The Mesencephalic Locomotor Region: Beyond Locomotor Control.

The mesencephalic locomotor region (MLR) was discovered several decades ago in the cat. It was functionally defined based on the ability of low threshold electrical stimuli within a region comprising the cuneiform and pedunculopontine nucleus to evoke locomotion. Since then, similar regions have been found in diverse vertebrate species, including the lamprey, skate, rodent, pig, monkey, and human. The MLR, while often viewed under the lens of locomotion, is involved in diverse processes involving the autonomic nervous system, respiratory system, and the state-dependent activation of motor systems. This review will discuss the pedunculopontine nucleus and cuneiform nucleus that comprises the MLR and examine their respective connectomes from both an anatomical and functional angle. From a functional perspective, the MLR primes the cardiovascular and respiratory systems before the locomotor activity occurs. Inputs from a variety of higher structures, and direct outputs to the monoaminergic nuclei, allow the MLR to be able to respond appropriately to state-dependent locomotion. These state-dependent effects are roughly divided into escape and exploratory behavior, and the MLR also can reinforce the selection of these locomotor behaviors through projections to adjacent structures such as the periaqueductal gray or to limbic and cortical regions. Findings from the rat, mouse, pig, and cat will be discussed to highlight similarities and differences among diverse species.

Combined neuromodulatory approaches in the central nervous system for treatment of spinal cord injury.

PURPOSE OF REVIEW: To report progress in neuromodulation following spinal cord injury (SCI) using combined brain and spinal neuromodulation.Neuromodulation refers to alterations in neuronal activity for therapeutic purposes. Beneficial effects are established in disease states such as Parkinson's Disease (PD), chronic pain, epilepsy, and SCI. The repertoire of neuromodulation and bioelectric medicine is rapidly expanding. After SCI, cohort studies have reported the benefits of epidural stimulation (ES) combined with training. Recently, we have explored combining ES with deep brain stimulation (DBS) to increase activation of descending motor systems to address limitations of ES in severe SCI. In this review, we describe the types of applied neuromodulation that could be combined in SCI to amplify efficacy to enable movement. These include ES, mesencephalic locomotor region (MLR) - DBS, noninvasive transcutaneous stimulation, transcranial magnetic stimulation, paired-pulse paradigms, and neuromodulatory drugs. We examine immediate and longer-term effects and what is known about: (1) induced neuroplastic changes, (2) potential safety concerns; (3) relevant outcome measures; (4) optimization of stimulation; (5) therapeutic limitations and prospects to overcome these. RECENT FINDINGS: DBS of the mesencephalic locomotor region is emerging as a potential clinical target to amplify supraspinal command circuits for locomotion. SUMMARY: Combinations of neuromodulatory methods may have additive value for restoration of function after spinal cord injury.

MR Tractography-Based Targeting and Physiological Identification of the Cuneiform Nucleus for Directional DBS in a Parkinson's Disease Patient With Levodopa-Resistant Freezing of Gait.

BACKGROUND: Freezing of gait (FOG) is a debilitating motor deficit in a subset of Parkinson's Disease (PD) patients that is poorly responsive to levodopa or deep brain stimulation (DBS) of established PD targets. The proposal of a DBS target in the midbrain, known as the pedunculopontine nucleus (PPN), to address FOG was based on its observed neuropathology in PD and its hypothesized involvement in locomotor control as a part of the mesencephalic locomotor region (MLR). Initial reports of PPN DBS were met with enthusiasm; however, subsequent studies reported mixed results. A closer review of the MLR basic science literature, suggests that the closely related cuneiform nucleus (CnF), dorsal to the PPN, may be a superior site to promote gait. Although suspected to have a conserved role in the control of gait in humans, deliberate stimulation of a homolog to the CnF in humans using directional DBS electrodes has not been attempted. METHODS: As part of an open-label Phase 1 clinical study, one PD patient with predominantly axial symptoms and severe FOG refractory to levodopa therapy was implanted with directional DBS electrodes (Boston Science Vercise CartesiaTM) targeting the CnF bilaterally. Since the CnF is a poorly defined reticular nucleus, targeting was guided both by diffusion tensor imaging (DTI) tractography and anatomical landmarks. Intraoperative stimulation and microelectrode recordings were performed near the targets with leg EMG surface recordings in the subject. RESULTS: Post-operative imaging revealed accurate targeting of both leads to the designated CnF. Intraoperative stimulation near the target at low thresholds in the awake patient evoked involuntary electromyography (EMG) oscillations in the legs with a peak power at the stimulation frequency, similar to observations with CnF DBS in animals. Oscillopsia was the primary side effect evoked at higher currents, especially when directed posterolaterally. Directional DBS could mitigate oscillopsia. CONCLUSION: DTI-based targeting and intraoperative stimulation to evoke limb EMG activity may be useful methods to help target the CnF accurately and safely in patients. Long term follow-up and detailed gait testing of patients undergoing CnF stimulation will be necessary to confirm the effects on FOG. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04218526.

Deep brain stimulation of the Cuneiform nucleus for levodopa-resistant freezing of gait in Parkinson's disease: study protocol for a prospective, pilot trial.

BACKGROUND: Freezing of gait (FOG) is a particularly debilitating motor deficit seen in a subset of Parkinson's disease (PD) patients that is poorly responsive to standard levodopa therapy or deep brain stimulation (DBS) of established PD targets such as the subthalamic nucleus and the globus pallidus interna. The proposal of a DBS target in the midbrain, known as the pedunculopontine nucleus (PPN) to address FOG, was based on its observed pathology in PD and its hypothesized involvement in locomotor control as a part of the mesencephalic locomotor region, a functionally defined area of the midbrain that elicits locomotion in both intact animals and decerebrate animal preparations with electrical stimulation. Initial reports of PPN DBS were met with much enthusiasm; however, subsequent studies produced mixed results, and recent meta-analysis results have been far less convincing than initially expected. A closer review of the extensive mesencephalic locomotor region (MLR) preclinical literature, including recent optogenetics studies, strongly suggests that the closely related cuneiform nucleus (CnF), just dorsal to the PPN, may be a superior target to promote gait initiation. METHODS: We will conduct a prospective, open-label, single-arm pilot study to assess safety and feasibility of CnF DBS in PD patients with levodopa-refractory FOG. Four patients will receive CnF DBS and have gait assessments with and without DBS during a 6-month follow-up. DISCUSSION: This paper presents the study design and rationale for a pilot study investigating a novel DBS target for gait dysfunction, including targeting considerations. This pilot study is intended to support future larger scale clinical trials investigating this target. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04218526 (registered January 6, 2020).

Deep brain stimulation of midbrain locomotor circuits in the freely moving pig.

BACKGROUND: Deep brain stimulation (DBS) of the mesencephalic locomotor region (MLR) has been studied as a therapeutic target in rodent models of stroke, parkinsonism, and spinal cord injury. Clinical DBS trials have targeted the closely related pedunculopontine nucleus in patients with Parkinson's disease as a therapy for gait dysfunction, with mixed reported outcomes. Recent studies suggest that optimizing the MLR target could improve its effectiveness. OBJECTIVE: We sought to determine if stereotaxic targeting and DBS in the midbrain of the pig, in a region anatomically similar to that previously identified as the MLR in other species, could initiate and modulate ongoing locomotion, as a step towards generating a large animal neuromodulation model of gait. METHODS: We implanted Medtronic 3389 electrodes into putative MLR structures in Yucatan micropigs to characterize the locomotor effects of acute DBS in this region, using EMG recordings, joint kinematics, and speed measurements on a manual treadmill. RESULTS: MLR DBS initiated and augmented locomotion in freely moving micropigs. Effective locomotor sites centered around the cuneiform nucleus and stimulation frequency controlled locomotor speed and stepping frequency. Off-target stimulation evoked defensive and aversive behaviors that precluded locomotion in the animals. CONCLUSION: Pigs appear to have an MLR and can be used to model neuromodulation of this gait-promoting center. These results indicate that the pig is a useful model to guide future clinical studies for optimizing MLR DBS in cases of gait deficiencies associated with such conditions as Parkinson's disease, spinal cord injury, or stroke.

In vivo Population Averaged Stereotaxic T2w MRI Brain Template for the Adult Yucatan Micropig.

Population averaged brain templates are an essential tool for imaging-based neuroscience research, providing investigators with information about the expected size and morphology of brain structures and the spatial relationships between them, within a demographic cross-section. This allows for a standardized comparison of neuroimaging data between subjects and provides neuroimaging software with a probabilistic framework upon which further processing and analysis can be based. Many different templates have been created to represent specific study populations and made publicly available for human and animal research. An increasingly studied animal model in the neurosciences that still lacks appropriate brain templates is the adult Yucatan micropig. In particular, T2-weighted templates are absent in this species as a whole. To address this need and provide a tool for neuroscientists wishing to pursue neuroimaging research in the adult micropig, we present the construction of population averaged (n = 16) T2-weighted MRI brain template for the adult Yucatan micropig. Additionally, we present initial analysis of T1-weighted (n = 3), and diffusion-weighted (n = 3) images through multimodal registration of these contrasts to our T2 template. The strategies used here may also be generalized to create similar templates for other study populations or species in need of template construction.

Dissecting Brainstem Locomotor Circuits: Converging Evidence for Cuneiform Nucleus Stimulation.

There are a pressing and unmet need for effective therapies for freezing of gait (FOG) and other neurological gait disorders. Deep brain stimulation (DBS) of a midbrain target known as the pedunculopontine nucleus (PPN) was proposed as a potential treatment based on its postulated involvement in locomotor control as part of the mesencephalic locomotor region (MLR). However, DBS trials fell short of expectations, leading many clinicians to abandon this strategy. Here, we discuss the potential reasons for this failure and review recent clinical data along with preclinical optogenetics evidence to argue that another nearby nucleus, the cuneiform nucleus (CnF), may be a superior target.

Activation of Brainstem Neurons During Mesencephalic Locomotor Region-Evoked Locomotion in the Cat.

The distribution of locomotor-activated neurons in the brainstem of the cat was studied by c-Fos immunohistochemistry in combination with antibody-based cellular phenotyping following electrical stimulation of the mesencephalic locomotor region (MLR) - the anatomical constituents of which remain debated today, primarily between the cuneiform (CnF) and the pedunculopontine tegmental nuclei (PPT). Effective MLR sites were co-extensive with the CnF nucleus. Animals subject to the locomotor task showed abundant Fos labeling in the CnF, parabrachial nuclei of the subcuneiform region, periaqueductal gray, locus ceruleus (LC)/subceruleus (SubC), Kölliker-Fuse, magnocellular and lateral tegmental fields, raphe, and the parapyramidal region. Labeled neurons were more abundant on the side of stimulation. In some animals, Fos-labeled cells were also observed in the ventral tegmental area, medial and intermediate vestibular nuclei, dorsal motor nucleus of the vagus, n. tractus solitarii, and retrofacial nucleus in the ventrolateral medulla. Many neurons in the reticular formation were innervated by serotonergic fibers. Numerous locomotor-activated neurons in the parabrachial nuclei and LC/SubC/Kölliker-Fuse were noradrenergic. Few cholinergic neurons within the PPT stained for Fos. In the medulla, serotonergic neurons within the parapyramidal region and the nucleus raphe magnus were positive for Fos. Control animals, not subject to locomotion, showed few Fos-labeled neurons in these areas. The current study provides positive evidence for a role for the CnF in the initiation of locomotion while providing little evidence for the participation of the PPT. The results also show that MLR-evoked locomotion involves the parallel activation of reticular and monoaminergic neurons in the pons/medulla, and provides the anatomical and functional basis for spinal monoamine release during evoked locomotion. Lastly, the results indicate that vestibular, cardiovascular, and respiratory centers are centrally activated during MLR-evoked locomotion. Altogether, the results show a complex pattern of neuromodulatory influences of brainstem neurons by electrical activation of the MLR.

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.

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.

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.

Spontaneous and electrically-evoked catecholamine secretion from long-term cultures of bovine adrenal chromaffin cells.

Catecholamine release was measured from bovine adrenal medullary chromaffin cell (CC) cultures maintained over a period of three months. Cells were plated over simple biocompatible cell platforms with electrical stimulation capability and at specified times transferred to an acrylic superfusion chamber designed to allow controlled flow of superfusate over the culture. Catecholamine release was measured from the superfusates using fast cyclic voltammetry before, during and after electrical stimulation of the cells. Immunocytochemical staining of CC cultures revealed that they were composed of epinephrine (EP) and/or norepinephrine (NE) type cells. Both spontaneous and evoked-release of catecholamines from CCs were observed throughout the testing period. EP predominated during spontaneous release, whereas NE was more prevalent during electrically-evoked release. Electrical stimulation for 20 s, increased total catecholamine release by 60-130% (measured over a period of 500 s) compared to that observed for an equivalent 20 s period of spontaneous release. Stimulus intensity was correlated with the amount of evoked release, up to a plateau which was observed near the highest intensities. Shorter intervals between stimulation trials did not significantly affect the initial amount of release, and the amount of evoked release was relatively stable over time and did not decrease significantly with age of the culture. The present study demonstrates long-term survival of CC cultures in vitro and describes a technique useful for rapid assessment of cell functionality and release properties of cultured monoaminergic cell types that later can be transplanted for neurotransmitter replacement following injury or disease.

Locomotor-activated neurons of the cat. II. Noradrenergic innervation and colocalization with NEα 1a or NEα 2b 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-ß-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.

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.

Serotonin concentrations in the lumbosacral spinal cord of the adult rat following microinjection or dorsal surface application.

Application of neuroactive substances, including monoamines, is common in studies examining the spinal mechanisms of sensation and behavior. However, affected regions and time courses of transmitter activity are uncertain. We measured the spatial and temporal distribution of serotonin [5-hydroxytryptamine (5-HT)] in the lumbosacral spinal cord of halothane-anesthetized adult rats, following its intraspinal microinjection or surface application. Carbon fiber microelectrodes (CFMEs) were positioned at various locations in the spinal cord and oxidation currents corresponding to extracellular 5-HT were measured by fast cyclic voltammetry. Intraspinal microinjection of 5-HT (100 microM, 1-3 microl) produced responses that were most pronounced at CFMEs positioned

Steady-state levels of monoamines in the rat lumbar spinal cord: spatial mapping and the effect of acute spinal cord injury.

Monoamines in the spinal cord are important in the regulation of locomotor rhythms, nociception, and motor reflexes. To gain further insight into the control of these functions, the steady-state extracellular distribution of monoamines was mapped in the anesthetized rat's lumbar spinal cord. The effect of acute spinal cord lesions at sites selected for high resting levels was determined over approximately 1 h to estimate contributions to resting levels from tonic descending activity and to delineate chemical changes that may influence the degree of pathology and recovery after spinal injury. Measurements employed fast cyclic voltammetry with carbon fiber microelectrodes to give high spatial resolution. Monoamine oxidation currents, sampled at equal vertical spacings within each segment, were displayed as contours over the boundaries delineated by histologically reconstructed electrode tracks. Monoamine oxidation currents were found in well defined foci, often confined within a single lamina. Larger currents were typically found in the dorsal or ventral horns and in the lateral aspect of the intermediate zone. Cooling of the low-thoracic spinal cord led to a decrease in the oxidation current (to 71-85% of control) in dorsal and ventral horns. Subsequent low-thoracic transection produced a transient increase in signal in some animals followed by a longer lasting decrease to levels similar to or below that with cooling (to 17-86% of control values). We conclude that descending fibers tonically release high amounts of monoamines in localized regions of the dorsal and ventral horn of the lumbar spinal cord at rest. Lower amounts of monoamines were detected in medial intermediate zone areas, where strong release may be needed for descending activation of locomotor rhythms.

Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region.

The synaptic pathways of mesencephalic locomotor region (MLR)-evoked excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) recorded from lumbar motoneurons of unanesthetized decerebrate cats during fictive locomotion were analyzed prior to, during, and after cold block of the medial reticular formation (MedRF) or the low thoracic ventral funiculus (VF). As others have shown, electrical stimulation of the MLR typically evoked short-latency excitatory or mixed excitatory/inhibitory PSPs in flexor and extensor motoneurons. The bulbospinal conduction velocities averaged approximately 88 m/s (range: 62-145 m/s) and segmental latencies for EPSPs ranged from 1.2 to 10.9 ms. The histogram of segmental latencies showed three peaks, suggesting di-, tri-, and polysynaptic linkages. Segmental latencies for IPSPs suggested trisynaptic or polysynaptic transmission. Most EPSPs (69/77) were significantly larger during the depolarized phase of the intracellular locomotor drive potential (LDP), and most IPSPs (35/46) were larger during the corresponding hyperpolarized phase. Bilateral cooling of the MedRF reversibly abolished locomotion of both hindlimbs as measured from the electroneurogram (ENG) activity of muscle nerves and simultaneously abolished or diminished the motoneuron PSPs and LDPs. Unilateral cooling of the VF blocked locomotion ipsilaterally and diminished it contralaterally with concomitant loss or decrease the motoneuron PSPs and LDPs. Relative to the side of motoneuron recording, cooling of the ipsilateral VF sometimes uncovered longer-latency EPSPs, whereas cooling of the contralateral VF abolished longer-latency EPSPs. It is concluded that MLR stimulation activates a pathway that relays in the MedRF and descends bilaterally in the VF to contact spinal interneurons that project to motoneurons. Local segmental pathways that activate or inhibit motoneurons during MLR-evoked fictive locomotion appear to be both ipsilateral and contralateral.

Temporal and spatial profiles of pontine-evoked monoamine release in the rat's spinal cord.

In the spinal cord, the monoamine neurotransmitter norepinephrine, which is released mainly from fibers descending from the dorsal pons, has major modulatory effects on nociception and locomotor rhythms. To map the spatial and temporal patterns of this release, changes in monoamine level were examined in laminae I-VIII of lumbar segments L3-L6 of halothane-anesthetized rats during pontine stimulation. The changes were measured through a carbon fiber microelectrode at 0.5-s intervals by fast cyclic voltammetry, which presently is the method of best spatiotemporal resolution. When different pontine sites were tested with 20-s pulse trains (50-to 200-microA amplitude, 0.5-ms pulse width, and 50-Hz frequency) during measurement in the dorsal horn (lamina IV), the largest consistent increases were produced by the locus ceruleus, although effective pontine sites extended 1.5 mm dorsally and ventral from the locus ceruleus. When the locus ceruleus stimulus was used to map the spinal cord, increased levels were always seen in lamina I and laminae IV-VIII, whereas 50% of sites in laminae II and III showed substantial decreases and the rest showed increases. These increases typically had short latencies [4.5 +/- 0.4 (SE) s] and variable decay times (5-200 s), with peaks occurring during the stimulus train (mean rise-time: 12.0 +/- 0.6 s). The mean peak level was 544 +/- 82 nM as estimated from postexperimental calibration with norepinephrine. Other significant laminar differences included higher mean peak concentrations (805 nM) and rise times (14.9 s) in lamina I and shorter latencies in lamina VI (3.2 s). Peak concentrations were inversely correlated with latency. When stimulation frequency was varied, increases were disproportionately larger with faster frequencies (> or =50 Hz), hence extrajunctional overflow probably contributed most of the signal. We conclude, generally, that pontine noradrenergic control is exerted on widespread spinal laminae with a significant component of paracrine transmission after several seconds of sustained activity. Relatively stronger effects prevail where nociceptive transmission (lamina I) and locomotor rhythm generation (lamina VI) occur.