Orexin/hypocretin receptor signalling: a functional perspective.

Multiple homeostatic systems are regulated by orexin (hypocretin) peptides and their two known GPCRs. Activation of orexin receptors promotes waking and is essential for expression of normal sleep and waking behaviour, with the sleep disorder narcolepsy resulting from the absence of orexin signalling. Orexin receptors also influence systems regulating appetite/metabolism, stress and reward, and are found in several peripheral tissues. Nevertheless, much remains unknown about the signalling pathways and targets engaged by native receptors. In this review, we integrate knowledge about the orexin receptor signalling capabilities obtained from studies in expression systems and various native cell types (as presented in Kukkonen and Leonard, this issue of British Journal of Pharmacology) with knowledge of orexin signalling in different tissues. The tissues reviewed include the CNS, the gastrointestinal tract, the pituitary gland, pancreas, adrenal gland, adipose tissue and the male reproductive system. We also summarize the findings in different native and recombinant cell lines, especially focusing on the different cascades in CHO cells, which is the most investigated cell line. This reveals that while a substantial gap exists between what is known about orexin receptor signalling and effectors in recombinant systems and native systems, mounting evidence suggests that orexin receptor signalling is more diverse than originally thought. Moreover, rather than being restricted to orexin receptor 'overexpressing' cells, this signalling diversity may be utilized by native receptors in a site-specific manner.

Orexin/hypocretin receptor signalling cascades.

Orexin (hypocretin) peptides and their two known G-protein-coupled receptors play essential roles in sleep-wake control and powerfully influence other systems regulating appetite/metabolism, stress and reward. Consequently, drugs that influence signalling by these receptors may provide novel therapeutic opportunities for treating sleep disorders, obesity and addiction. It is therefore critical to understand how these receptors operate, the nature of the signalling cascades they engage and their physiological targets. In this review, we evaluate what is currently known about orexin receptor signalling cascades, while a sister review (Leonard & Kukkonen, this issue) focuses on tissue-specific responses. The evidence suggests that orexin receptor signalling is multifaceted and is substantially more diverse than originally thought. Indeed, orexin receptors are able to couple to members of at least three G-protein families and possibly other proteins, through which they regulate non-selective cation channels, phospholipases, adenylyl cyclase, and protein and lipid kinases. In the central nervous system, orexin receptors produce neuroexcitation by postsynaptic depolarization via activation of non-selective cation channels, inhibition of K⁺ channels and activation of Na⁺/Ca²âº exchange, but they also can stimulate the release of neurotransmitters by presynaptic actions and modulate synaptic plasticity. Ca²âº signalling is also prominently influenced by these receptors, both via the classical phospholipase C-Ca²âº release pathway and via Ca²âº influx, mediated by several pathways. Upon longer-lasting stimulation, plastic effects are observed in some cell types, while others, especially cancer cells, are stimulated to die. Thus, orexin receptor signals appear highly tunable, depending on the milieu in which they are operating.

Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons.

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild-type (WT) mice. This was region-specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.

Dual orexin actions on dorsal raphe and laterodorsal tegmentum neurons: noisy cation current activation and selective enhancement of Ca2+ transients mediated by L-type calcium channels.

The hypocretin/orexins (Hcrt/Orxs) are hypothalamic neuropeptides that regulate stress, addiction, feeding, and arousal behaviors. They depolarize many types of central neurons and can increase [Ca2+]i in some, including those of the dorsal raphe (DR) and laterodorsal tegmental (LDT) nuclei-two structures likely to contribute to the behavioral actions of Hcrt/Orx. In this study, we used simultaneous whole cell and Ca2+-imaging methods in mouse brain slices to compare the Hcrt/Orx-activated current in DR and LDT neurons and to determine whether it contributes to the Ca2+ influx evoked by Hcrt/Orx. We found Hcrt/Orx activates a similar noisy cation current that reversed near 0 mV in both cell types. Contrary to our expectation, this current did not contribute to the somatic Ca2+ influx evoked by Hcrt/Orx. In contrast, Hcrt/Orx enhanced the Ca2+ transients produced by voltage steps (-60 to -30 mV) by approximately 30% even in neurons lacking an inward current. This effect was abolished by nifedipine, augmented by Bay-K and abolished by bisindolylmaleimide I. Thus Hcrt/Orx has two independent actions: activation of noisy cation channels that generate depolarization and activation of a protein kinase C (PKC)-dependent enhancement of Ca2+ transients mediated by L-type Ca2+ channels. Immunocytochemistry verified that both these actions occurred in serotonergic and cholinergic neurons, indicating that Hcrt/Orx can function as a neuromodulator in these key neurons of the reticular activating system. Because regulation of Ca2+ transients mediated by L-channels is often linked to the control of transcriptional signaling, our findings imply that Hcrt/Orxs may also function in the regulation of long-term homeostatic or trophic processes.

Activity-dependent nitric oxide concentration dynamics in the laterodorsal tegmental nucleus in vitro.

The behavioral-state related firing of mesopontine cholinergic neurons of the laterodorsal tegmental nucleus appears pivotal for generating both arousal and rapid-eye-movement sleep. Since these neurons express high levels of nitric oxide synthase, we investigated whether their firing increases local extracellular nitric oxide levels. We measured nitric oxide in the laterodorsal tegmental nucleus with a selective electrochemical microprobe (35 microm diam) in brain slices. Local electrical stimulation at 10 or 100 Hz produced electrochemical responses that were attributable to nitric oxide. Stimulus trains (100 Hz; 1 s) produced biphasic increases in nitric oxide that reached a mean peak concentration of 33 +/- 2 (SE) nM at 4.8 +/- 0.4 s after train onset and decayed to a plateau concentration of 8 +/- 1 nM that lasted an average of 157 +/- 23.4 s (n = 14). These responses were inhibited by N(G)-nitro-L-arginine-methyl-ester (1 mM; 92% reduction of peak; n = 3) and depended on extracellular Ca(2+). Chemically reduced hemoglobin attenuated both the electrically evoked responses and those produced by authentic nitric oxide. Application of the precursor, L-arginine (5 mM) augmented the duration of the electrically evoked response, while tetrodotoxin (1 microM) abolished it. Analysis of the stimulus-evoked field potentials indicated that electrically evoked nitric oxide production resulted from a direct, rather than synaptic, activation of laterodorsal tegmental neurons because neither nitric oxide production nor the field potentials were blocked by ionotropic glutamate receptor inhibitors. Nevertheless, application of N-methyl-D-aspartate also increased local nitric oxide concentration by 39 +/- 14 nM (n = 8). Collectively, these data demonstrate that laterodorsal tegmental neuron activity elevates extracellular nitric oxide concentration probably via somatodendritic nitric oxide production. These data support the hypothesis that nitric oxide can function as a local paracrine signal during the states of arousal and rapid-eye-movement sleep when the firing of mesopontine cholinergic neurons are highest.

Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins.

Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

Serotonergic inhibition of action potential evoked calcium transients in NOS-containing mesopontine cholinergic neurons.

Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca(2+)](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca(2+)](i) was monitored optically from neurons filled with Ca(2+) indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca(2+)](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca(2+)](i) that were related to firing rate. TTX (1 microM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca(2+) spiking on depolarization, restored the change in [Ca(2+)](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca(2+)](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca(2+)](i), suppressed both current- and NMDA-evoked increases in [Ca(2+)](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca(2+) transients by approximately 40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca(2+) currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca(2+)](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca(2+)](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca(2+) signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca(2+) signals may function to trigger Ca(2+)-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.

Function of specific K(+) channels in sustained high-frequency firing of fast-spiking neocortical interneurons.

Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (

Recovery of cable properties through active and passive modeling of subthreshold membrane responses from laterodorsal tegmental neurons.

The laterodorsal tegmental nucleus (LDT) is located in the dorsolateral pontine reticular formation. Cholinergic neurons in the LDT and the adjacent pedunculopontine tegmental nucleus (PPT) are hypothesized to play a critical role in the generation of the electroencephalographic-desynchronized states of wakefulness and rapid eye movement sleep. A quantitative analysis of the cable properties of these cells was undertaken to provide a more detailed understanding of their integrative behavior. The data used in this analysis were the morphologies of intracellularly labeled guinea pig LDT neurons and the voltage responses of these cells to somatic current injection. Initial attempts to model the membrane behavior near resting potential and in the presence of tetrodotoxin (TTX, 1 microM) as purely passive produced fits that did not capture many features of the experimental data. Moreover, the recovered values of membrane conductance or intracellular resistivity were often very far from those reported for other neurons, suggesting that a passive description of cell behavior near rest was not adequate. An active membrane model that included a subthreshold A-type K+ current and/or a hyperpolarization-activated cation current (H-current) then was used to model cell behavior. The voltage traces calculated using this model were better able to reproduce the experimental data, and the cable parameters determined using this methodology were more consistent with those reported for other cells. Additionally, the use of the active model parameter extraction methodology eliminated a problem encountered with the passive model in which parameter sets with widely varying values, sometimes spanning an order of magnitude or more, would produce effectively indistinguishable fits to the data. The use of an active model to directly fit the experimentally measured voltage responses to both long and short current pulses is a novel approach that is of general utility.

Preservation of ultrastructure and antigenicity for EM immunocytochemistry following intracellular recording and labeling of single cortical neurons in brain slices.

Knowledge of the distribution of neurotransmitters, neuromodulators, and transmitter receptors operating at specific synaptic sites on cortical neurons is essential for understanding the precise mechanisms that underlie the dynamic properties of cortical microcircuitry. We report on a new combination of techniques for analyzing chemically-specified synaptic input to individual cortical neurons first electrophysiologically characterized in the in vitro brain slice preparation. We tested the feasibility of this approach by performing intracellular recordings and biocytin injections in guinea pig medial prefrontal cortex slices and then by performing dual preembedding immunocytochemistry in order to localize neuronal nitric oxide synthase relative to single biocytin-filled neurons. The recorded cell and nitric oxide synthase immunoreactivity were visualized by light and electron microscopy utilizing both peroxidase and silver intensified gold stains. Single neurons were also dually visualized with fluorescence for light microscopy and with silver intensified gold for electron microscopy. Our findings indicate that both antigenicity and ultrastructure can be well preserved in tissue first used for in vitro slice experiments. This combination of methods should be widely applicable for analyzing the subcellular distribution of neuronal molecules such as receptors, channels and enzymes on physiologically characterized mammalian neurons.

Voltage-clamp analysis and computer simulation of a novel cesium-resistant A-current in guinea pig laterodorsal tegmental neurons.

Increased firing of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) plays a critical role in generating the behavioral states of arousal and rapid eye movement sleep. The majority of these neurons exhibit a prominent transient potassium current (IA) that shapes firing but the properties of which have not been examined in detail. Although IA has been reported to be blocked by intracellular cesium, the IA in LDT neurons appeared resistant to intracellular cesium. The present study compared the properties of this cesium-resistant current to those typically ascribed to IA. Whole cell recordings were obtained from LDT neurons (n = 67) in brain slices with potassium- or cesium-containing pipette solutions. A transient current was observed in cells dialyzed with each solution (KGluc-85%; CsGluc-79%). However, in cesium-dialyzed neurons, the transient current was inward at test potentials negative to about -35 mV. Extracellular 4-aminopyridine (4-AP; 2-5 mM) blocked both inward and outward current, suggesting the inward current was reversed IA rather than an unmasked transient calcium current as previously suggested. This conclusion was supported by increasing [K]o from 5 to 15 mM, which shifted the reversal potential positively for both inward and outward current (+17.89 +/- 0.41 mV; mean +/- SE). Moreover, recovery from inactivation was rapid (tau = 15.5 +/- 4 ms; n = 4), as reported for IA, and both inward and outward transient current persisted in calcium-free solution [0 calcium/4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid; n = 4] and during cadmium-blockade of calcium currents (n = 3). Finally, the transient current was blocked by intracellular 4-AP indicating that adequate dialysis occurred during the recordings. Thus the Cs-resistant current is a subthreshold IA. We also estimated the voltage-dependence of activation (V1/2 = -45.8 +/- 2 mV, k = 5.21 +/- 0.62 mV, n = 6) and inactivation (V1/2 = -59. 0 +/- 2.38 mV, k = -5.4 +/- 0.49 mV, n = 3) of this current. Computer simulations using a morphologically accurate model cell indicated that except for the extreme case of only distal A-channels and a high intracellular resistivity, our parameter estimates were good approximations. In conclusion, guinea pig LDT neurons express subthreshold A-channels that are resistant to intracellular cesium ions. This suggests that these channels differ fundamentally in their ion permeation mechanism from those previously studied. It remains to be determined if Cs+ resistance is common among brain A-channels or if this property is conferred by known A-channel subunits.

Spike train encoding by regular-spiking cells of the visual cortex.

1. To study the encoding of input currents into output spike trains by regular-spiking cells, we recorded intracellularly from slices of the guinea pig visual cortex while injecting step, sinusoidal, and broadband noise currents. 2. When measured with sinusoidal currents, the frequency tuning of the spike responses was markedly band-pass. The preferred frequency was between 8 and 30 Hz, and grew with stimulus amplitude and mean intensity. 3. Stimulation with broadband noise currents dramatically enhanced the gain of the spike responses at low and high frequencies, yielding an essentially flat frequency tuning between 0.1 and 130 Hz. 4. The averaged spike responses to sinusoidal currents exhibited two nonlinearities: rectification and spike synchronization. By contrast, no nonlinearity was evident in the averaged responses to broadband noise stimuli. 5. These properties of the spike responses were not present in the membrane potential responses. The latter were roughly linear, and their frequency tuning was low-pass and well fit by a single-compartment passive model of the cell membrane composed of a resistance and a capacitance in parallel (RC circuit). 6. To account for the spike responses, we used a "sandwich model" consisting of a low-pass linear filter (the RC circuit), a rectification nonlinearity, and a high-pass linear filter. The model is described by six parameters and predicts analog firing rates rather than discrete spikes. It provided satisfactory fits to the firing rate responses to steps, sinusoids, and broadband noise currents. 7. The properties of spike encoding are consistent with temporal nonlinearities of the visual responses in V1, such as the dependence of response frequency tuning and latency on stimulus contrast and bandwidth. We speculate that one of the roles of the high-frequency membrane potential fluctuations observed in vivo could be to amplify and linearize the responses to lower, stimulus-related frequencies.

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 microns; 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) microns2, 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 (< or = 5%), while larger errors were seen for electrotonic length (< or = 21%) and intracellular resistivity (< or = 5%). 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.

NMDA-receptor-mediated synaptic currents in guinea pig laterodorsal tegmental neurons in vitro.

1. Whole cell voltage-clamp techniques were used to record glutamate-receptor-mediated synaptic currents from neurons of the laterodorsal tegmental nucleus (LDT). The principal cells of the LDT contain acetylcholine and nitric oxide synthase, and are believed to be involved in the control of sleep-waking behavior via widespread projections to the thalamus and brain stem. LDT cells were recorded from slices of mature guinea pig brain stem with patch pipette solutions containing cesium as the primary cation. 2. Application of N-methyl-D-aspartate (NMDA) elicited currents that were strongly voltage dependent with a mean reversal potential of +16.3 mV. Peak currents occurred near -15 mV, and a region of negative slope conductance was seen at more negative potentials. Application of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid evoked currents that exhibited a nearly linear current-voltage relation with a mean reversal potential of -3.4 mV. 3. Electrical stimulation of local afferents elicited dual-component excitatory postsynaptic currents (EPSCs) with decays that were well fitted by the sum of two exponentials. Mean decay time constants at -60 mV were 8.77 ms for the faster component and 129.4 ms for the slower component. The faster component displayed a linear current-voltage relation and was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitroquinoxaline-2,3-dione, indicating that it was mediated by non-NMDA receptors, whereas the slower component displayed a voltage dependence similar to that for NMDA-evoked currents and was blocked by 2-amino-5-phosphonopentanoic acid (AP-5), indicating its mediation by NMDA receptors. 4. The fractional contribution of NMDA receptors to the EPSC was estimated from double-exponential curve fits to the decay phases. With this method, NMDA receptors were estimated on average to carry 10.1% of the total peak EPSC at -60 mV. Blockade of the non-NMDA-receptor-mediated component with CNQX revealed a residual EPSC whose amplitude was 14.4% of the control value, whereas AP-5 alone reduced the control EPSC peak by 16.1%, both values were comparable with those obtained from curve fit estimates. 5. Previous work has shown that the presence of 4-aminopyridine-sensitive, A-like transient current in LDT cells is correlated with the cholinergic phenotype. The majority of cells in this study exhibited A-like transient currents that were blocked by 4-amino-pyridine, suggesting that the majority of the data were obtained from the cholinergic and NOS-containing neurons of the LDT nucleus. 6. These experiments demonstrate the synaptic activation of functional NMDA and non-NMDA receptors in LDT neurons, and indicate that NMDA receptors contribute to fast excitatory transmission in these cells. The results suggest that afferents releasing excitatory amino acids may play an important role in controlling the state-dependent activity of LDT neurons.

Interdigitation of nitric oxide synthase-, tyrosine hydroxylase-, and serotonin-containing neurons in and around the laterodorsal and pedunculopontine tegmental nuclei of the guinea pig.

The topography of neurons containing nitric oxide synthase (NOS) and monoamines was investigated in the guinea pig mesopontine tegmentum. NOS-containing neurons were identified with NADPH-diaphorase (NADPH-d) histochemistry, and monoamine-containing neurons were identified with tyrosine hydroxylase (TH) and serotonin (5-HT) immunocytochemistry. The distribution of NADPH-d positive cells was centered on the laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei. Diaphorase-containing cells had a mean soma diameter of 23.0 +/- 4.1 microns (n = 160) and were distributed inhomogeneously, with numerous cells found within densely packed clusters. A nearest-neighbor analysis revealed that these cells were closely spaced, with up to 20% within one cell diameter and more than 50% within two cell diameters of a neighboring NADPH-d cell. Within the LDT and PPT, NADPH-d positive cells were mixed with smaller, diaphorase-negative cells (diam: 12.8 +/- 3.3 microns; n = 182; P << 0.01). TH-containing cells were not organized into a compact LC as in rat and their distribution more closely resembled that observed in cat. On average, TH-containing cells (diam: 21.2 +/- 4.8 microns; n = 160) were smaller than NADPH-d cells (P < 0.01). 5-HT-containing cells were mainly located in the raphe nuclei, as in other species. 5-HT-containing cells (diam: 18.2 +/- 4.4 microns; n = 161) were smaller on average than both the NADPH-d (P < 0.01) and TH-containing cells (P < 0.01). An analysis of the overlap in soma distributions revealed that TH-containing cells were largely interdigitated with NADPH-d containing cells. As much as 78% of the area occupied by the NADPH-d cells of LDT was contained within the area occupied by TH cells. Substantial numbers of TH and 5-HT immunoreactive processes were seen in both LDT and PPT. Varicose 5-HT and TH-containing fibers, as well as thicker, possibly dendritic processes containing TH were often seen in close apposition to NADPH-d containing somata and proximal dendrites. These results support the hypothesis that NADPH-d cells of both the PPT and LDT receive input from TH and 5-HT cells. Moreover, the clustered substructure of LDT and PPT and the extensive overlap of NADPH-d and TH-containing somata raise the possibility that the membrane permeable messenger nitric oxide plays a role in modulating TH-containing somata and their processes as well as 5-HT-containing processes in the LDT and PPT.

NMDA receptor-mediated synaptic input to nitric oxide synthase-containing neurons of the guinea pig mesopontine tegmentum in vitro.

Intracellular recordings were obtained from laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) neurons in a slice preparation to determine if excitatory synaptic input to the nitric oxide synthase (NOS)-containing cells was mediated by NMDA receptors. NOS cells were identified by intracellular injection of biocytin and NADPH-diaphorase histochemistry. Application of NMDA produced membrane depolarization, and EPSPs were reversibly reduced by an average of 46.3 +/- 16.6% with AP-5 (50 microM). These results indicate that post-synaptic NMDA receptors participate in synaptic transmission to NOS-containing neurons in LDT and PPT and are consistent with the hypothesis that synaptic NMDA receptors may control NO production in these cells.

Serotonergic and cholinergic inhibition of mesopontine cholinergic neurons controlling REM sleep: an in vitro electrophysiological study.

Intracellular recordings were obtained from neurons of the laterodorsal tegmental and pedunculopontine tegmental nuclei in a brain-slice preparation. The action of exogenously applied 5-hydroxytryptamine and acetylcholine was studied on NADPH-diaphorase-labeled cells which contain nitric oxide synthase and are presumed to be cholinergic. Our results indicated that these cells were hyperpolarized by both 5-hydroxytryptamine and acetylcholine; the ionic mechanism of this inhibition was investigated using current and voltage clamp methods. Cells voltage-clamped at resting membrane potential exhibited a net outward current and an increased membrane conductance during 5-hydroxytryptamine and acetylcholine mediated inhibition. The membrane hyperpolarization and outward current generated by this paradigm reversed near the expected K equilibrium potential and was blocked by low concentrations of extracellular Ba. The 5-hydroxytryptamine- and acetylcholine-dependent currents showed inward rectification and the reversal potential shifted in the depolarizing direction by about 15 mV for a doubling of extracellular K, indicating that both 5-hydroxytryptamine and acetylcholine activate inwardly rectifying, potassium-selective conductances. The 5-hydroxytryptamine-evoked hyperpolarization was antagonized by spiperone and mimicked by (+)8-hydroxy-2-(Di-N-propylamino)-tetralin suggesting the presence of a 5-hydroxytryptamine1A receptor while the acetylcholine-evoked hyperpolarization was blocked by atropine and only high concentrations of pirenzepine, suggesting a muscarinic M2 receptor. The outward currents evoked by 5-hydroxytryptamine and acetylcholine were not additive, suggesting that both receptors are coupled to an overlapping pool of K channels as has been observed in several systems in which receptors are coupled to effectors by G-proteins. These results indicate that the dominant actions of 5-hydroxytryptamine and acetylcholine relate to the inhibition of mesopontine cholinergic neurons via activation of an overlapping pool of inwardly rectifying K channels. Cholinergic neurons of these nuclei are thought to play an instrumental role in the induction and maintenance of rapid eye movement sleep. It has been previously hypothesized that acetylcholine would be excitatory and that 5-hydroxytryptamine would be inhibitory to these cells in the context of rapid eye movement sleep. [McCarley R. and Massaquoi S. (1986) Am. J. Physiol. 251, R1011-R1029; McCarley R. W. et al. (1975) Science 189, 58-60]. Our results are consistent with the proposed inhibitory action of 5-hydroxytryptamine but indicate recurrent input to cholinergic neurons would be inhibitory. Accordingly, models of the neural substrate underlying rapid eye movement sleep production need to be changed to reflect this inhibitory action of acetylcholine on cholinergic neurons.

The rostral dorsal cap and ventrolateral outgrowth of the rabbit inferior olive receive a GABAergic input from dorsal group Y and the ventral dentate nucleus.

The dorsal cap and ventrolateral outgrowth of the inferior olive are involved in the control of eye movements. The caudal dorsal cap is predominantly involved in the horizontal optokinetic reflex; it receives most of its GABAergic input from the nucleus prepositus hypoglossi. In the present study, we determined the source of a major inhibitory input to the rostral dorsal cap and the ventrolateral outgrowth, which are the olivary subnuclei mainly involved in the "vertical" optokinetic reflexes. We studied these subnuclei in the rabbit with the use of retrograde tracing of horseradish peroxidase and anterograde tracing of wheat germ agglutinin-coupled horseradish peroxidase combined with postembedding immunocytochemistry. The ventral dentate nucleus of the cerebellum and dorsal group y project contralaterally to the rostral dorsal cap and ventrolateral outgrowth; this projection is entirely GABAergic. The terminals of this input form predominantly symmetric synapses with extraglomerular and intraglomerular dendrites; the remaining terminals are axosomatic. In addition, the dorsal cap and ventrolateral outgrowth contain significantly more crest synapses than any other olivary subnucleus. The terminals that form these crest synapses are derived from dorsal group y and/or the ventral dentate nucleus. None of the terminals in the dorsal cap or ventrolateral outgrowth was glycinergic.

Representations of ocular rotations in the cerebellar flocculus of the rabbit.

The climbing fibres (CFs) of the rabbit flocculus that respond in a speed- and direction-selective manner to retinal image slip produced by eye rotations can be divided into three classes on the basis of the orientation of the rotation axis associated with their greatest modulation (the preferred axis). The similarity of the orientations of these axes to those of the eye rotation axes of the extraocular muscles suggests that a simple geometrical correspondence may exist between the eye rotation associated with the preferred axis of a given class of CFs and the eye rotation produced by activation of the Purkinje cells upon which that class of CFs synapse. To pursue this possibility, the axes of the eye rotations evoked by electrical microstimulation of the alert rabbit's flocculus were determined simultaneously for both eyes in three dimensions using two orthogonal search coils on each eye. A limited number of slow eye movement response patterns were found, and of these, two predominated. The most common response was a counterclockwise (CCW) rotation of the ipsilateral (left) eye around an axis close to the horizontal plane and at about 140 degrees posterior to the nose. The other predominant response was abduction of the ipsilateral eye. These two response patterns, together with the smaller conjugate components for the contralateral eye, are consonant with the orientations of the preferred CF axes. In addition, a clear CCW rotation of the contralateral (right) eye about its 135 degrees axis was also evoked from some stimulation sites. This response, which occurred either alone or as a component of an upward rotation about the nasal-occipital (roll) axis, is at variance with the orientations of the preferred CF axis. However, the latencies of the CCW contralateral 135 degrees component (80-140 ms) were greater than those of the CW contralateral 45 degrees component, the CCW ipsilateral 135 degrees component and the ipsilateral abduction component (8-48 ms). These latency differences may distinguish stimulation of Purkinje cells from stimulation of other neurones.

The accessory optic system of rabbit. II. Spatial organization of direction selectivity.

1. To compare the spatial organization of the direction selectivity of neurons in the medial terminal nucleus (MTN) of the accessory optic system with that of neurons in the adjacent ventral tegmentum, extracellular single-unit recordings were made in the anesthetized rabbit. The ventral tegmental neurons were located in a region called the visual tegmental relay zone (VTRZ), which is defined by the ventral tegmental terminal field of contralaterally projecting MTN neurons. 2. Some of the present sample of MTN neurons (5 of 34) had monocular receptive fields composed of two parts distinguished by a marked difference in the orientation of their respective direction-selective tuning curves. For one part of the receptive field the preferred excitatory direction was "up," while for the other part it was "down." Such receptive fields for one eye were called bipartite, whereas the more usually encountered MTN receptive fields, which could be characterized by a single direction-selective tuning curve, were called uniform. 3. Of the 16 neurons recorded from the VTRZ, all but one were binocular. For these neurons, both uniform and bipartite receptive fields were found for each eye alone. The only monocular neuron encountered in the VTRZ had a contralateral, bipartite receptive field. 4. The spatial organization of the direction selectivity of bipartite receptive fields strongly suggests that they are suited to represent rotation of the visual field about a horizontal axis located in the vertical plane that divides the receptive field into two parts. 5. The boundary between the two parts of the bipartite receptive fields was found using handheld visual stimuli at one of two azimuthal locations, either close to 45 degrees or between 95 and 125 degrees (the 0 degree reference was rostral in the midsagittal plane). This particular structure of the bipartite receptive fields suggests that their preferred rotation axes have a close spatial relation to the best-response axes of the semicircular canals. 6. Seven VTRZ neurons were antidromically activated by electrical stimulation of the ipsilateral dorsal cap of the inferior olive. Since the receptive fields of VTRZ neurons have many of the structural features characteristic of the receptive fields of rostral dorsal cap neurons we conclude that the spatial organization of the receptive fields of dorsal cap neurons is, for the most part, synthesized prior to the inferior olive.(ABSTRACT TRUNCATED AT 400 WORDS)