Sustained and potent analgesia with negligible side effects enabled by adaptive individualized granular stimulation in rat brainstem.

Objectives.To clarify if an adaptive current stimulation protocol, in which current amplitude is modulated during continuous stimulation, provides better efficacy than constant current stimulation protocol with respect to analgesia caused by individualized stimulation in rat periaqueductal gray matter (PAG) /dorsal raphe nuclei (DRN).Approach.Ultrathin microelectrodes adapted for recording (n= 6) and stimulation (n= 16) were implanted in rat primary somatosensory cortex and PAG/DRN, respectively. In each animal included (n= 12), a subset of PAG/DRN microelectrodes (n= 1-3 per animal) was selected that on simultaneous stimulation blocked nociceptive withdrawal reflexes in awake unrestrained animals without noticeable side effects. Analgesic effects were subsequently assessed from both nociceptive withdrawal reflexes and intracortical pain-related responses on CO2laser hind paw stimulation. The analgesic effects of adaptive current PAG/DRN stimulation comprising incremental increases of 5µA/microelectrode (initial median current 30µA/microelectrode) when effects declined were compared to the effects of constant current stimulation. Behavioral effects and brain state related changes were analyzed using quantitative movement analysis and electrocorticography (recorded on top of the dura mater), respectively. Tissue reactions and probe placement in PAG/DRN were assessed with immunohistochemistry.Main results.Powerful and sustained (4 h) analgesia was achieved with the adaptive current protocol within a rather wide area of PAG/DRN. Analgesic after-effects were seen for up to 30 min. Behavioral and brain state related side effects were minimal. Moreover, 6 weeks after implantation, there were no traces of bleedings, only small glial reactions and small but not statistically significant loss of neurons nearby indicating that the microelectrode stimulation employed is biocompatible.Significance.The results indicate that sustained and powerful analgesia with minimal side effects can be achieved by granular and individualized stimulation in PAG/DRN using an adaptive current stimulation protocol. This microelectrode technology and stimulation paradigm thus has the potential of providing a highly efficient and safe pain therapy.

Microelectrode clusters enable therapeutic deep brain stimulation without noticeable side-effects in a rodent model of Parkinson's disease.

BACKGROUND: Deep Brain Stimulation (DBS) is an established treatment for motor symptoms in Parkinson's disease (PD). However, side effects often limit the usefulness of the treatment. NEW METHOD: To mitigate this problem, we developed a novel cluster of ultrathin platinum-iridium microelectrodes (n = 16) embedded in a needle shaped gelatin vehicle. In an established rodent PD-model (6-OHDA unilateral lesion), the clusters were implanted in the subthalamic area for up to 8 weeks. In an open field setting, combinations of microelectrodes yielding therapeutic effects were identified using statistical methods. Immunofluorescence techniques were used for histological assessments of biocompatibility. RESULTS: In all rats tested (n = 5), we found subsets of 3-4 microelectrodes which, upon stimulation (160 Hz, 60 µs pulse width, 25-40 µA/microelectrode), prompted normal movements without noticeable side effects. Other microelectrode subsets often caused side effects such as rotation, dyskinesia and tremor. The threshold (per microelectrode) to elicit normal movements strongly depended on the number of activated microelectrodes in the selected subset. The histological analysis revealed viable neurons close to the electrode contacts, minor microglial and astrocytic reactions and no major changes in the vasculature, indicating high biocompatibility. COMPARISON TO EXISTING METHODS AND CONCLUSION: By contrast to the continuous and relatively large stimulation fields produced by existing DBS electrodes, the developed microelectrode cluster enables a fine-tuned granular and individualized microstimulation. This granular type of stimulation pattern provided powerful and specific therapeutic effects, free of noticeable side effects, in a PD animal model.

3D microelectrode cluster and stimulation paradigm yield powerful analgesia without noticeable adverse effects.

The lack of satisfactory treatment for persistent pain profoundly impairs the quality of life for many patients. Stimulation of brainstem pain control systems can trigger powerful analgesia, but their complex network organization frequently prevents separation of analgesia from side effects. To overcome this long-standing challenge, we developed a biocompatible gelatin-embedded cluster of ultrathin microelectrodes that enables fine-tuned, high-definition three-dimensional stimulation in periaqueductal gray/dorsal raphe nucleus in awake rats. Analgesia was assessed from both motor reactions and intracortical signals, corresponding to pain-related signals in humans. We could select an individual-specific subset of microelectrodes in each animal that reliably provided strong pain inhibition during normal and hyperalgesia conditions, without noticeable behavioral side effects. Gait, spontaneous cortical activity at rest, and cortical tactile responses were minimally affected, indicating a highly selective action. In conclusion, our developed biocompatible microelectrode cluster and stimulation paradigm reliably enabled powerful, fine-tuned, and selective analgesia without noticeable side effects.

Hindlimb motor responses to unilateral brain injury: spinal cord encoding and left-right asymmetry.

Mechanisms of motor deficits (e.g. hemiparesis and hemiplegia) secondary to stroke and traumatic brain injury remain poorly understood. In early animal studies, a unilateral lesion to the cerebellum produced postural asymmetry with ipsilateral hindlimb flexion that was retained after complete spinal cord transection. Here we demonstrate that hindlimb postural asymmetry in rats is induced by a unilateral injury of the hindlimb sensorimotor cortex, and characterize this phenomenon as a model of spinal neuroplasticity underlying asymmetric motor deficits. After cortical lesion, the asymmetry was developed due to the contralesional hindlimb flexion and persisted after decerebration and complete spinal cord transection. The asymmetry induced by the left-side brain injury was eliminated by bilateral lumbar dorsal rhizotomy, but surprisingly, the asymmetry after the right-side brain lesion was resistant to deafferentation. Pancuronium, a curare-mimetic muscle relaxant, abolished the asymmetry after the right-side lesion suggesting its dependence on the efferent drive. The contra- and ipsilesional hindlimbs displayed different musculo-articular resistance to stretch after the left but not right-side injury. The nociceptive withdrawal reflexes evoked by electrical stimulation and recorded with EMG technique were different between the left and right hindlimbs in the spinalized decerebrate rats. On this asymmetric background, a brain injury resulted in greater reflex activation on the contra- versus ipsilesional side; the difference between the limbs was higher after the right-side brain lesion. The unilateral brain injury modified expression of neuroplasticity genes analysed as readout of plastic changes, as well as robustly impaired coordination of their expression within and between the ipsi- and contralesional halves of lumbar spinal cord; the effects were more pronounced after the left side compared to the right-side injury. Our data suggest that changes in the hindlimb posture, resistance to stretch and nociceptive withdrawal reflexes are encoded by neuroplastic processes in lumbar spinal circuits induced by a unilateral brain injury. Two mechanisms, one dependent on and one independent of afferent input may mediate asymmetric hindlimb motor responses. The latter, deafferentation resistant mechanism may be based on sustained muscle contractions which often occur in patients with central lesions and which are not evoked by afferent stimulation. The unusual feature of these mechanisms is their lateralization in the spinal cord.

Ice coating -A new method of brain device insertion to mitigate acute injuries.

BACKGROUND: Reduction of insertion injury is likely important to approach physiological conditions in the vicinity of implanted devices intended to interface with the surrounding brain. NEW METHODS: We have developed a novel, low-friction coating around frozen, gelatin embedded needles. By introducing a layer of thawing ice onto the gelatin, decreasing surface friction, we mitigate damage caused by the implantation. RESULTS AND COMPARISON WITH EXISTING METHODS: The acute effects of a transient stab on neuronal density and glial reactions were assessed 1 and 7 days post stab in rat cortex and striatum both within and outside the insertion track using immunohistochemical staining. The addition of a coat of melting ice to the frozen gelatin embedded needles reduced the insertion force with around 50 %, substantially reduced the loss neurons (i.e. reduced neuronal void), and yielded near normal levels of astrocytes within the insertion track 1 day after insertion, as compared to gelatin coated probes of the same temperature without ice coating. There were negligible effects on glial reactions and neuronal density immediately outside the insertion track of both ice coated and cold gelatin embedded needles. This new method of implantation presents a considerable improvement compared to existing modes of device insertion. CONCLUSIONS: Acute brain injuries following insertion of e.g. ultra-flexible electrodes, can be reduced by providing an outer coat of ultra-slippery thawing ice. No adverse effect of lowered implant temperature was found, opening the possibility of locking fragile electrode construct configurations in frozen gelatin, prior to implantation into the brain.

Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.

Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.

A mouse model of the 15q13.3 microdeletion syndrome shows prefrontal neurophysiological dysfunctions and attentional impairment.

RATIONALE: A microdeletion at locus 15q13.3 is associated with high incidence rates of psychopathology, including schizophrenia. A mouse model of the 15q13.3 microdeletion syndrome has been generated (Df[h15q13]/+) with translational utility for modelling schizophrenia-like pathology. Among other deficits, schizophrenia is characterised by dysfunctions in prefrontal cortical (PFC) inhibitory circuitry and attention. OBJECTIVES: The objective of this study is to assess PFC-dependent functioning in the Df(h15q13)/+ mouse using electrophysiological, pharmacological, and behavioural assays. METHOD: Experiments 1-2 investigated baseline firing and auditory-evoked responses of PFC interneurons and pyramidal neurons. Experiment 3 measured pyramidal firing in response to intra-PFC GABAA receptor antagonism. Experiments 4-6 assessed PFC-dependent attentional functioning through the touchscreen 5-choice serial reaction time task (5-CSRTT). Experiments 7-12 assessed reversal learning, paired-associate learning, extinction learning, progressive ratio, trial-unique non-match to sample, and object recognition. RESULTS: In experiments 1-3, the Df(h15q13)/+ mouse showed reduced baseline firing rate of fast-spiking interneurons and in the ability of the GABAA receptor antagonist gabazine to increase the firing rate of pyramidal neurons. In assays of auditory-evoked responses, PFC interneurons in the Df(h15q13)/+ mouse had reduced detection amplitudes and increased detection latencies, while pyramidal neurons showed increased detection latencies. In experiments 4-6, the Df(h15q13)/+ mouse showed a stimulus duration-dependent decrease in percent accuracy in the 5-CSRTT. The impairment was insensitive to treatment with the partial α7nAChR agonist EVP-6124. The Df(h15q13)/+ mouse showed no cognitive impairments in experiments 7-12. CONCLUSION: The Df(h15q13)/+ mouse has multiple dysfunctions converging on disrupted PFC processing as measured by several independent assays of inhibitory transmission and attentional function.

Nanowire-based electrode for acute in vivo neural recordings in the brain.

We present an electrode, based on structurally controlled nanowires, as a first step towards developing a useful nanostructured device for neurophysiological measurements in vivo. The sensing part of the electrode is made of a metal film deposited on top of an array of epitaxially grown gallium phosphide nanowires. We achieved the first functional testing of the nanowire-based electrode by performing acute in vivo recordings in the rat cerebral cortex and withstanding multiple brain implantations. Due to the controllable geometry of the nanowires, this type of electrode can be used as a model system for further analysis of the functional properties of nanostructured neuronal interfaces in vivo.

Can histology solve the riddle of the nonfunctioning electrode? Factors influencing the biocompatibility of brain machine interfaces.

Neural interfaces hold great promise to become invaluable clinical and diagnostic tools in the near future. However, the biocompatibility and the long-term stability of the implanted interfaces are far from optimized. There are several factors that need to be addressed and standardized when improving the long-term success of an implanted electrode. We have chosen to focus on three key factors when evaluating the evoked tissue responses after electrode implantation into the brain: implant size, fixation mode, and evaluation period. Further, we show results from an ultrathin multichannel wire electrode that has been implanted in the rat cerebral cortex for 1 year. To improve biocompatibility of implanted electrodes, we would like to suggest that free-floating, very small, flexible, and, in time, wireless electrodes would elicit a diminished cell encapsulation. We would also like to suggest standardized methods for the electrode design, the electrode implantation method, and the analyses of cell reactions after implantation into the CNS in order to improve the long-term success of implanted neural interfaces.

Implant size and fixation mode strongly influence tissue reactions in the CNS.

The function of chronic brain machine interfaces depends on stable electrical contact between neurons and electrodes. A key step in the development of interfaces is therefore to identify implant configurations that minimize adverse long-term tissue reactions. To this end, we here characterized the separate and combined effects of implant size and fixation mode at 6 and 12 weeks post implantation in rat (n = 24) cerebral cortex. Neurons and activated microglia and astrocytes were visualized using NeuN, ED1 and GFAP immunofluorescence microscopy, respectively. The contributions of individual experimental variables to the tissue response were quantified. Implants tethered to the skull caused larger tissue reactions than un-tethered implants. Small diameter (50 µm) implants elicited smaller tissue reactions and resulted in the survival of larger numbers of neurons than did large diameter (200 µm) implants. In addition, tethering resulted in an oval-shaped cavity, with a cross-section area larger than that of the implant itself, and in marked changes in morphology and organization of neurons in the region closest to the tissue interface. Most importantly, for implants that were both large diameter and tethered, glia activation was still ongoing 12 weeks after implantation, as indicated by an increase in GFAP staining between week 6 and 12, while this pattern was not observed for un-tethered, small diameter implants. Our findings therefore clearly indicate that the combined small diameter, un-tethered implants cause the smallest tissue reactions.

Gelatine-embedded electrodes--a novel biocompatible vehicle allowing implantation of highly flexible microelectrodes.

Chronic neural interfaces that are both structurally and functionally stable inside the brain over years or decades hold great promise to become an invaluable clinical tool in the near future. A key flaw in the current electrode interfaces is that their recording capabilities deteriorate over time, possibly due to the lack of flexibility, which causes movements in relation to the neural tissue that result in small inflammations and loss of electrode function. We have developed a new neural probe using the stabilizing property of gelatine that allows the implantation of ultra-thin and flexible electrodes into the central nervous system. The microglial and astrocytic reactions evoked by implanted gelatine needles, as well as the wire bundles in combination with gelatine, were investigated using immunohistochemistry and fluorescence microscopy up to 12 weeks after implantation. The results indicate that pure gelatine needles were stiff enough to penetrate the brain tissue on their own, and evoked a significantly smaller chronic scar than stab wounds. Moreover, gelatine embedding appeared to reduce the acute reactions caused by the implants and we found no adverse effects of gelatine or gelatine-embedded electrodes. Successful electrophysiological recordings were made from very thin electrodes implanted in this fashion.

Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains.

BACKGROUND: Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR) system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1) if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2) if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR. The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP) in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO2 laser heat pulses. RESULTS: While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different. CONCLUSION: Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting.

Developmental learning in a pain-related system: evidence for a cross-modality mechanism.

The nociceptive spinal reflex system performs highly precise sensorimotor transformations that require functionally specified synaptic strengths. The specification is gradually attained during early development and appears to be learning dependent. Here we determine the time course of this specification for heat-nociceptive tail withdrawal reflexes and analyze which types of primary afferents are important for the learning by applying various forms of noninvasive sensory deprivations. The percentage of erroneous heat-nociceptive tail withdrawal reflexes (i.e., movements directed toward the stimulation) decreased gradually from 64.1 +/- 2.5% (mean +/- SEM) to <10% during postnatal days 10-21. This improvement was completely blocked by anesthetizing the tail during the adaptation period, confirming that an experience-dependent mechanism is involved in the specification of synaptic strengths. However, the results show that the adaptation occurs to a significant extent despite local analgesia and protection of the tail from noxious input, provided that tactile sensitivity is preserved. Therefore, it appears that a nociceptive input is not necessary for the adaptation, and that input from tactile receptors can be used to guide the nociceptive synaptic organization during development. Sensory deprivation in the adult rat failed to affect the heat-nociceptive withdrawal reflex system, indicating that the adaptation has a "critical period" during early development. These findings provide a key to the puzzle of how pain-related systems can be functionally adapted through experience despite the rare occurrence of noxious input during early life.

Heat nociception is severely reduced in a mutant mouse deficient for the L1 adhesion molecule.

Recent findings indicate that the spatial organization of the spinal nociceptive reflex system is adjusted postnatally through experience-dependent mechanisms. The cellular and molecular mechanisms underlying this tuning are not known. Because the adhesion molecule L1 is known to play an important role in neural development and synaptic plasticity, we studied the nociceptive withdrawal reflexes in awake adult mutant mice deficient in L1. Withdrawal reflexes were elicited by a CO(2) laser (heat stimulation) and von Frey monofilaments (tactile stimulation). L1-deficient mice (n=10) had an abnormally high nociceptive heat-reflex threshold compared with wild-type mice (n=11), except for the nose. Other behavioral signs of heat pain, such as vocalization, were either absent or strongly reduced in L1-deficient mice. Tactile thresholds for withdrawal reflexes were increased in L1-deficient mice when compared with wild-types except for the tail. By contrast, the spatial organization of the withdrawal reflexes appeared normal indicating that the L1 adhesion molecule is not essential for the spatial adjustments of reflex connections during development. The termination patterns of thin primary afferent fibers in the superficial dorsal horn, visualized using intra-plantar injections of WGA-HRP, were normal, suggesting that decreased nociceptive heat sensitivity in L1-deficient mice is mainly due to altered central processing. In view of the known interactions between L1 and some of the NMDA-receptor subtypes, and the prominent role of NMDA receptors in nociception and plasticity, it is conceivable that the hypoalgesia seen in L1 mutants is due, in part, to disturbed NMDA-receptor function.