Lamina-specific population encoding of cutaneous signals in the spinal dorsal horn using multi-electrode arrays.

KEY POINTS: Traditional, widely used in vivo electrophysiological techniques for the investigation of spinal processing of somatosensory information fail to account for the diverse functions of each lamina. To overcome this oversimplification, we have used multi-electrode arrays, in vivo, to simultaneously record neuronal activity across all laminae of the spinal dorsal horn. Multi-electrode arrays are sensitive enough to detect lamina- and region-specific encoding of different subtypes of afferent fibres and to detect short-lived changes in synaptic plasticity as measured by the application of cutaneous electrical stimulation of varying intensity and frequency. Differential encoding of innocuous and noxious thermal and mechanical stimuli were also detected across the laminae with the technique, as were the effects of the application of capsaicin. This new approach to the study of the dorsal spinal cord produces significantly more information per experiment, permitting accelerated research whilst also permitting the effects of pharmacological tools to modulate network responses. ABSTRACT: The dorsal horn (DH) of the spinal cord is a complex laminar structure integrating peripheral signals into the central nervous system. Spinal somatosensory processing is commonly measured electrophysiologically in vivo by recording the activity of individual wide-dynamic-range neurons in the deep DH and extrapolating their behaviour to all cells in every lamina. This fails to account for the specialized processes that occur in each lamina and the considerable heterogeneity in cellular phenotype within and between laminae. Here we overcome this oversimplification by employing linear multi-electrode arrays (MEAs) in the DH of anaesthetized rats to simultaneously measure activity across all laminae. The MEAs, comprising 16 channels, were inserted into the lumbar dorsal horn and peripheral neurons activated electrically via transcutaneous electrodes and ethologically with von Frey hairs (vFHs) or an aluminium heating block. Ascending electrical stimuli showed fibre thresholds with distinct dorsoventral innervation profiles. Wind up was observed across the DH during the C-fibre and post-discharge latencies following 0.5 Hz stimulation. Intrathecal application of morphine (5 ng/50 µl) significantly reduced Aδ- and C-fibre-evoked activity in deep and superficial DH. Light vFHs (≤10 g) predominantly activated intermediate and deep laminae whereas noxious vFHs (26 g) also activated the superficial laminae. Noxious heat (55°C) induced significantly greater activity in the superficial and deep laminae than the innocuous control (30°C). The application of these arrays produced the first description of the processing of innocuous and noxious stimuli throughout the intact DH.

Age-dependent plasticity in endocannabinoid modulation of pain processing through postnatal development.

Significant age- and experience-dependent remodelling of spinal and supraspinal neural networks occur, resulting in altered pain responses in early life. In adults, endogenous opioid peptide and endocannabinoid (ECs) pain control systems exist which modify pain responses, but the role they play in acute responses to pain and postnatal neurodevelopment is unknown. Here, we have studied the changing role of the ECs in the brainstem nuclei essential for the control of nociception from birth to adulthood in both rats and humans. Using in vivo electrophysiology, we show that substantial functional changes occur in the effect of microinjection of ECs receptor agonists and antagonists in the periaqueductal grey (PAG) and rostroventral medulla (RVM), both of which play central roles in the supraspinal control of pain and the maintenance of chronic pain states in adulthood. We show that in immature PAG and RVM, the orphan receptor, GPR55, is able to mediate profound analgesia which is absent in adults. We show that tissue levels of endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol, within the PAG and RVM are developmentally regulated (using mass spectrometry). The expression patterns and levels of ECs enzymes and receptors were assessed using quantitative PCR and immunohistochemistry. In human brainstem, we show age-related alterations in the expression of key enzymes and receptors involved in ECs function using PCR and in situ hybridisation. These data reveal that significant changes on ECs that to this point have been unknown and which shed new light into the complex neurochemical changes that permit normal, mature responses to pain.

Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function.

Optical imaging with voltage-sensitive dyes enables the visualization of extensive yet highly transient coalitions of neurons (assemblies) operating throughout the brain on a subsecond time scale. We suggest that operating at the mesoscale level of brain organization, neuronal assemblies may provide a functional link between "bottom-up" cellular mechanisms and "top-down" cognitive ones within anatomically defined regions. We demonstrate in ex vivo rat brain slices how varying spatiotemporal dynamics of assemblies reveal differences not previously appreciated between: different stages of development in cortical versus subcortical brain areas, different sensory modalities (hearing versus vision), different classes of psychoactive drugs (anesthetics versus analgesics), different effects of anesthesia linked to hyperbaric conditions and, in vivo, depths of anesthesia. The strategy of voltage-sensitive dye imaging is therefore as powerful as it is versatile and as such can now be applied to the evaluation of neurochemical signaling systems and the screening of related new drugs, as well as to mathematical modeling and, eventually, even theories of consciousness.

(II) Physiological profiling of an endogenous peptide in the basal forebrain: Age-related bioactivity and blockade with a novel modulator.

Previous studies have suggested that neurodegeneration is an aberrant form of development, mediated by a novel peptide from the C-terminus of acetylcholinesterase (AChE). Using voltage-sensitive dye imaging we have investigated the effects of a synthetic version of this peptide in the in vitro rat basal forebrain, a key site of degeneration in Alzheimer's disease. The brain slice preparation enables direct visualisation in real-time of sub-second meso-scale neuronal coalitions ('Neuronal Assemblies') that serve as a powerful index of brain functional activity. Here we show that (1) assemblies are site-specific in their activity profile with the cortex displaying a significantly more extensive network activity than the sub-cortical basal forebrain; (2) there is an age-dependency, in both cortical and sub-cortical sites, with the younger brain (p14 rats) exhibiting more conspicuous assemblies over space and time compared to their older counterparts (p35-40 rats). (3) AChE-derived peptide significantly modulates the dynamics of neuronal assemblies in the basal forebrain of the p14 rat with the degree of modulation negatively correlated with age, (4) the differential in assembly size with age parallels the level of endogenous peptide in the brain, which also declines with maturity, and (5) this effect is completely reversed by a cyclised variant of AChE-peptide, 'NBP14'. These observations are attributed to an enhanced calcium entry that, according to developmental stage, could be either trophic or toxic, and as such may provide insight into the basic neurodegenerative process as well as an eventual therapeutic intervention.

Risk-based learning games improve long-term retention of information among school pupils.

Risk heightens motivation and, if used appropriately, may have the potential to improve engagement in the classroom. We have developed a risk-based learning game for school pupils in order to test whether such learning games can improve later recall of information. The study was performed during a series of public engagement workshops delivered by undergraduate students. Undergraduate neuroscience students delivered 90-minute science workshops to 9-10 year old school pupils (n = 448) that were divided into 'Risk', 'No risk' and 'Control' classes. 'Risk' classes received periodic multiple-choice questions (MCQs) during the workshops which required small teams of pupils to assign tokens to the answer(s) they believed to be correct. Tokens assigned to the correct answer were returned to the group and an equal number given back as a prize; tokens assigned to incorrect answers were lost. Participation was incentivised by the promise of a brain-related prize to the team with the most tokens at the end of the workshop. 'No risk' classes received MCQs without the risk component whilst the 'Control' classes received no MCQs. When presented with a neuroscience quiz based on workshop content at the end of the workshop, pupils in the 'Risk' classes exhibited significantly greater recall of information one week later. Quiz scores were higher than scores from the day of the workshop which suggested pupils may have discussed the workshop content outside of the classroom, thereby increasing knowledge over and above what was learned during the workshop. This is supported by feedback from pupils in 'Risk' classes which indicated that 'Risk' workshops were more interesting than 'No risk' and 'Control' workshops. These data suggest that there is a role for risk in the classroom but further investigations are required to elucidate the causal mechanisms of improved retention of information.

Postnatal maturation of endogenous opioid systems within the periaqueductal grey and spinal dorsal horn of the rat.

Significant opioid-dependent changes occur during the fourth postnatal week in supraspinal sites (rostroventral medulla [RVM], periaqueductal grey [PAG]) that are involved in the descending control of spinal excitability via the dorsal horn (DH). Here we report developmentally regulated changes in the opioidergic signalling within the PAG and DH, which further increase our understanding of pain processing during early life. Microinjection of the µ-opioid receptor (MOR) agonist DAMGO (30 ng) into the PAG of Sprague-Dawley rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120 ng, MOR antagonist), which lowered mechanical thresholds and increased spinal reflex excitability. Spinal administration of DAMGO inhibited spinal excitability in all ages, yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan real-time polymerase chain reaction and immunohistochemistry. We found that pro-opiomelanocortin peaked at P21 in the ventral PAG, and MOR increased significantly in the DH as the animals aged. Enkephalin mRNA transcripts preceded the increase in enkephalin immunoreactive fibres in the superficial dorsal horn from P21 onwards. These results illustrate that profound differences in the endogenous opioidergic signalling system occur throughout postnatal development.

Neurovascular coupling is brain region-dependent.

Despite recent advances in alternative brain imaging technologies, functional magnetic resonance imaging (fMRI) remains the workhorse for both medical diagnosis and primary research. Indeed, the number of research articles that utilise fMRI have continued to rise unabated since its conception in 1991, despite the limitation that recorded signals originate from the cerebral vasculature rather than neural tissue. Consequently, understanding the relationship between brain activity and the resultant changes in metabolism and blood flow (neurovascular coupling) remains a vital area of research. In the past, technical constraints have restricted investigations of neurovascular coupling to cortical sites and have led to the assumption that coupling in non-cortical structures is the same as in the cortex, despite the lack of any evidence. The current study investigated neurovascular coupling in the rat using whole-brain blood oxygenation level-dependent (BOLD) fMRI and multi-channel electrophysiological recordings and measured the response to a sensory stimulus as it proceeded through brainstem, thalamic and cortical processing sites - the so-called whisker-to-barrel pathway. We found marked regional differences in the amplitude of BOLD activation in the pathway and non-linear neurovascular coupling relationships in non-cortical sites. The findings have important implications for studies that use functional brain imaging to investigate sub-cortical function and caution against the use of simple, linear mapping of imaging signals onto neural activity.

From scientific theory to classroom practice.

The importance of neuroscience in education is becoming widely recognized by both neuroscientists and educators. However, to date, there has been little effective collaboration between the two groups, resulting in the spread of ideas in education poorly based on neuroscience. For their part, educators are often too busy to develop sufficient scientific literacy, and neuroscientists are put off collaborations with risk of overinterpretation of their work. We designed and led a successful 6-month collaborative project between educators and neuroscientists. The project consisted of a series of seminars on topics chosen by both parties such as the neuroscience of attention, learning, and memory and aimed to create a dialog between the two. Here, we report that all teachers found the seminars relevant to their practice and that the majority felt the information was presented in an accessible manner. Such was the success of the project that teachers felt there were direct changes in their classroom practice as a consequence and that the course should be more widely available. We suggest that this format of co-constructed dialog allows for lucrative collaborations between neuroscientists and educators and may be a step to bridging the waters that separate these intrinsically linked disciplines.

Teaching medical students basic neurotransmitter pharmacology using primary research resources.

Teaching pharmacology to medical students has long been seen as a challenge, and one to which a number of innovative approaches have been taken. In this article, we describe and evaluate the use of primary research articles in teaching second-year medical students both in terms of the information learned and the use of the papers themselves. We designed a seminar where small groups of students worked on different neurotransmitters before contributing information to a plenary session. Student feedback suggested that when the information was largely novel, students learned considerably more. Crucially, this improvement in knowledge was seen even when they had not directly studied a particular transmitter in their work groups, suggesting a shared learning experience. Moreover, the majority of students reported that using primary research papers was easy and useful, with over half stating that they would use them in future study.

Neuroscience: viable applications in education?

As a relatively young science, neuroscience is still finding its feet in potential collaborations with other disciplines. One such discipline is education, with the field of neuroeducation being on the horizon since the 1960s. However, although its achievements are now growing, the partnership has not been as successful as first hopes suggested it should be. Here the authors discuss the theoretical barriers and potential solutions to this, which have been suggested previously, with particular focus on levels of research in neuroscience and their applicability to education. Moreover, they propose that these theoretical barriers are driven and maintained by practical barriers surrounding common language and research literacy. They propose that by overcoming these practical barriers through appropriate training and shared experience, neuroeducation can reach its full potential.

Effects of urethane anaesthesia on sensory processing in the rat barrel cortex revealed by combined optical imaging and electrophysiology.

The spatiotemporal dynamics of neuronal assemblies evoked by sensory stimuli have not yet been fully characterised, especially the extent to which they are modulated by prevailing brain states. In order to examine this issue, we induced different levels of anaesthesia, distinguished by specific electroencephalographic indices, and compared somatosensory-evoked potentials (SEPs) with voltage-sensitive dye imaging (VSDI) responses in the rat barrel cortex evoked by whisker deflection. At deeper levels of anaesthesia, all responses were reduced in amplitude but, surprisingly, only VSDI responses exhibited prolonged activation resulting in a delayed return to baseline. Further analysis of the optical signal demonstrated that the reduction in response amplitude was constant across the area of activation, resulting in a global down-scaling of the population response. The manner in which the optical signal relates to the various neuronal generators that produce the SEP signal is also discussed. These data provide information regarding the impact of anaesthetic agents on the brain, and show the value of combining spatial analyses from neuroimaging approaches with more traditional electrophysiological techniques.

A dynamic model of neurovascular coupling: implications for blood vessel dilation and constriction.

Neurovascular coupling in response to stimulation of the rat barrel cortex was investigated using concurrent multichannel electrophysiology and laser Doppler flowmetry. The data were used to build a linear dynamic model relating neural activity to blood flow. Local field potential time series were subject to current source density analysis, and the time series of a layer IV sink of the barrel cortex was used as the input to the model. The model output was the time series of the changes in regional cerebral blood flow (CBF). We show that this model can provide excellent fit of the CBF responses for stimulus durations of up to 16 s. The structure of the model consisted of two coupled components representing vascular dilation and constriction. The complex temporal characteristics of the CBF time series were reproduced by the relatively simple balance of these two components. We show that the impulse response obtained under the 16-s duration stimulation condition generalised to provide a good prediction to the data from the shorter duration stimulation conditions. Furthermore, by optimising three out of the total of nine model parameters, the variability in the data can be well accounted for over a wide range of stimulus conditions. By establishing linearity, classic system analysis methods can be used to generate and explore a range of equivalent model structures (e.g., feed-forward or feedback) to guide the experimental investigation of the control of vascular dilation and constriction following stimulation.

A dynamic causal model of the coupling between pulse stimulation and neural activity.

We present a dynamic causal model that can explain context-dependent changes in neural responses, in the rat barrel cortex, to an electrical whisker stimulation at different frequencies. Neural responses were measured in terms of local field potentials. These were converted into current source density (CSD) data, and the time series of the CSD sink was extracted to provide a time series response train. The model structure consists of three layers (approximating the responses from the brain stem to the thalamus and then the barrel cortex), and the latter two layers contain nonlinearly coupled modules of linear second-order dynamic systems. The interaction of these modules forms a nonlinear regulatory system that determines the temporal structure of the neural response amplitude for the thalamic and cortical layers. The model is based on the measured population dynamics of neurons rather than the dynamics of a single neuron and was evaluated against CSD data from experiments with varying stimulation frequency (1-40 Hz), random pulse trains, and awake and anesthetized animals. The model parameters obtained by optimization for different physiological conditions (anesthetized or awake) were significantly different. Following Friston, Mechelli, Turner, and Price (2000), this work is part of a formal mathematical system currently being developed (Zheng et al., 2005) that links stimulation to the blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal through neural activity and hemodynamic variables. The importance of the model described here is that it can be used to invert the hemodynamic measurements of changes in blood flow to estimate the underlying neural activity.

Altered neurovascular coupling during information-processing states.

Brain imaging techniques rely on changes in blood flow, volume and oxygenation to infer the loci and magnitude of changes in activity. Although progress has been made in understanding the link between stimulus-evoked neural activity and haemodynamics, the extent to which neurovascular-coupling relationships remain constant during different states of baseline cortical activity is poorly understood. Optical imaging spectroscopy, laser Doppler flowmetry and electrophysiology were used to measure haemodynamics and neural activity in the barrel cortex of anaesthetized rats. The responses to stimulation of the whisker pad were recorded during quiescence and cortical desynchronization produced by stimulation of the brainstem. Cortical desynchronization was accompanied by increases in baseline blood flow, volume and oxygenation. Haemodynamic responses to low-frequency whisker stimuli (1 Hz) were attenuated during arousal compared with that observed during quiescence. During arousal it was possible to increase stimulus-evoked haemodynamics by increasing the frequency of the stimulus. Neural responses to low-frequency stimuli were also attenuated but to a far lesser extent than the reduction in the accompanying haemodynamics. In contrast, neuronal activity evoked by high-frequency stimuli (40 Hz) was enhanced during arousal, but induced haemodynamic responses of a similar magnitude compared with that observed for the same high-frequency stimulus presented during quiescence. These data suggest that there may be differences in stimulus-evoked neural activity and accompanying haemodynamics during different information-processing states.

Investigating the coupling between stimulation and neural activity: a dynamic modeling approach.

The objective of the present study was to build a dynamic model relating changes in neural responses in rat barrel cortex to an electrical whisker stimulation pulse train of varying frequencies. This work is part of a formal mathematical system currently being developed, which links stimulation to the Blood Oxygen Level Dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) signal. Neural responses were measured in terms of local field potentials, which were then converted into current source density (CSD) data. Responses were found to be strongly suppressed immediately following the first stimulus pulse, before recovering to a steady state, which was maintained throughout the rest of the stimulation. The amplitude of this steady state decreases as the stimulation frequency increases. The model structure is based on the physiological pathway from the rat sensory organ to the cortex. Dynamic linear second order systems are used to model the excitatory as well as the suppressive components of the neural response. The interactions between components contain nonlinear modulations. The model was evaluated against CSD data from experiments with varying stimulation frequency (1-40 Hz), and shows a plausible fit. The model parameters obtained by optimization for different physiological conditions (anaesthetized or awake) were significantly different. Although this is a descriptive model, it may well have some physiological implications.

Simultaneous laser Doppler flowmetry and arterial spin labeling MRI for measurement of functional perfusion changes in the cortex.

This study compares laser Doppler flowmetry (LDF) and arterial spin labeling (ASL) for the measurement of functional changes in cerebral blood flow (CBF). The two methods were applied concurrently in a paradigm of electrical whisker stimulation in the anaesthetised rat. Multi-channel LDF was used, with each channel corresponding to different fiber separation (and thus measurement depth). Continuous ASL was applied using separate imaging and labeling coils at 3 T. Careful experimental set up ensured that both techniques recorded from spatially concordant regions of the barrel cortex, where functional responses were maximal. Strong correlations were demonstrated between CBF changes measured by each LDF channel and ASL in terms of maximum response magnitude and response time-course within a 6-s-long temporal resolution imposed by ASL. Quantitatively, the measurements of the most superficial LDF channels agreed strongly with those of ASL, whereas the deeper LDF channels underestimated consistently the ASL measurement. It was thus confirmed that LDF quantifies CBF changes consistently at a superficial level, and for this case the two methods provided concordant measures of functional CBF changes, despite their essentially different physical principles and spatiotemporal characteristics.