Neural Activity Pattern in Different Brain Stem Structures during Eye-Head Movements

Rhesus monkeys were trained to track a visual target with head and eye movements in order to study central vestibular neurons under natural conditions. Single unit recordings of cells in the vestibular nuclei were obtained during active head rotations in the horizontal plane, and also during passive copies of these self-induced movements. Most cells exhibited secondary responses immediately following the primary vestibular responses to active or passive rapid head movements. They were of opposite polarity to the primary responses, and generally rate enhancements of secondary responses were of greater amplitude than rate suppressions. In addition, vestibular nuclei cells also encoded tonic neck position. The corresponding signal consisted of a variation in the basal discharge rate as a function of neck, and thus head, position. These observations prove for the first time that dynamic and static response characteristics recorded earlier from lesioned brains and under anaesthesia are, at least qualitatively, representative for the normally behaving vestibulomotor system in primates.

Transneuronal labeling in hamster brainstem following lingual injections with Herpes simplex virus-1

In the present study, the dendritic organisation of neurones in the normal human basal forebrain was analysed as a prerequisite for the evaluation of pathological changes occurring in Alzheimer's disease and related conditions (see other Arendt et al. papers in this issue). Neurones in the basal nucleus of Meynert (NbM), the nucleus of the vertical limb of the diagonal band, and the medial septal nucleus were examined after Golgi impregnation. Cells were classified according to the dendritic branching pattern and soma shape as either reticular neurones or multipolar giant neurones. The reticular type of neurones constitutes more than 90% of neurones in the BnM. Cholinergic neurones also belong to this cell type. Reticular neurones were further subdivided into four subtypes. Morphological features and arrangement of reticular basal forebrain neurones were identical to those described for "reticular formation cells" or "isodendritic" neurones. Dendritic trees of reticular neurones show a spatial orientation perpendicular to passing fibres as well as a high degree of overlap, both of which are hallmarks of "open nuclei." The qualitative classification of Golgi-impregnated basal forebrain neurones was substantiated by a computer-based three-dimensional analysis. Topologic and metric parameters of the dendritic tree were calculated for each type of neurone to characterise the degree of dendritic branching, the shape and orientation of the dendritic arborisation, the spatial extension of the dendritic tree, and soma size. The classification criteria were evaluated according to their power of discrimination between different cell types by means of a discriminant analysis. The quantitative approach applied in the present study not only provides an objective measure for the description and comparison of the structure of various types of neurones but also makes it possible to elucidate fine structural changes that might occur under pathologic conditions and that are not evident during qualitative studies alone.

The reticular formation is composed of heterogeneous cell populations with multiple functions. Among these multiple functions is the processing of sensory information in the context of behavior. The purpose of the present study was to identify and characterize neurons in the reticular formation of the rat that receive auditory input. In order to do so, we combined intracellular electrophysiology in vivo with intracellular injection of horseradish peroxidase, enabling us to correlate electrophysiology unequivocally with anatomy at the single cell level. We found that many neurons in the caudal pontine reticular nucleus (PnC), which we analyzed intracellularly, responded to acoustic stimuli and were excited at short latency (mean EPSP latency: 2.6 ms; mean spike latency: 5.2 ms). This short latency suggests a direct input from the cochlear nucleus, the first central nucleus of the auditory pathway. The morphology revealed that the acoustically driven PnC neurons have very large somata (mean diameter: 44.0 microns). They can therefore be referred to as "giant PnC neurons." Complex dendritic arbors extended from these neurons into the reticular formation and thus formed a large membrane surface for the integration of multimodal inputs. Most of the giant PnC neurons sent their axons caudally into the medial longitudinal fasciculus and can therefore be regarded as reticulospinal neurons. The results demonstrate that the giant reticulospinal PnC neurons are in a position to transmit acoustic information very quickly to spinal cord neurons and to receive converging input from other parts of the brain. They are thus good candidates for participation in the mediation and modulation of acoustically elicited behaviors, such as the short latency acoustic startle response.

The course and cerebellar termination of the axons of the cells in the central cervical nucleus (CCN) was studied in five cats after injections of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the C1-4 segments involving the CCN. In two cats a hemisection was performed ipsilateral to and above the injections in order to prevent transport of WGA-HRP from cerebellar-projecting neurons in lamina VI, just dorsal to the CCN. Labeled axons were found in the brain stem contralateral to the injections just lateral to the spinal trigeminal tract and the vestibular nuclei, and in the reticular formation. Some fibers terminated in the vestibular nuclei (especially group X), the reticular formation, and the inferior olive. The axons entered the cerebellum via the superior cerebellar peduncle. In the cerebellum mossy fiber terminals were found bilaterally in the deep vermal parts of lobules I-VIII. Only in cases without lesions were terminals found in the paramedian lobule ipsilateral to the injection, suggesting that neurons in lamina VI, but not in the CCN, project to the paramedian lobule. In the brain stem retrogradely labeled neurons (possible afferents to the CCN) were found in the vestibular (lateral, inferior and medial) nuclei, the reticular formation, and the trigeminal (spinal and mesencephalic) nuclei. The cerebellar distribution of mossy fiber terminals suggests that spinocerebellar fibers from lower parts of the spinal cord passing through the injection area do not transport WGA-HRP to their terminals.

To generate movements, the motor cortex acts on an intricate network of intermediate neurons in the brainstem and spinal cord, termed ‘premotor neurons’. Premotor neurons activate the motoneurons driving the muscles (Chakrabarti & Schwarz, 2014). Specifically, premotor networks are involved in the generation of reflexes, and form central pattern generators (CPGs) that contribute to stereotyped movement patterns. The precise anatomical organization of these premotor networks is poorly understood, because traditional tract tracing methods lack the specificity to trace tangled networks and are unable to reveal multi-synaptic networks. In this issue of EJN, Sreenivasan et al. (2015) describe evidence that extends previous work by Takatoh et al. (2013), and identify key components of the premotor networks in developing mouse pups. The authors used an elegant neuroanatomical technique called monosynaptic retrograde tracing (MRT) (Wickersham et al., 2007). MRT employs a genetically defective rabies virus that requires a glycoprotein complement in order for the virus to cross a synapse retrogradely and infect the presynaptic neuron. An essential component of this method is the use of genetic engineering to express the required glycoprotein exclusively in postsynaptic neurons, thereby allowing the virus to pass exactly one synapse and exclusively label ‘first-order’ presynaptic neurons. Applied in combination with immunohistochemistry to visualize transmitter content, MRT serves as a powerful tool with which to dissect the anatomical organization of premotor networks. The current study by Sreenivasan et al., and the former study by Takotah et al., investigated the rodent vibrissal system. Whisking movements consist of rhythmic protractions and retractions that are generated by the activation of two antagonistic muscle groups, the intrinsic and extrinsic muscles, respectively. Upon injection of the defective rabies virus into the antagonistic sets of whisker muscles, both studies revealed a fairly consistent anatomical organization of clusters of first-order ‘whisking’ premotor neurons. Among them are known CPGs in the reticular formation, reticular formation cell clusters with unknown function, cells in the spinal trigeminal nuclei, cells in the vestibular nuclei, and, interestingly, also motor cortex neurons. These results contribute greatly to our understanding of this specialized motor system in rodents. Moreover, the current work indicated a fundamental distinction between premotor networks driving extrinsic muscle groups and those driving intrinsic muscle groups. Extrinsic motoneurons are targeted predominantly by excitatory spinal trigeminal and vestibular neurons, whereas intrinsic motoneurons receive relatively more inputs from whisking CPGs. Sreenivasan et al. also combined MRT with anterograde tracing from primary sensorimotor whisker-related cortices [motor, whisker motor cortex (wM1); tactile, primary whisker somatosensory cortex (wS1)] to show that the target profiles of wM1 and wS1 complement the innervation pattern seen for the extrinsic vs. intrinsic premotor systems: wM1 preferentially controls the intrinsic muscle group via the whisking CPG, whereas wS1 controls the extrinsic muscle group via the spinal trigeminal nuclei. Furthermore, the authors could elicit distinct evoked whisker movements after optogenetic activation of the two areas: stimulation in wM1 initiated rhythmic movement, whereas in wS1 it evoked whisker retraction. Although this observation clearly points to differences in motor control between the two cortical areas, it is questionable whether these evoked movements reflect specialization in terms of movement types. First, wM1 is subdivided in several functionally distinct areas, at least in rats (Chakrabarti & Schwarz, 2014). Second, the interpretation of results from bulk activation of motor structures requires caution. Movements evoked from wS1 may merely reflect modulatory action on the whisking brainstem reflex arc (Nguyen & Kleinfeld, 2005), or alternatively its involvement in corticobulbar control of active touch (Furuta et al., 2010). Consistent with these assumptions, we used bulk stimulation to identify the rhythmic whisking subarea of the rat wM1 (Haiss & Schwarz, 2005), and found that it codes for both whisker position and whisking frequency. These effects occurred on the scale of seconds, and therefore possibly reflect indirect control of movement through various premotor networks (Gerdjikov et al., 2013). Rather than attributing distinct types of movement to specific cortical areas, it may be more informative to focus on the differential access of the sensorimotor cortex to specific premotor subsystems, such as CPGs and reflex arcs. In summary, the findings of Sreenivasan et al. (2015), together with the recent discovery of the whisking CPG (Moore et al., 2013), constitute considerable progress towards the description of the whisker-related premotor network. More work will be required to classify the premotor neuron clusters identified so far into functional subsystems. For example, some of the premotor neurons in reticular formation and spinal trigeminal nuclei may well be components of the brainstem reflex arc (Nguyen & Kleinfeld, 2005). Disentangling the entire polysynaptic premotor network will probably require a multifaceted strategy, including additional ingenious genetic trickery, tracing polysynaptic networks in adult animals, and old-fashioned monitoring of the activity of premotor neurons during whisking behavior.

The effects of ginkgolide B (BN52021) on guinea pig vestibular nucleus neurons in vitro: importance of controlling for effects of dimethylsulphoxide (DMSO) vehicles

Orienting-related eye-neck neurons of the medial ponto-bulbar reticular formation do not participate in horizontal canal-dependent vestibular reflexes of alert cats

Inhibition of central vestibular neurons from the contralateral labyrinth and its mediating pathway.

Neurons in the medullary reticular formation are involved in the control of postural and locomotor behaviors in all vertebrates. Reticulospinal neurons in this brain region provide one of the major descending projections to the spinal cord. Although neurons in the newt medullary reticular formation have been extensively studied using in vivo extracellular recordings, little is known of their intrinsic biophysical properties or of the underlying circuitry of this region. Using whole cell patch-clamp recordings in brain slices containing the rostromedial reticular formation from adult male newts, we observed spontaneous miniature outward currents (SMOCs) in ~2/3 of neurons. Although SMOCs superficially resembled inhibitory postsynaptic currents (IPSCs), they had slower risetimes and decay times than spontaneous IPSCs. SMOCs required intracellular Ca2+ release from ryanodine receptors and were also dependent on the influx of extracellular Ca2+. SMOCs were unaffected by apamin but were partially blocked by iberiotoxin and charybdotoxin, indicating that SMOCs were mediated by big-conductance Ca2+-activated K+ channels. Application of the sarco/endoplasmic Ca2+ ATPase inhibitor cyclopiazonic acid blocked the generation of SMOCs and also increased neural excitability. Neurons with SMOCs had significantly broader action potentials, slower membrane time constants, and higher input resistance than neurons without SMOCs. Thus, SMOCs may serve as a mechanism to regulate action potential threshold in a majority of neurons within the newt medullary reticular formation. NEW & NOTEWORTHY The medullary reticular formation exerts a powerful influence on sensorimotor integration and subsequent motor behavior, yet little is known about the neurons involved. In this study, we identify a transient potassium current that regulates action potential threshold in a majority of medullary reticular neurons.

Glutamatergic reticulospinal neurons in the gigantocellular reticular nucleus (GRN) of the medullary reticular formation can function as command neurons, transmitting motor commands to spinal cord circuits to instruct movement. Recent advances in our understanding of this neuron-dense region have been facilitated by the discovery of expression of the transcriptional regulator, Chx10, in excitatory reticulospinal neurons. Here, we address the capacity of local circuitry in the GRN to contribute to reticulospinal output. We define two subpopulations of Chx10-expressing neurons in this region, based on distinct electrophysiological properties and soma size (small and large), and show that these populations correspond to local interneurons and reticulospinal neurons, respectively. Using focal release of caged glutamate combined with patch clamp recordings, we demonstrated that Chx10 neurons form microcircuits in which the Chx10 local interneurons project to and facilitate the firing of Chx10 reticulospinal neurons. We discuss the implications of these microcircuits in terms of movement selection.NEW & NOTEWORTHY Reticulospinal neurons in the medullary reticular formation integrate inputs from higher regions to effectively instruct spinal motor circuits. Using photoactivation of neurons in brainstem slices, we studied connectivity of reticular formation neurons that express the transcriptional regulator, Chx10. We show that a subpopulation of these neurons functions as local interneurons that affect descending commands. The results shed light on the internal organization and microcircuit formation of reticular formation neurons.

Kynurenate in the pontine reticular formation inhibits acoustic and trigeminal nucleus-evoked startle, but not vestibular nucleus-evoked startle

Pontine reticular formation (PRF) neurons provide tonic excitatory drive to sympathetic nerves and are involved in cardiovascular control [K. Hayes and L. C. Weaver. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1567-H1575, 1992]. However, connections between the PRF and the well-known vasomotor region in the rostral ventrolateral medulla (RVLM) are unknown. In propofol (Diprivan)- anesthetized rats we investigated arterial pressure, heart rate, and renal nerve responses to microinjection of glycine (1.0 M, 60 nl) into the PRF before and after injection of the synaptic blocking agent cobalt chloride (4.0 mM, 200 nl) into the RVLM. Glycine injections into the PRF caused decreases in arterial pressure, heart rate, and discharge of renal sympathetic nerves. Synaptic blockade of the RVLM almost eliminated cardiovascular and sympathetic responses to glycine injections into the PRF and blocked somatosympathetic reflexes in the renal nerve. Cobalt injections into the RVLM had very small effects on basal renal nerve firing, arterial pressure, or heart rate. These results suggest that the neurons within the RVLM relay influences from the PRF to sympathetic preganglionic neurons. Because injections of the excitatory amino acid antagonist, kynurenate, into the RVLM also interrupted responses to blockade of the PRF and blocked somatosympathetic reflexes, glutamate is a likely neurotransmitter from the PRF to the RVLM and for somatosympathetic reflexes.

Previous physiological investigations have suggested the existence of a neural circuit that coordinates activation of motor and autonomic efferents before or at the onset of exercise. Traditionally these circuits have been postulated to involve forebrain areas. However, overlapping populations of medullary reticular formation neurons that participate in motor or autonomic control have been described previously, suggesting that individual pontomedullary reticular formation neurons may coordinate both motor and autonomic responses. We tested this hypothesis by conducting transneuronal retrograde tracing of motor and sympathetic nervous system pathways in rats using recombinant strains of pseudorabies virus (PRV). A PRV strain expressing the green fluorescent protein (PRV-152) was injected into the left gastrocnemius muscle, which was surgically sympathectomized, whereas another recombinant (PRV-BaBlu) was injected into the left adrenal gland. Immunofluorescence methods using monospecific antisera and distinct fluorophores identified neurons infected with one or both of the recombinants. Brainstem neurons coinfected with both PRV recombinants, which presumably had collateralized projections to both adrenal sympathetic preganglionic neurons and gastrocnemius motoneurons, were observed in several areas of the pontomedullary reticular formation. The largest number of such neurons was located in the rostral ventromedial medulla within the ventral gigantocellular nucleus, gigantocellular nucleus pars alpha, raphe obscurus, and raphe magnus. These neurons are candidates for relaying central command signals to the spinal cord.

The first sections of this paper survey the history and recent developments relevant to the major neurotransmitters and neuromodulators involved in REM sleep control. The last portion of this paper proposes a structural model of cellular interaction that produces the REM sleep cycle, and constitutes a further revision of the reciprocal interaction model This paper proposes seven criteria to define a causal role in REM sleep control for putative neuro-transmitters/modulators. The principal criteria are measurements during behavioral state changes of the extracellular concentrations of the putative substances, and electrophysiological recording of their neuronal source. A cautionary note is that, while pharmacological manipulations are suggestive, they alone do not provide definitive causal evidence. The extensive body of in vivo and in vitro evidence supporting cholinergic promotion of REM sleep via LDT/PPT neuronal activity is surveyed. An interesting question raised by some studies is whether cholinergic influences in rat are less puissant than in cat. At least some of the apparent lesser REM-inducing effect of carbachol in the rat may be due to incomplete control of circadian influences; almost all experiments have been run only in the daytime, inactive period, when REM sleep is more prominent, rather than in the REM-sparse nighttime inactive period. Monoaminergic inhibition of cholinergic neurons, once thought to be the most shaky proposal of the reciprocal interaction model, now enjoys considerable support from both in vivo and in vitro data. However, the observed time course of monoaminergic neurons, their "turning off" discharge activity as REM sleep is approached and entered would seem to be difficult to produce from feedback inhibition, as originally postulated by the reciprocal interaction model. New data suggest the possibility that GABAergic inhibition of Locus Coeruleus and Dorsal Raphe monoaminergic neurons may account for the "REM-off" neurons turning off. However, the source(s) of GABAergic influences suggested by anatomical studies has yet to be definitively identified by electrophysiological recordings of GABAergic neurons that show the requisite inverse time course of activity relative to monoaminergic neurons. New and still preliminary microdialysis data suggest that reticular formation neurons, the effector neurons for REM sleep phenomena, might be disinhibited during REM sleep by decreased GABAergic influence, perhaps stemming from REM-on cholinergic neuronal inhibition of reticular formation GABAergic neurons. Whether the postulated cholinergic inhibition of GABAergic neurons is present is testable with in vitro recordings and double labeling. Taking into account the observed data on neuro-modulators/transmitters, a structural model incorporating interaction of REM-on and REM-off neurons and GABAergic influences is proposed. Finally, with respect to orexin and REM sleep, it is hypothesized that orexinergic activity may be a principal factor controlling REM sleep's absence from the active period in strongly circadian animals such as rat and man.