Brian R. Noga

Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord

Central nervous system axons regenerate into a Schwann cell implant placed in the transected thoracic spinal cord of an adult rat. The present study was designed to test whether these regenerated axons are capable of conducting action potentials. Following the transection and removal of a 4‐ to 5‐mm segment of the thoracic spinal cord (T8–T9), a polymer guidance channel filled with a mixture of adult rat Schwann cells and Matrigel was grafted into a 4‐ to 5‐mm‐long gap in the transected thoracic spinal cord. The two cut ends of the spinal cord were eased into the guidance channel openings. Transected control animals received a channel containing Matrigel only. Three months after implantation, electrophysiological studies were performed. Tungsten microelectrodes were used for monopolar stimulation of regenerated axons within the Schwann cell graft. Glass microelectrodes were used to record responses in the spinal cord rostral to the stimulation site. Evoked responses to electrical stimulation of the axon cable were found in two out of nine Schwann cell‐grafted animals. These responses had approximate latencies in the range of those of myelinated axons. No responses were seen in any of the Matrigel‐grafted animals. Histological analysis revealed that the two cases that showed evoked potentials had the largest number of myelinated axons present in the cable. This study demonstrates that axons regenerating through Schwann cell grafts in the complete transected spinal cord can produce measurable evoked responses following electrical stimulation. J. Neurosci. Res. 64:533–541, 2001.

Locomotor-Activated Neurons of the Cat. I. Serotonergic Innervation and Co-Localization of 5-HT7, 5-HT2A, and 5-HT1A Receptors in the Thoraco-Lumbar Spinal Cord

Monoamines are strong modulators and/or activators of spinal locomotor networks. Thus monoaminergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the serotonergic innervation of locomotor-activated neurons within the thoraco-lumbar spinal cord following induction of hindlimb locomotion. This was determined by immunohistochemical co-localization of serotonin (5-HT) fibers or 5-HT(7)/5-HT2A/5-HT1A receptors with cells expressing the activity-dependent marker c-fos. Experiments were performed on paralyzed, decerebrate cats in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. Abundant c-fos immunoreactive cells were observed in laminae VII and VIII throughout the thoraco-lumbar segments of locomotor animals. Control sections from the same segments showed significantly fewer labeled neurons, mostly within the dorsal horn. Multiple serotonergic boutons were found in close apposition to the majority (80-100%) of locomotor cells, which were most abundant in lumbar segments L3-7. 5-HT7 receptor immunoreactivity was observed on cells across the thoraco-lumbar segments (T7-L7), in a dorsoventral gradient. Most locomotor-activated cells co-localized with 5-HT7, 5-HT2A, and 5-HT1A receptors, with largest numbers in laminae VII and VIII. Co-localization of c-fos and 5-HT7 receptor was highest in the L5-L7 segments (>90%) and decreased rostrally (to approximately 50%) due to the absence of receptors on cells within the intermediolateral nucleus. In contrast, 60-80 and 35-80% of c-fos immunoreactive cells stained positive for 5-HT2A and 5-HT1A receptors, respectively, with no rostrocaudal gradient. These results indicate that serotonergic modulation of locomotion likely involves 5-HT(7)/5-HT2A/5-HT1A receptors located on the soma and proximal dendrites of serotonergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments.

Spatial and temporal patterns of serotonin release in the rat’s lumbar spinal cord following electrical stimulation of the nucleus raphe magnus

The monoamine neurotransmitter serotonin is released from spinal terminals of nucleus raphe magnus (NRM) neurons and important in sensory and motor control, but its pattern of release has remained unclear. Serotonin was measured by the high-resolution method of fast cyclic voltammetry (2 Hz) with carbon-fiber microelectrodes in lumbar segments (L3-L6) of halothane-anesthetized rats during electrical stimulation of the NRM. Because sites of serotonin release are often histologically remote from membrane transporters and receptors, rapid emergence into aggregate extracellular space was expected. Increased monoamine oxidation currents were found in 94% of trials of 50-Hz, 20-s NRM stimulation across all laminae. The estimated peak serotonin concentration averaged 37.8 nM (maximum 287 nM), and was greater in dorsal and ventral laminae (I-III and VIII-IX) than in intermediate laminae (IV-VI). When measured near NRM-evoked changes, basal monoamine levels (relative to dorsal white matter) were highest in intermediate laminae, while changes in norepinephrine level produced by locus ceruleus (LC) stimulation were lowest in laminae II/III and VII. The NRM-evoked monoamine peak was linearly proportional to stimulus frequency (10-100 Hz). The peak often occurred before the stimulus ended (mean 15.6 s at 50 Hz, range 4-35 s) regardless of frequency, suggesting that release per impulse was constant during the rise but fell later. The latency from stimulus onset to electrochemical signal detection (mean 4.2 s, range 1-23 s) was inversely correlated with peak amplitude and directly correlated with time-to-peak. Quantitative modeling suggested that shorter latencies mostly reflected the time below detection threshold (5-10 nM), so that extrasynaptic serotonin was significantly elevated well within 1 s. Longer latencies (>5 s), which were confined to intermediate laminae, appeared mainly to be due to diffusion from distant sources. In conclusion, except possibly in intermediate laminae, serotonergic volume transmission is a significant mode of spinal control by the NRM.

Locomotor-activated neurons of the cat. II. Noradrenergic innervation and colocalization with NEα1a or NEα2b receptors in the thoraco-lumbar spinal cord

Norepinephrine (NE) is a strong modulator and/or activator of spinal locomotor networks. Thus noradrenergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the noradrenergic innervation of functionally related, locomotor-activated neurons within the thoraco-lumbar spinal cord. This was accomplished by immunohistochemical colocalization of noradrenergic fibers using dopamine-β-hydroxylase or NEα(1A) and NEα(2B) receptors with cells expressing the c-fos gene activity-dependent marker Fos. Experiments were performed on paralyzed, precollicular-postmamillary decerebrate cats, in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. The majority of Fos labeled neurons, especially abundant in laminae VII and VIII throughout the thoraco-lumbar (T13-L7) region of locomotor animals, showed close contacts with multiple noradrenergic boutons. A small percentage (10-40%) of Fos neurons in the T7-L7 segments showed colocalization with NEα(1A) receptors. In contrast, NEα(2B) receptor immunoreactivity was observed in 70-90% of Fos cells, with no obvious rostrocaudal gradient. In comparison with results obtained from our previous study on the same animals, a significantly smaller proportion of Fos labeled neurons were innervated by noradrenergic than serotonergic fibers, with significant differences observed for laminae VII and VIII in some segments. In lamina VII of the lumbar segments, the degree of monoaminergic receptor subtype/Fos colocalization examined statistically generally fell into the following order: NEα(2B) = 5-HT(2A) ≥ 5-HT(7) = 5-HT(1A) > NEα(1A). These results suggest that noradrenergic modulation of locomotion involves NEα(1A)/NEα(2B) receptors on noradrenergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments. Further study of the functional role of these receptors in locomotion is warranted.

LFP Oscillations in the Mesencephalic Locomotor Region during Voluntary Locomotion

Oscillatory rhythms in local field potentials (LFPs) are thought to coherently bind cooperating neuronal ensembles to produce behaviors, including locomotion. LFPs recorded from sites that trigger locomotion have been used as a basis for identification of appropriate targets for deep brain stimulation (DBS) to enhance locomotor recovery in patients with gait disorders. Theta band activity (6–12 Hz) is associated with locomotor activity in locomotion-inducing sites in the hypothalamus and in the hippocampus, but the LFPs that occur in the functionally defined mesencephalic locomotor region (MLR) during locomotion have not been determined. Here we record the oscillatory activity during treadmill locomotion in MLR sites effective for inducing locomotion with electrical stimulation in rats. The results show the presence of oscillatory theta rhythms in the LFPs recorded from the most effective MLR stimulus sites (at threshold ≤60 μA). Theta activity increased at the onset of locomotion, and its power was correlated with the speed of locomotion. In animals with higher thresholds (>60 μA), the correlation between locomotor speed and theta LFP oscillations was less robust. Changes in the gamma band (previously recorded in vitro in the pedunculopontine nucleus (PPN), thought to be a part of the MLR) were relatively small. Controlled locomotion was best achieved at 10–20 Hz frequencies of MLR stimulation. Our results indicate that theta and not delta or gamma band oscillation is a suitable biomarker for identifying the functional MLR sites.

Monoamine release in the cat lumbar spinal cord during fictive locomotion evoked by the mesencephalic locomotor region

Spinal cord neurons active during locomotion are innervated by descending axons that release the monoamines serotonin (5-HT) and norepinephrine (NE) and these neurons express monoaminergic receptor subtypes implicated in the control of locomotion. The timing, level and spinal locations of release of these two substances during centrally-generated locomotor activity should therefore be critical to this control. These variables were measured in real time by fast-cyclic voltammetry in the decerebrate cat’s lumbar spinal cord during fictive locomotion, which was evoked by electrical stimulation of the mesencephalic locomotor region (MLR) and registered as integrated activity in bilateral peripheral nerves to hindlimb muscles. Monoamine release was observed in dorsal horn (DH), intermediate zone/ventral horn (IZ/VH) and adjacent white matter (WM) during evoked locomotion. Extracellular peak levels (all sites) increased above baseline by 138 ± 232.5 nM and 35.6 ± 94.4 nM (mean ± SD) for NE and 5-HT, respectively. For both substances, release usually began prior to the onset of locomotion typically earliest in the IZ/VH and peaks were positively correlated with net activity in peripheral nerves. Monoamine levels gradually returned to baseline levels or below at the end of stimulation in most trials. Monoamine oxidase and uptake inhibitors increased the release magnitude, time-to-peak (TTP) and decline-to-baseline. These results demonstrate that spinal monoamine release is modulated on a timescale of seconds, in tandem with centrally-generated locomotion and indicate that MLR-evoked locomotor activity involves concurrent activation of descending monoaminergic and reticulospinal pathways. These gradual changes in space and time of monoamine concentrations high enough to strongly activate various receptors subtypes on locomotor activated neurons further suggest that during MLR-evoked locomotion, monoamine action is, in part, mediated by extrasynaptic neurotransmission in the spinal cord.

The Hierarchical Circuit for Executive Control of Movement

A major challenge in neuroscience research is the understanding of the vast universe of human brain and its mind. In this context, the ability of the mind to control behavior relies upon the executive control of movement. Herein, we focus on the hierarchical circuitry of the brain that exercises the executive control of movement. This executive mechanism spans hierarchically over frontal/parietal/temporal cortices, subcortical structures in basal ganglia and thalamus, brainstem and spinal cord. To address the hierarchical executive mechanism of movement, we will examine its frontal/parietal cortical microcircuits interconnected in thalamo-cortical loops via cortico-striatal projections, and further to the mesencephalic locomotor region and central pattern generators in the spinal cord for locomotor control. Spinal locomotor circuits are enabled by parallel activation/modulation from descending reticulospinal and monoaminergic pathways. The use of various stimulation approaches developed recently is examined in terms of preclinical (animal experiments) and clinical applications to human brain disorders. Future clinical studies on the pathological aspects of movement will employ novel, deep brain stimulation that is capable to function in a closed-loop manner, adjusting therapy delivery to the patient’s level of disease impairment.

From Symmetry to Symmetry-Breaking in Locomotion

The locomotor system is a hierarchical mechanism consisting of several functional components, including the decision mechanism, navigation map, locomotion command, central pattern generators and the EMG muscle activity patterns. In this chapter we discuss the role of symmetry/asymmetry and symmetry breaking of neural states during the emergence of locomotion and movement. We review recent results that show that inhibition plays a critical role in decision making, in the formation of grid cells and place cells for navigation, the locomotor command and central pattern generators. We employ the analogy with symmetry breaking in physical systems where at a bifurcation point on the phase diagram, infinitesimal perturbations result in a transition to a new global attractor state. This observation may have major implications for both understanding normal locomotion and therapeutics of spinal cord injury, as triggered by neuromodulatory/inhibitory causes.

Symmetry Breaking in Cognitive Disorders

In this chapter we discuss the effect of symmetry breaking and the emergence of brain/psychiatric disorders such as Alzheimer disease (AD), autism spectrum disorder (ASD), schizophrenia (SCZ), aging, and drug addiction. A common denominator in these brain disorders seems to be a faulty inhibition mechanism that plays a key role in the optimal functioning of neuronal microcircuits, loops and networks. When inhibition becomes suboptimal a transition from healthy mental states to pathological states starts to occur. A perspective that views mental disorders from the standpoint of symmetrical probability distributions may offer a paradigmatic shift for both diagnosis and therapeutical interventions.

What Is the Evidence for Inter-laminar Integration in a Prefrontal Cortical Minicolumn?

The objective of this perspective article is to examine columnar inter-laminar integration during the executive control of behavior. The integration hypothesis posits that perceptual and behavioral signals are integrated within the prefrontal cortical inter-laminar microcircuits. Inter-laminar minicolumnar activity previously recorded from the dorsolateral prefrontal cortex (dlPFC) of nonhuman primates, trained in a visual delay match-to-sample (DMS) task, was re-assessed from an integrative perspective. Biomorphic multielectrode arrays (MEAs) played a unique role in the in vivo recording of columnar cell firing in the dlPFC layers 2/3 and 5/6. Several integrative aspects stem from these experiments: 1. Functional integration of perceptual and behavioral signals across cortical layers during executive control. The integrative effect of dlPFC minicolumns was shown by: (i) increased correlated firing on correct vs. error trials; (ii) decreased correlated firing when the number of non-matching images increased; and (iii) similar spatial firing preference across cortical-striatal cells during spatial-trials, and less on object-trials. 2. Causal relations to integration of cognitive signals by the minicolumnar turbo-engines. The inter-laminar integration between the perceptual and executive circuits was facilitated by stimulating the infra-granular layers with firing patterns obtained from supra-granular layers that enhanced spatial preference of percent correct performance on spatial trials. 3. Integration across hierarchical levels of the brain. The integration of intention signals (visual spatial, direction) with movement preparation (timing, velocity) in striatum and with the motor command and posture in midbrain is also discussed. These findings provide evidence for inter-laminar integration of executive control signals within brain’s prefrontal cortical microcircuits.