Medial Pontine Research Articles

Quantitative morphology of physiologically identified and intracellularly labeled neurons from the guinea-pig laterodorsal tegmental nucleus in vitro

Mesopontine cholinergic neurons have been implicated in the initiation and maintenance of rapid eye movement sleep via their efferent connections to the thalamus and the medial pontine reticular formation. As a first step toward understanding how these modulatory neurons integrate synaptic input, we have investigated the dendritic architecture of laterodorsal tegmental nucleus neurons. The principal cells of the guinea-pig laterodorsal tegmental nucleus were identified electrophysiologically in a brain slice preparation, then were intracellularly injected with biocytin and reconstructed using a computer-aided tracing system. The somata were large (27 ± 3 μm; n = 11) and gave rise to an average of 4.8 primary dendrites which, in most cases, emerged from the soma in a pattern that was radially symmetric in the plane of the slice. Primary dendrites had an average of 3.7 endings. A single axon arose from either the soma or a proximal dendrite and exited the nucleus with a medial and/or lateral trajectory. Some axons also gave rise to a local terminal plexus composed of fine fibers bearing numerous punctate swellings that ramified profusely within the neuron's dendritic field. Total dendritic area averaged about 10 5 μm 2, and therefore the average contribution of the soma to the total surface area (20%) was significantly larger than the values reported for many other cell types. Dendritic diameters were non-uniform in three respects. Some processes were sparsely spiny. Most processes were varicose, with the degree of varicosity increasing substantially in secondary and tertiary dendritic segments. There was also a large degree of taper in dendritic processes; those processes with a non-negative taper had an average diameter decrease of 40 ± 25%. Dendritic processes deviated from the criteria necessary for a Rall equivalent cylinder approximation due to non-uniformity in morphotonic path length, failure to conform to the Rall 3/2 branching rule and non-uniformity of dendritic diameter. An analysis was done to assess the impact of dendritic varicosities on the extraction of cable parameters for these cells. Voltage traces were simulated by solving the cable equation for a varicose dendrite and then membrane parameters were recovered using an equivalent cylinder model. Errors in the extracted values of specific membrane conductance and specific membrane capacitance were quite small (≤5%), while larger errors were seen for electrotonic length (≤21%) and intracellular resistivity (≤50%). These data indicate that the principal cells of the laterodorsal tegmental nucleus, while possessing a relatively simple dendritic structure in terms of number and branchiness of dendrites, display a heterogeneity of dendritic process types. Processes range from smooth to markedly varicose, and can be aspiny or sparsely spiny. The possibility that the dendritic varicosities function as sites of either electrical or chemical compartmentalization is discussed. The degree of error resulting from a Rall equivalent cylinder approximation in light of these varicosities indicated that a generalized cable model approach may prove more effective in estimating their cable parameters.

Nociceptive neurones in rat superior colliculus. II. Effects of lesions to the contralateral descending output pathway on nocifensive behaviours.

A wealth of evidence implicates the crossed descending projection from the superior colliculus (SC) in orientation and approach behaviours directed towards novel, non-noxious stimuli. In our preceding paper, we identified a population of nociceptive neurones in the rat SC that have axons that project to the contralateral brainstem via this output pathway. The purpose of the present study was, therefore, to evaluate the prediction that the crossed descending projection of the SC is also involved in the control of orientation and approach movements of the head and mouth made during the localisation of persistent noxious stimuli. An independent-groups design was used to test the effects of interrupting the contralateral descending projection from the SC on the behavioural reactions elicited by noxious mechanical stimuli presented to the tail and hindpaws. In different groups of animals, a microwire knife was used to cut the contralateral descending fibres at two different locations: (1) a sagittal cut at the level of the dorsal tegmental decussation; (2) a bilateral coronal cut of the predorsal bundle at the level of the medial pontine reticular formation. Retrograde anatomical tracing techniques were then used to evaluate the effectiveness of the cuts and to assess possible involvement of non-collicular fibre systems in both lesioned and control animals. Additional behavioural procedures were performed to test for general neurological status and responsiveness of animals to non-noxious stimuli. Anatomical tracing data indicated that the largest population of neurones with fibres severed by both cuts were the cells-of-origin of the contralateral descending projection in the intermediate white layer of the SC. Behavioural results showed that significantly more animals in both lesion groups failed to locate and bite a mechanical clip placed on the tail. Instead of switching to motor behaviours to localise and remove noxious stimuli, they persisted with defensive reactions, which included freezing, vocalisation or forward and backward escape. In contrast, when the clip was placed on the hindpaws, it was successfully localised by most lesioned and control animals; however, lesioned animals had reliably longer latencies and spent less time in close contact with the clip. Consistent with the established role of the contralateral descending projection in non-noxious orientation, lesioned animals also showed orienting deficits to a range of non-noxious sensory stimuli. These data suggest that, under certain behavioural circumstances, nociceptive information from the SC is integral to the elaboration of orienting and approach movements of the head and mouth elicited by persistent noxious stimuli.

Pontine cholinergic neurons show Fos-like immunoreactivity associated with cholinergically induced REM sleep.

Recently, we showed c-fos expression in pontine nuclei in association with cholinergically induced REM sleep (REMc). Pontine cholinergic mechanisms have been implicated in the orchestration of the phasic and tonic events underlying REM sleep. Therefore, in the present study, we examined whether pontine cholinergic neurons demonstrate Fos-like immunoreactivity (Fos-LI) following cholinergically induced, sustained rapid-eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 2) or carbachol (n = 3; 2.0 micrograms/0.25 microliter) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations ranging from 27 to 40.1 min. Animals were killed immediately after the end of REMc. Compared to vehicle treated animals (0.9% saline), the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions implicated in REM sleep generation. More importantly, 11.2% (SEM +/- 0.83) of cholinergic neurons in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei were determined to be also Fos-LI positive. In the vehicle treated animals very few Fos-LI cells were found and none of these were found to be cholinergic. These findings indicate that during REMc a transcriptional cascade involving c-fos occurs in a subpopulation of pontine cholinergic neurons.

Pontine Cholinergic Mechanisms Modulate the Cortical Electroencephalographic Spindles of Halothane Anesthesia

Halothane anesthesia causes spindles in the electroencephalogram (EEG), but the cellular and molecular mechanisms generating these spindles remain incompletely understood. The current study tested the hypothesis that halothane-induced EEG spindles are regulated, in part, by pontine cholinergic mechanisms. Adult male cats were implanted with EEG electrodes and trained to sleep in the laboratory. Approximately 1 month after surgery, animals were anesthetized with halothane and a microdialysis probe was stereotaxically placed in the medial pontine reticular formation (mPRF). Simultaneous measurements were made of mPRF acetylcholine release and number of cortical EEG spindles during halothane anesthesia and subsequent wakefulness. In additional experiments, carbachol (88 mM) ws microinjected in the the mPRF before halothane anesthesia to determine whether enhanced cholinergic neurotransmission in the MPRF would block the ability of halothane to induce cortical EEG spindles. During wakefulness, mPRF acetylcholine release averaged 0.43 pmol/10 min of dialysis. Halothane at 1 minimum alveolar concentration decreased acetylcholine release (0.25 pmol/10 min) while significantly increasing the number of cortical EEG spindles. Cortical EEG spindles caused by 1 minimum alveolar concentration halothane were not significantly different in waveform, amplitude, or number from the EEG spindles of nonrapid eye movement sleep. Microinjection of carbachol into the mPRF before halothane administration caused a significant reduction in number of halothane-induced EEG spindles. Laterodorsal and pedunculopontine tegmental neurons, which provide cholinergic input to the mPRF, play a causal role in generating the EEG spindles of halothane anesthesia.

Evidence for GAP-43 within descending spinal axons in the North American opossum, Didelphis virginiana.

We have shown previously that GAP-43, a growth associated protein characteristically present in growing and regenerating axons, is relatively abundant in the spinal cord of adult opossums. In the present study, we combined the orthograde transport of the fluorescent marker Fluoro-Ruby with immunofluorescence for GAP-43 to determine if any of it is present within descending spinal axons. When Fluoro-Ruby was injected into the red nucleus and midbrain tegmentum, the medial pontine or medullary reticular formation, the medullary raphe or the lateral vestibular nucleus, axons were labeled in the expected areas of the spinal cord, but in most cases none showed evidence for GAP-43. In two of the four cases with rubral injections, however, a few labeled axons within the rubrospinal tract showed GAP-43 immunofluorescence, and in one case with an injection of the gigantocellular reticular nucleus and adjacent raphe, labeled axons within lamina IX immunostained for the protein. Since serotoninergic neurons are present within the gigantocellular reticular nucleus and adjacent raphe, and axons of the same phenotype are abundant within lamina IX, we asked whether serotoninergic axons contain GAP-43. When sections of the spinal cord were immunostained for both serotonin and GAP-43, many axons within lamina IX showed evidence for both substances. Such axons appeared to contact presumptive motoneurons. In cases with Fluoro-Ruby injections of the forelimb motor cortex, labeled axons were present within the pyramidal tract, and some of them showed evidence for GAP-43.

Efferents from the optic tectum to the brain stem in the Japanese quail (Coturnix japonica). Anterogradely biocytin method.

Efferents from the optic tectum to the brain stem in the Japanese quail (Coturnix japonica) were studied with the anterogradely biocytin method. After injection of biocytin into the ipsilateral optic tectum, labeled terminals were seen in the rotund nucleus (Rt), neuropil part of the ventral lateral geniculate nucleus (GLnv), principal part of the dorsal lateral geniculate nucleus, lateral part of the dorsolateral thalamic nucleus, triangular nucleus (T), superficial parvocellular nucleus (SPC), pretectal nucleus, pretectal area (PA), subpretectal nucleus, central gray matter (GC), isthimo optic nucleus (ION), magnocellular and parvocellular parts of the isthimo nuclei (Imc and Ipc), semilunar nucleus (SLu), lateral and medial pontine nuclei and reticular formation (FRM) of the medulla, ipsilaterally. Labeled fibers were seen in the septomesencephalic tract nucleus, FRM, interstitio-paraetecto-subpraetectal nucleus, and the dorsal and ventral tectoreticular tracts (TRd and TRv). In the contralateral brain stem, labeled terminals were seen in the Rt, T. FRM, PA and paramedian nucleus. The contralateral terminals were remarkably fewer than those of the ipsilateral side. The present findings of the labeled terminals of the SPC and the GC at the level of the mesencephalic nucleus of the trigeminal nerve (MnT), and the topographic projection from optic tectum to the Rt in the thalamus, were original observations in the avian. The labeled terminals in the GLnv, Ipc, Imc and ION showed topographical projections from the optic tectum. Pathways to the contralateral brain stem were via the commissure posterior, ventral supraoptic decussation, and the predorsal bundle. The present results suggest that tectofugal impulses in the quail relate to various functions with special relation to the function of the GC at the level of the MnT as well as a visual function.

Orthograde axonal transport studies of projections from the zona incerta and pretectum to the basilar pontine nuclei in the rat.

This study employed orthogradely transported axonal tracers to demonstrate, in the rat, projections that reach the basilar pontine nuclei from the zona incerta or pretectal nuclei. Except for the most rostral levels, all subdivisions of the zona incerta give rise to substantive basilar pontine projections. Although some topographic differences exist among the temination patterns of various subdivisions, no clear somatotopically organized scheme is apparent. Most incertopontine axons descend to the basilar pons in association with fibers of the medial lemniscus or crus cerebri and reach ipsilateral ventral and medial pontine gray regions. A sparse number of terminals are evident in the contralateral medial pontine gray. The anterior pretectal axons also descend with the medial lemniscus and crus cerebri to enter exclusively the ipsilateral basilar pons where they terminate most densely in ventral and medial regions. Dual orthograde labeling experiments indicate that some pretectal terminal fields in the pontine gray are shared with incertopontine projections and with afferents from the dorsal column nuclei. This potential convergence of basilar pontine afferent projections is significant in light of 1) the known somatosensory input to the zona incerta and pretectum and 2), the fractured somatotopy of peripheral cutaneous inputs that arrive in the cerebellar cortex via mossy fibers. The present studies also employed electron microscopy to identify synaptic boutons formed by incerto- and pretectopontine axons, and they proved to be remarkably similar. Each is a medium to small-sized bouton that contains spheroidal synaptic vesicles and forms asymmetric membrane specializations. Most incerto- and pretectopontine boutons participate in glomerular synaptic complexes that include a single, centrally located bouton contacted on its perimeter by several types of dendritic profiles including shafts and spine-like appendages. A relatively small number of labeled boutons of either type contacts single, isolated dendritic elements in the neuropil. Taken together, these findings suggest that some basilar pontine neurons might receive convergent inputs from the zona incerta and pretectum as well as other somatosensory-related systems such as the dorsal column nuclei and sensorimotor cortex.

Pertussis toxin-sensitive G proteins mediate carbachol-induced REM sleep and respiratory depression.

Microinjecting cholinomimetics into the medial pontine reticular formation (mPRF) of conscious cats causes a rapid eye movement (REM) sleep-like state and state-dependent respiratory depression. Muscarinic receptors within the mPRF have been shown to mediate this state-dependent respiratory depression, but the specific signal transduction mechanisms remain poorly understood. This study tested the hypothesis that the cholinergically induced REM sleep-like state and state-dependent respiratory depression are mediated by guanine nucleotide binding proteins (G proteins). Cholera toxin, pertussis toxin, 5'-guanylylimidodiphosphate, and forskolin were microinjected alone and in combination with carbachol into the mPRF of intact unanesthetized cats. All of the G protein-altering compounds significantly reduced the ability of carbachol to produce the REM sleep-like state. Pertussis toxin caused the greatest decrease in the percent of time spent in the carbachol-evoked REM sleep-like state, showing for the first time mediation by a pertussis toxin-sensitive (Gi- or G(o)-like) G protein. Cholera toxin blocked the carbachol-induced respiratory depression, indicating mediation by a Gs-like G protein. Forskolin significantly decreased carbachol-evoked REM sleep. These data provide the first demonstration that adenylyl cyclase within the mPRF contributes to the carbachol induction of REM sleep and respiratory depression.

Stimulus effects of the medial pontine reticular formation and the mesencephalic locomotor region upon medullary reticulospinal neurons in acute decerebrate cats

In acute decerebrate cats, medial pontine reticular formation (mPRF) and the mesencephalic locomotor region (MLR) were stimulated and their stimulus effects upon 250 medullary reticulospinal neurons (RSNs) were studied. One hundred and twenty-six RSNs were mono- and disynaptically activated. From the response patterns of the RSNs, they were divided into the mPRF-activated RSNs ( n = 67) and the MLR-activated RSNs ( n = 59). The former group of RSNs was located in the nucleus reticularis gigantocellularis (NRGc), while the latter group of RSNs was distributed in both the NRGc and the nucleus reticularis magnocellularis (NRMc). The activity of MLR-excited 12 RSNs was suppressed with the preceding mPRF stimulation. These RSNs were mainly located in the NRMc. Most mPRF-excited RSNs increased their discharge rates during mPRF-evoked suppression of postural muscle tone, and most MLR-excited RSNs increased their discharge rates during MLR-evoked locomotion. With mPRF stimulation, MLR-evoked locomotion was suppressed with cessation of MLR-excited RSNs activity. These results suggest that mPRF stimulation suppresses the activity of the locomotor rhythm generating system at the levels of not only the spinal cord but also the medullary output cells.

Nitric oxide synthase inhibition decreases pontine acetylcholine release.

This study tested the hypothesis that inhibition of nitric oxide synthase (NOS) in the medial pontine reticular formation (mPRF) would cause decreased acetylcholine (ACh) release. Microdialysis of cat mPRF permitted measurement of ACh during states of wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. ACh release during microdialysis with Ringers (control) was compared to ACh release during microdialysis with 10 mM NG-nitro-L-arginine (NLA). The NOS inhibitor NLA caused a significant reduction in ACh released from the mPRF during wakefulness, NREM sleep, and REM sleep. This reduction in mPRF ACh release elicited by NLA suggests that nitric oxide (NO) contributes to cholinergic neurotransmission in the pontine reticular formation.

Time course of Fos-like immunoreactivity associated with cholinergically induced REM sleep

Now that the pharmacology and neuronal connectivity underlying REM sleep is beginning to be understood, it is important to begin investigations that elucidate the transcriptional response related to the REM sleep process. The present study focuses on determining the temporal development of Fos-like immunoreactivity (Fos-LI) in the dorsolateral pons following cholinergically induced, sustained rapid eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 3) or carbachol (0.2-4.0 micrograms/0.25 microliters) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations from 0 min to 120 min. Animals were killed either immediately or at various intervals after the end of REMc. Compared to vehicle- and carbachol-treated animals without REMc, the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions that have been implicated in REM sleep generation. Regions with REMc-associated Fos-LI increases included the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei; the locus coeruleus; the dorsal raphe; and the medial pontine reticular formation. More Fos-LI cells were found with longer REMc bouts than with shorter-duration REMc bouts. However, with 2 hr long REMc bouts the number of Fos-LI cells returned to control levels, suggesting that the c-fos transcriptional cascade is turned off once a threshold of REMc has been reached. These findings indicate that pontine neuronal populations implicated in REM sleep express more c-fos in the course of REMc, and that the extent of expression is related to the duration of the state.

Altered pattern of immunohistochemical staining for glial fibrillary acidic protein (GFAP) in the forebrain and cerebellum of the mutant spastic rat

The spastic rat is a neurological mutant of the Han-Wistar strain with prominent spasticity, tremor, and ataxia. Neurodegeneration is found in the CA3 sector of the hippocampus and in Purkinje cells of the cerebellum. We examined the forebrain and cerebellum of spastic rats for glial reactions by using immunolabelling for the astrocytic marker, glial fibrillary acidic protein (GFAP). First, a map of the GFAP-distribution was made representing a systematic series of frontal sections in controls. Reactive astrocytes with increased GFAP should occur in the areas with established neuronal degeneration, but they could also demarcate further regions with pathology in this rat strain. Since the baseline levels of GFAP-immunoreactivity differ between brain regions, control rats and clinically normal littermates served as controls to judge relative increases in major structures. In the CA3 sector and hilus of the dorsal hippocampus, a massive gliosis was detected. In the cerebellum, a patchy increase of GFAP labelling in Bergmann glia was found. Further increases of GFAP-labelling in reactive astrocytes occurred in fiber tracts, the ventral thalamic nuclei, medial geniculate nuclei, pontine region and optic layer of the superior colliculus. Inconsistent changes were noted in cortex and pallidum. No defects of glial labelling or malformations in glial architectonics were found. The reactive changes of astroglial cells in hippocampus and cerebellum are in proportion to the neuronal degeneration. The glial reactions in the other brain regions possibly reflect a reaction to fiber degeneration and incipient neuronal degeneration or functional alterations of glial cells in response to neuronal dysfunction.

Opioid inhibition of rapid eye movement sleep by a specific mu receptor agonist

Patients receiving opioids report feeling sleepy, but opioids actually inhibit the rapid eye movement phase of sleep (REM). Inhibition of REM sleep is followed by a rebound increase in REM sleep associated with cardiopulmonary complications. The medial pontine reticular formation (mPRF) is a brain region from which morphine can inhibit REM sleep. The present study tested the hypothesis that specific subtypes of opioid receptors within the mPRF mediate inhibition of REM sleep. Synthetic opioid agonists selective for mu, delta and kappa subtypes were microinjected into the mPRF of four awake cats and polygraphic recordings of sleep and breathing were obtained. An enkephalinase inhibitor was microinjected into the mPRF to assess the contribution of endogenous opioids to the control of sleep and breathing. Only the mu agonist significantly inhibited REM sleep, and no opioid depressed breathing. These results demonstrate that opioid-induced REM sleep inhibition is mediated by mu receptor subtypes in the mPRF.

Respiratory nuclei share synaptic connectivity with pontine reticular regions regulating REM sleep.

Injection of cholinomimetics into the medial pontine reticular formation (mPRF) of intact, unanesthetized cat causes a rapid eye movement (REM) sleep-like state and respiratory depression. The mPRF contains no concentrations of respiratory neurons, and this study examined the hypothesis that respiratory depression evoked from the mPRF is synaptically mediated. The mPRF of conscious cats was injected with bethanechol to define an mPRF zone causing state-dependent respiratory depression. Bethanechol caused a 361% increase in the REM sleep-like state and a 37% decrease in minute ventilation. Additional cats were injected with the retrograde fluorescent tracers True Blue and either Fluoro-Gold or Diamidino Yellow aimed for the cholinoceptive mPRF or for the pontine respiratory group (PRG). After mPRF dye injection, 1) labeling was observed in the PRG, dorsal respiratory group (DRG), and ventral respiratory group (VRG); and 2) double-labeled cells were observed in the VRG and PRG. Dye injections into the PRG produced contralateral and ipsilateral fluorescent labeling of the mPRF, DRG, and VRG. Thus cholinoceptive regions of the mPRF involved in REM sleep generation have reciprocal monosynaptic connections with the PRG and receive monosynaptic projections from the DRG and VRG.

Unrestrained walking in cats with medial pontine reticular lesions

The early postoperative effects of lesions, aimed to destroy the caudal pole of the nucleus reticularis pontis oralis (NRPO) and the rostral pole of the nucleus reticularis pontis caudalis (NRPC), were tested in freely moving cats, walking at moderate speed (0.4–1.0 m/s). In cats in which these structures were partly or completely destroyed, the main effect of lesions was an impairment of fore-hindlimb coordination, as shown by a change in the relationships between the lateral and diagonal time shift durations and the step cycle duration. In the second week after the surgery the values of the slopes of linear regressions relating these variables were markedly changed as compared to the preoperative data. The results suggest that the NRPO and NRPC are involved in maintaining the proper forehindlimb coordination during unrestrained locomotion in cats.

Multi-segmental and generalized suppression of postural muscle tone evoked by stimulating the dorsal tegmental field and the medial pontine reticular formation in acute decerebrate cats

In acute decerebrate cat, stimulation of the dorsal tegmental field (DTF) of the caudal pons along its midline and the medial pontine reticular formation (mPRF) evokes generalized suppression of postural muscle tone. In this study, the effects of stimulation of the DTF area were compared with those evoked by stimulating the mPRF unilaterally or bilaterally. By stimulating the mPRF bilaterally, simultaneous suppression of tonic activities in the neck, lumbar back, forelimb and hindlimb muscles, bilaterally, was evoked as that evoked by stimulating the DTF area alone.

The mechanism of noradrenergic alpha 1 excitatory modulation of pontine reticular formation neurons

The alpha 1 adrenergic receptor occurs in all major divisions of the CNS and is thought to play a role in all behaviors influenced by norepinephrine (NE). In the medial pontine reticular formation (mPRF), the proposed site of adrenergic enhancement of startle responses (Davis, 1984), alpha 1 agonists excite most neurons (Gerber et al., 1990). We here report that alpha 1 excitation results from a reduction of a voltage- and calcium-dependent potassium current, not previously recognized as ligand-modulated. The calcium sensitivity is suggested by its antagonism with Mg2+, Cd2+, Ba2+, low concentrations of tetraethylammonium, and charybdotoxin. The voltage sensitivity of this conductance falls within the membrane potential range critical to action potential generation. Based on this voltage sensitivity, the change in repetitive firing characteristics may be predicted according to a mathematical model of the mPRF neuronal electrophysiology. The predicted response to a 50% decrease in the phenylephrine (PE)-sensitive conductance is similar to the observed responses, with respect to both the current response under voltage-clamp conditions and alterations of the AHP and frequency/current curve. In contrast, modeling a reduction of a voltage-insensitive leak current predicts none of these changes. Thus, the noradrenergic reduction of this current depolarizes the membrane, increases the likelihood of an initial response to depolarizing input, and increases firing rate during sustained depolarization in a manner consistent with an NE role as an excitatory neuromodulator of the mPRF.

Brainstem network controlling descending drive to phrenic motoneurons in rat.

Contraction of the diaphragm is controlled by phrenic motoneurons that receive input from sources that are not fully established. Bulbospinal (second-order) neurons projecting to phrenic motoneurons and propriobulbar (third-order) neurons projecting to these bulbspinal neurons were investigated in rat by transsynaptic transport of the neuroinvasive pseudorabies virus. Bulbospinal neurons were located predominantly in the medullary lateral tegmental field in two functionally described regions, the ventral respiratory group and Bötzinger complex. An intervening region, the pre-Bötzinger complex, contained essentially no phrenic premotoneurons. Bulbospinal neurons were also located in ventral, interstitial, and ventrolateral subnuclei of the solitary tract, and gigantocellular, Kölliker-Fuse, parabrachial, and medullary raphe nuclei. A monosynaptic pathway to phrenic motoneurons from the nucleus of the solitary tract was confirmed; monosynaptic pathways from upper cervical spinal cord, spinal trigeminal nucleus, medical and lateral vestibular nuclei, and medial pontine tegmentum were not verified. Locations of third-order neurons were consistent with described projections to the ventral respiratory group, from contralateral ventral respiratory group, Bötzinger complex, A5 noradrenergic cell group, and the following nuclei; solitary, raphe, Kölliker-Fuse, parabrachial, retrotrapezoid, and paragigantocellular. Novel findings included a projection from locus coeruleus to respiratory premotoneurons and the lack of previously described pathways from area postrema and spinal trigeminal nucleus. These second- and third-order neurons from the output network for diphragm motor control which includes numerous behaviors (e.g., respiration, phonation, defecation). Of the premotoneurons, the rostral ventral respiratory group is the primary population controlling phrenic motoneurons.

Extracellular levels of serotonin in the medial pontine reticular formation in acute decerebrate cats with a microdialysis technique

Extracellular levels of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were measured in the medial pontine reticular formation of acute decerebrate cats. The mean basal levels of 5-HT and 5-HIAA were 26 fmol 20 μ l and 15 pmol 20 μ l . Perfusion of the dialysis probe with high K + and Ca 2+-free Ringer solution for 60 min resulted in 4.8–8.5 × increase and 25–48% decrease in the extracellular levels of 5-HT, respectively, in comparison to the basal 5-HT levels. Perfusion with TTX-added Ringer solution for 60 min resulted in a consistent decrease in the extracellular levels of 5-HT.

Distribution of serotonin cells projecting to the pontomedullary reticular formation in the cat

Using immunohistochemistry and retrograde transport techniques, this study demonstrates that serotonin (5-HT) cells in the dorsal tegmental gray of the pons at rostrocaudal from P1 to P6 levels are sources of bilateral serotonergic projections to the gigantocellular tegmental field of the medial pontine and medullary reticular formation in the cat. The cells observed were a relatively homogeneous population of small (30 μm) to medium-sized (40 μm) cells, oval, fusiform or spindle-shaped. Rostrally and caudally located 5-HT cells in the dorsal tegmental gray tended to project to the pontine and medullary reticular formation respectively. The dendrites of these cells spread in a plane perpendicular to the long axis of the brainstem. Thin 5-HT fibers with a series of fine varicosities (0.5–1.0 μm in diameter) surrounded reticular neurons, and some of these varicosities closely apposed the proximal dendrites and somata of the reticular neurons. Both large (50 μm) and small (20–30 μm) reticular neurons were uniformly innervated by 5-HT fibers. As revealed in the present study, the serotonergic system, together with the monoaminergic and cholinergic systems, seems to be involved in behavioral state regulation.