 4 and for some stimuli, in infragranular Layer 5. The observed enrichment-induced effect is consistent with the postulate that enrichment causes shift in cortical excitatory/inhibitory balance, and we demonstrate this is greatest in supragranular layers. However, we also report that the effects are non-selective for stimulus parameters across a range of stimuli except for one modeling the likely use of whiskers by the rats in the enriched housing.

Cortical hypoexcitation defines neuronal responses in the immediate aftermath of traumatic brain injury.

Traumatic brain injury (TBI) from a blow to the head is often associated with complex patterns of brain abnormalities that accompany deficits in cognitive and motor function. Previously we reported that a long-term consequence of TBI, induced with a closed-head injury method modelling human car and sporting accidents, is neuronal hyper-excitation in the rat sensory barrel cortex that receives tactile input from the face whiskers. Hyper-excitation occurred only in supra-granular layers and was stronger to complex than simple stimuli. We now examine changes in the immediate aftermath of TBI induced with same injury method. At 24 hours post-trauma significant sensorimotor deficits were observed and characterisation of the cortical population neuronal responses at that time revealed a depth-dependent suppression of neuronal responses, with reduced responses from supragranular layers through to input layer IV, but not in infragranular layers. In addition, increased spontaneous firing rate was recorded in cortical layers IV and V. We postulate that this early post-injury suppression of cortical processing of sensory input accounts for immediate post-trauma sensory morbidity and sets into train events that resolve into long-term cortical hyper-excitability in upper sensory cortex layers that may account for long-term sensory hyper-sensitivity in humans with TBI.

Diffuse traumatic brain injury and the sensory brain.

In this review we discuss the consequences to the brain's cortex, specifically to the sensory cortex, of traumatic brain injury. The thesis underlying this approach is that long-term deficits in cognition seen after brain damage in humans are likely underpinned by an impaired cortical processing of the sensory information needed to drive cognition or to be used by cognitive processes to produce a response. We take it here that the impairment to sensory processing does not arise from damage to peripheral sensory systems, but from disordered brain processing of sensory input.

Sensory cortex underpinnings of traumatic brain injury deficits.

Traumatic brain injury (TBI) can result in persistent sensorimotor and cognitive deficits including long-term altered sensory processing. The few animal models of sensory cortical processing effects of TBI have been limited to examination of effects immediately after TBI and only in some layers of cortex. We have now used the rat whisker tactile system and the cortex processing whisker-derived input to provide a highly detailed description of TBI-induced long-term changes in neuronal responses across the entire columnar network in primary sensory cortex. Brain injury (n=19) was induced using an impact acceleration method and sham controls received surgery only (n=15). Animals were tested in a range of sensorimotor behaviour tasks prior to and up to 6 weeks post-injury when there were still significant sensorimotor behaviour deficits. At 8-10 weeks post-trauma, in terminal experiments, extracellular recordings were obtained from barrel cortex neurons in response to whisker motion, including motion that mimicked whisker motion observed in awake animals undertaking different tasks. In cortex, there were lamina-specific neuronal response alterations that appeared to reflect local circuit changes. Hyper-excitation was found only in supragranular layers involved in intra-areal processing and long-range integration, and only for stimulation with complex, naturalistic whisker motion patterns and not for stimulation with simple trapezoidal whisker motion. Thus TBI induces long-term directional changes in integrative sensory cortical layers that depend on the complexity of the incoming sensory information. The nature of these changes allow predictions as to what types of sensory processes may be affected in TBI and contribute to post-trauma sensorimotor deficits.