Confounders and Pharmacological Characterization When Using the QT, JTp, and Tpe Intervals in Beagle Dogs.

INTRODUCTION: Corrected QT (QTc) interval is an essential proarrhythmic risk biomarker, but recent data have identified limitations to its use. The J to T-peak (JTp) interval is an alternative biomarker for evaluating drug-induced proarrhythmic risk. The aim of this study was to evaluate pharmacological effects using spatial magnitude leads and DII electrocardiogram (ECG) leads and common ECG confounders (ie, stress and body temperature changes) on covariate adjusted QT (QTca), covariate adjusted JTp (JTpca), and covariate adjusted T-peak to T-end (Tpeca) intervals. METHODS: Beagle dogs were exposed to body hyper- (42 °C) or hypothermic (33 °C) conditions or were administered epinephrine to assess confounding effects on heart rate corrected QTca, JTpca, and Tpeca intervals. Dofetilide (0.1, 0.3, 1.0 mg/kg), ranolazine (100, 140, 200 mg/kg), and verapamil (7, 15, 30, 43, 62.5 mg/kg) were administered to evaluate pharmacological effects. RESULTS: Covariate adjusted QT (slope -12.57 ms/°C) and JTpca (-14.79 ms/°C) were negatively correlated with body temperature but Tpeca was minimally affected. Epinephrine was associated with QTca and JTpca shortening, which could be related to undercorrection in the presence of tachycardia, while minimal effects were observed for Tpeca. There were no significant ECG change following ranolazine administration. Verapamil decreased QTca and JTpca intervals and increased Tpeca, whereas dofetilide increased QTca and JTpca intervals but had inconsistent effects on Tpeca. CONCLUSION: Results highlight potential confounders on QTc interval, but also on JTpca and Tpeca intervals in nonclinical studies. These potential confounding effects may be relevant to the interpretation of ECG data obtained from nonclinical drug safety studies with Beagle dogs.

A Proof-of-Concept Evaluation of JTPc and Tp-Tec as Proarrhythmia Biomarkers in Preclinical Species: A Retrospective Analysis by an HESI-Sponsored Consortium.

INTRODUCTION: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. METHODS: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). RESULTS: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. DISCUSSION: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

Short-term depression of external globus pallidus-subthalamic nucleus synaptic transmission and implications for patterning subthalamic activity.

The frequency and pattern of activity in the reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN) are closely related to motor function. Although phasic, unitary GPe-STN inputs powerfully pattern STN activity ex vivo, correlated GPe-STN activity is not normally observed in vivo. To test the hypothesis that the GPe's influence is constrained by short-term synaptic depression, unitary GPe-STN inputs were stimulated in rat and mouse brain slices at rates and in patterns that mimicked GPe activity in vivo. Together with connectivity estimates these data were then used to simulate GPe-STN transmission. Unitary GPe-STN synaptic connections initially generated large conductances and transmitted reliably. However, the amplitude and reliability of transmission declined rapidly (τ = 0.6 ± 0.5 s) to <10% of their initial values when connections were stimulated at the mean rate of GPe activity in vivo (33 Hz). Recovery from depression (τ = 17.3 ± 18.9 s) was also longer than pauses in tonic GPe activity in vivo. Depression was the result of the limited supply of release-ready vesicles and was in sharp contrast to Calyx of Held transmission, which exhibited 100% reliability. Injection of simulated GPe-STN conductances revealed that synaptic depression caused tonic, nonsynchronized GPe-STN activity to disrupt rather than abolish autonomous STN activity. Furthermore, synchronous inhibition of tonically active GPe-STN neurons or phasic activity of GPe-STN neurons reliably patterned STN activity through disinhibition and inhibition, respectively. Together, these data argue that the frequency and pattern of GPe activity profoundly influence its transmission to the STN.

Chapter 2--the spinal generation of phases and cycle duration.

During walking, an increase in speed is accompanied by a decrease in the stance phase duration while the swing phase remains relatively invariant. By definition, the rhythm generator in the lumbar spinal cord controls cycle period, phase durations, and phase transitions. Our first aim was to determine if this asymmetry in the control of locomotor cycles is an inherent property of the central pattern generator (CPG). We recorded episodes of fictive locomotion, that is, locomotor patterns in absence of reafference, in decerebrate cats with or without a complete spinal transection (acute or chronic). In fictive locomotion, stance and swing phases typically correspond to extension and flexion, respectively. In the vast majority of locomotor episodes, cycle period varied more with extensor phase duration. This could be observed without phasic sensory feedback or supraspinal structures or pharmacology. In a few experiments, we stimulated the mesencephalic locomotor region or selected peripheral nerves during fictive locomotion and both could alter the phase/cycle period relationship. We conclude that there is a built-in asymmetry within the spinal rhythm generator for locomotion, which can be modified by extraneous factors. Locomotor and scratching rhythms are characterized by alternation of flexion and extension phases within one hindlimb, which are mediated by rhythm-generating circuitry within the spinal cord. Our second aim was to determine if rhythm generators for locomotion and scratch have similar control mechanisms in adult decerebrate cats. The regulation of cycle period during fictive scratching was evaluated, as were the effects of specific sensory inputs on phase durations and transitions during pinna-evoked fictive scratching. Results show that cycle period during fictive scratching varied predominantly with flexion phase duration, contrary to spontaneous fictive locomotion. Ankle dorsiflexion greatly increased extension phase duration and cycle period during fictive locomotion but did not alter cycle period during scratching. These data indicate that cycle period, phase durations, and phase transitions are not regulated similarly during fictive locomotion and scratching, with or without sensory inputs, providing evidence for the existence of distinct interneuronal components of rhythm generation within the mammalian spinal cord.

Sparse but selective and potent synaptic transmission from the globus pallidus to the subthalamic nucleus.

The reciprocally connected GABAergic globus pallidus (GP)-glutamatergic subthalamic nucleus (STN) network is critical for voluntary movement and an important site of dysfunction in movement disorders such as Parkinson's disease. Although the GP is a key determinant of STN activity, correlated GP-STN activity is rare under normal conditions. Here we define fundamental features of the GP-STN connection that contribute to poorly correlated GP-STN activity. Juxtacellular labeling of single GP neurons in vivo and stereological estimation of the total number of GABAergic GP-STN synapses suggest that the GP-STN connection is surprisingly sparse: single GP neurons maximally contact only 2% of STN neurons and single STN neurons maximally receive input from 2% of GP neurons. However, GP-STN connectivity may be considerably more selective than even these estimates imply. Light and electron microscopic analyses revealed that single GP axons give rise to sparsely distributed terminal clusters, many of which correspond to multiple synapses with individual STN neurons. Application of the minimal stimulation technique in brain slices confirmed that STN neurons receive multisynaptic unitary inputs and that these inputs largely arise from different sets of GABAergic axons. Finally, the dynamic-clamp technique was applied to quantify the impact of GP-STN inputs on STN activity. Small fractions of GP-STN input were sufficiently powerful to inhibit and synchronize the autonomous activity of STN neurons. Together these data are consistent with the conclusion that the rarity of correlated GP-STN activity in vivo is due to the sparsity and selectivity, rather than the potency, of GP-STN synaptic connections.

Diencephalic locomotor region in the lamprey--afferents and efferent control.

In vertebrates, locomotion can be initiated by stimulation of the diencephalic locomotor region (DLR). Little is known of the different forebrain regions that provide input to the neurons in DLR. In the lamprey, it had been shown previously that DLR provides monosynaptic input to reticulospinal neurons, which in turn elicit rhythmic ventral root activity at the spinal level. To show that actual locomotor movements are produced from DLR, we use a semi-intact preparation in which the brain stem is exposed and the head fixed, while the body is left to generate actual swimming movements. DLR stimulation induced symmetric locomotor movements with an undulatory wave transmitted along the body. To explore if DLR is under tonic GABAergic input under resting conditions, as in mammals, GABAergic antagonists and agonists were locally administered into DLR. Injections of GABA agonists inhibited locomotion, whereas GABA antagonists facilitated the induction of locomotion. These findings suggest that GABAergic projections provide tonic inhibition that once turned off can release locomotion. Double-labeling experiments were carried out to identify GABAergic projections to the DLR. Populations of GABAergic projection neurons to DLR originated in the caudoventral portion of the medial pallium, the lateral and dorsal pallium, and the striatal area. These different GABAergic projection neurons, which also project to other brain stem motor centers, may represent the basal ganglia output to DLR. Moreover, electrical stimulation of striatum induced long-lasting plateau potentials in reticulospinal cells and associated locomotor episodes dependent on DLR being intact, suggesting that striatum may act via the basal ganglia output identified here.

Initiation of locomotion in lampreys.

The spinal circuitry underlying the generation of basic locomotor synergies has been described in substantial detail in lampreys and the cellular mechanisms have been identified. The initiation of locomotion, on the other hand, relies on supraspinal networks and the cellular mechanisms involved are only beginning to be understood. This review examines some of the findings relative to the neural mechanisms involved in the initiation of locomotion of lampreys. Locomotion can be elicited by sensory stimulation or by internal cues associated with fundamental needs of the animal such as food seeking, exploration, and mating. We have described mechanisms by which escape swimming is elicited in lampreys in response to mechanical skin stimulation. A rather simple neural connectivity is involved, including sensory and relay neurons, as well as the brainstem rhombencephalic reticulospinal cells, which act as command neurons. We have shown that reticulospinal cells have intrinsic membrane properties that allow them to transform a short duration sensory input into a long-lasting excitatory command that activates the spinal locomotor networks. These mechanisms constitute an important feature for the activation of escape swimming. Other sensory inputs can also elicit locomotion in lampreys. For instance, we have recently shown that olfactory signals evoke sustained depolarizations in reticulospinal neurons and chemical activation of the olfactory bulbs with local injections of glutamate induces fictive locomotion. The mechanisms by which internal cues initiate locomotion are less understood. Our research has focused on one particular locomotor center in the brainstem, the mesencephalic locomotor region (MLR). The MLR is believed to channel inputs from many brain regions to generate goal-directed locomotion. It activates reticulospinal cells to elicit locomotor output in a graded fashion contrary to escape locomotor bouts, which are all-or-none. MLR inputs to reticulospinal cells use both glutamatergic and cholinergic transmission; nicotinic receptors on reticulospinal cells are involved. MLR excitatory inputs to reticulospinal cells in the middle (MRRN) are larger than those in the posterior rhombencephalic reticular nucleus (PRRN). Moreover at low stimulation strength, reticulospinal cells in the MRRN are activated first, whereas those in the PRRN require stronger stimulation strengths. The output from the MLR on one side activates reticulospinal neurons on both sides in a highly symmetrical fashion. This could account for the symmetrical bilateral locomotor output evoked during unilateral stimulation of the MLR in all animal species tested to date. Interestingly, muscarinic receptor activation reduces sensory inputs to reticulospinal neurons and, under natural conditions, the activation of MLR cholinergic neurons will likely reduce sensory inflow. Moreover, exposing the brainstem to muscarinic agonists generates sustained recurring depolarizations in reticulospinal neurons through pre-reticular effects. Cells in the caudal half of the rhombencephalon appear to be involved and we propose that the activation of these muscarinoceptive cells could provide additional excitation to reticulospinal cells when the MLR is activated under natural conditions. One important question relates to sources of inputs to the MLR. We found that substance P excites the MLR, whereas GABA inputs tonically maintain the MLR inhibited and removal of this inhibition initiates locomotion. Other locomotor centers exist such as a region in the ventral thalamus projecting directly to reticulospinal cells. This region, referred to as the diencephalic locomotor region, receives inputs from several areas in the forebrain and is likely important for goal-directed locomotion. In summary, this review focuses on the most recent findings relative to initiation of lamprey locomotion in response to sensory and internal cues in lampreys.

GABA distribution in lamprey is phylogenetically conserved.

The localization of gamma-aminobutyric acid (GABA) has been well described in most classes of vertebrates but not in adult lampreys. The question if the GABA distribution is similar throughout the vertebrate subphylum is therefore still to be addressed. We here investigate two lamprey species, the sea lamprey, Petromyzon marinus, and the river lamprey, Lampetra fluviatilis, and compare the GABA pattern with that of other vertebrates. The present immunohistochemical study provides an anatomical basis for the general distribution and precise localization of GABAergic neurons in the adult lamprey forebrain and brainstem. GABA-immunoreactive cells were organized in a virtually identical manner in the two species. They were found throughout the brain, with the following regions being of particular interest: the granular cell layer of the olfactory bulb, the nucleus of the anterior commissure, the septum, the lateral and medial pallia, the striatum, the nucleus of the postoptic commissure, the thalamus, the hypothalamus, and pretectal areas, the optic tectum, the torus semicircularis, the mesencephalic tegmentum, restricted regions of the rhombencephalic tegmentum, the octavolateral area, and the dorsal column nucleus. The GABA distribution found in cyclostomes is very similar to that of other classes of vertebrates, including mammals. Since the lamprey diverged from the main vertebrate line around 450 million years ago, this implies that already at that time the basic vertebrate plan for the GABA innervation in different parts of the brain had been developed.

Tectal control of locomotion, steering, and eye movements in lamprey.

The intrinsic function of the brain stem-spinal cord networks eliciting the locomotor synergy is well described in the lamprey-a vertebrate model system. This study addresses the role of tectum in integrating eye, body orientation, and locomotor movements as in steering and goal-directed behavior. Electrical stimuli were applied to different areas within the optic tectum in head-restrained semi-intact lampreys (n = 40). Motions of the eyes and body were recorded simultaneously (videotaped). Brief pulse trains (<0.5 s) elicited only eye movements, but with longer stimuli (>0.5 s) lateral bending movements of the body (orientation movements) were added, and with even longer stimuli locomotor movements were initiated. Depending on the tectal area stimulated, four characteristic response patterns were observed. In a lateral area conjugate horizontal eye movements combined with lateral bending movements of the body and locomotor movements were elicited, depending on stimulus duration. The amplitude of the eye movement and bending movements was site specific within this region. In a rostromedial area, bilateral downward vertical eye movements occurred. In a caudomedial tectal area, large-amplitude undulatory body movements akin to struggling behavior were elicited, combined with large-amplitude eye movements that were antiphasic to the body movements. The alternating eye movements were not dependent on vestibuloocular reflexes. Finally, in a caudolateral area locomotor movements without eye or bending movements could be elicited. These results show that tectum can provide integrated motor responses of eye, body orientation, and locomotion of the type that would be required in goal-directed locomotion.

Descending GABAergic projections to the mesencephalic locomotor region in the lamprey Petromyzon marinus.

The mesencephalic locomotor region (MLR) plays a significant role in the control of locomotion in all vertebrate species investigated. Forebrain neurons are likely to modulate MLR activity, but little is known about their inputs. Descending GABAergic projections to the MLR were identified by double-labeling neurons using Neurobiotin injected into the MLR combined with immunofluorescence against GABA. Several GABAergic projections to the MLR were identified in the telencephalon and diencephalon. The most abundant GABAergic projection to the MLR came from the caudal portion of the medial pallium, a region that may have similarities with the amygdala of higher vertebrates. A small population of GABAergic cells projecting to the MLR was found in the striatum and the ventral portion of the lateral pallium, which could respectively correspond to the input and output components of the basal ganglia thought to be involved in the selection of motor programs. Other GABAergic projections were found to come from the thalamus and the hypothalamus, which could take part in the motivational aspect of motor behavior in lampreys. Electrophysiological experiments were also carried out to examine the effects of GABA agonists and antagonists injected into the MLR in a semi-intact lamprey preparation. The GABA agonist inhibited locomotion, whereas the GABA antagonist initiated it. These results suggest that the GABAergic projections to the MLR modulate the activity of MLR neurons, which would be inhibited by GABA at rest.

Afferents of the lamprey optic tectum with special reference to the GABA input: combined tracing and immunohistochemical study.

The optic tectum in the lamprey midbrain, homologue of the superior colliculus in mammals, is important for eye movement control and orienting responses. There is, however, only limited information regarding the afferent input to the optic tectum except for that from the eyes. The objective of this study was to define specifically the gamma-aminobutyric acid (GABA)-ergic projections to the optic tectum in the river lamprey (Lampetra fluviatilis) and also to describe the tectal afferent input in general. The origin of afferents to the optic tectum was studied by using the neuronal tracer neurobiotin. Injection of neurobiotin into the optic tectum resulted in retrograde labelling of cell groups in all major subdivisions of the brain. The main areas shown to project to the optic tectum were the following: the caudoventral part of the medial pallium, the area of the ventral thalamus and dorsal thalamus, the nucleus of the posterior commissure, the torus semicircularis, the mesencephalic M5 nucleus of Schober, the mesencephalic reticular area, the ishtmic area, and the octavolateral nuclei. GABAergic projections to the optic tectum were identified by combining neurobiotin tracing and GABA immunohistochemistry. On the basis of these double-labelling experiments, it was shown that the optic tectum receives a GABAergic input from the caudoventral part of the medial pallium, the dorsal and ventral thalamus, the nucleus of M5, and the torus semicircularis. The afferent input to the optic tectum in the lamprey brain is similar to that described for other vertebrate species, which is of particular interest considering its position in phylogeny.

Mechanisms for selection of basic motor programs--roles for the striatum and pallidum.

The nervous system contains a toolbox of motor programs in the brainstem and spinal cord--that is, neuronal networks designed to handle the basic motor repertoire required for survival, including locomotion, posture, eye movements, breathing, chewing, swallowing and expression of emotions. The neural mechanisms responsible for selecting which motor program should be recruited at a given instant are the focus of this review. Motor programs are kept under tonic inhibition by GABAergic pallidal neurons (the output nuclei of the basal ganglia). The motor programs can be relieved from pallidal inhibition through activation of striatal neurons at the input stage of the basal ganglia. It is argued that the striatum has a prominent role in selecting which motor program should be called into action.

Spinal cats on the treadmill: changes in load pathways.

Treadmill training and clonidine, an alpha-2 noradrenergic agonist, have been shown to improve locomotion after spinal cord injury. We speculate that transmission in load pathways, which are involved in body support during stance, is specifically modified by training. This was evaluated by comparing two groups of spinal cats; one group (n = 11) was trained to walk until full-weight-bearing (3-4 weeks), and the other (shams; n = 7) was not. During an acute experiment, changes in group I pathways, monosynaptic excitation, disynaptic inhibition, and polysynaptic excitation were investigated by measuring the response amplitude in extensor motoneurons before and after clonidine injection. Monosynaptic excitation was not modified by clonidine but was decreased significantly by training. Disynaptic inhibition was significantly decreased by clonidine in both groups, but more significantly in trained cats, and significantly reduced by training after clonidine. Also, clonidine could reverse group IB inhibition into polysynaptic excitation in both groups but more frequently in trained cats. We also investigated whether fictive stepping revealed additional changes. In trained cats, the phase-dependent modulation of all three responses was similar to patterns reported previously, but in shams, modulation of monosynaptic and polysynaptic responses was not. Overall, training appears to decrease monosynaptic excitation and enhance the effects of clonidine in the reduction of disynaptic inhibition and reversal to polysynaptic excitation. Because it is believed that polysynaptic excitatory group I pathways transmit locomotor drive to extensor motoneurons, we suggest that the latter changes would facilitate the recruitment of extensor muscles for recovering weight-bearing during stepping.

Modulation of monosynaptic transmission by presynaptic inhibition during fictive locomotion in the cat.

The effect of multisensory inputs onto the presynaptic inhibitory pathways affecting IA terminals was studied during fictive locomotion in decerebrated cats. The effect was evaluated from changes in amplitude of the monosynaptic excitatory postsynaptic potential (EPSP) measured in lumbosacral motoneurones. Responses were grouped and averaged according to their timing within the step cycle divided into five bins. Presynaptic inhibition was evoked by stimulating group I afferents from the posterior biceps-semitendinosus (PBSt) muscles and one of three cutaneous nerves: superficial peroneal (SP), sural and saphenous. Statistical analysis was applied to compare (1) EPSPs conditioned by PBSt input alone and those conditioned by the combined PBSt and cutaneous inputs, and (2) each bin dividing the step cycle to disclose phase-dependent changes. Results from 19 motoneurones showed that: (1) there was a significant phase-dependent modulation in EPSP amplitude (by 25%) with the maximum usually occurring during the depolarized phase; (2) PBSt alone reduced the EPSP amplitude (by 21%) in 3.2 bins on average; (3) combined PBSt and cutaneous stimuli further modified (up or down) the EPSP amplitude in half the trials but only in one to two bins; and (4) the most efficient cutaneous nerve (SP) usually decreased the PBSt-evoked reduction in EPSP size. Minimal changes in membrane input resistance suggest that the EPSP modifications were mostly due to presynaptic inhibition. Results indicate that muscle afferents can induce an important phase-dependent presynaptic inhibition of monosynaptic transmission and that concomitant activation of cutaneous afferents can alter this inhibition but only for a restricted part of the step cycle.

Sensory integration in presynaptic inhibitory pathways during fictive locomotion in the cat.

The aim of this study is to understand how sensory inputs of different modalities are integrated into spinal cord pathways controlling presynaptic inhibition during locomotion. Primary afferent depolarization (PAD), an estimate of presynaptic inhibition, was recorded intra-axonally in group I afferents (n = 31) from seven hindlimb muscles in L(6)-S(1) segments during fictive locomotion in the decerebrate cat. PADs were evoked by stimulating alternatively low-threshold afferents from a flexor nerve, a cutaneous nerve and a combination of both. The fictive step cycle was divided in five bins and PADs were averaged in each bin and their amplitude compared. PADs evoked by muscle stimuli alone showed a significant phase-dependent modulation in 20/31 group I afferents. In 12/20 afferents, the cutaneous stimuli alone evoked a phase-dependent modulation of primary afferent hyperpolarization (PAH, n = 9) or of PADs (n = 3). Combining the two sensory modalities showed that cutaneous volleys could significantly modify the amplitude of PADs evoked by muscle stimuli in at least one part (bin) of the step cycle in 17/31 (55%) of group I afferents. The most common effect (13/17) was a decrease in the PAD amplitude by 35% on average, whereas it was increased by 17% on average in the others (4/17). Moreover, in 8/13 afferents, the PAD reduction was obtained in 4/5 bins i.e., for most of the duration of the step cycle. These effects were seen in group I afferents from all seven muscles. On the other hand, we found that different cutaneous nerves had quite different efficacy; the superficial peroneal (SP) being the most efficient (85% of trials) followed by Saphenous (60%) and caudal sural (44%) nerves. The results indicate that cutaneous interneurons may act, in part, by modulating the transmission in PAD pathways activated by group I muscle afferents. We conclude that cutaneous input, especially from the skin area on the dorsum of the paw (SP), could subtract presynaptic inhibition in some group I afferents during perturbations of stepping (e.g., hitting an obstacle) and could thus adjust the influence of proprioceptive feedback onto motoneuronal excitability.