Kristi A. Kohlmeier

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.

Nicotinic Activation of Laterodorsal Tegmental Neurons: Implications for Addiction to Nicotine

Identifying the neurological mechanisms underlying nicotine reinforcement is a healthcare imperative, if society is to effectively combat tobacco addiction. The majority of studies of the neurobiology of addiction have focused on dopamine (DA)-containing neurons of the ventral tegmental area (VTA). However, recent data suggest that neurons of the laterodorsal tegmental (LDT) nucleus, which sends cholinergic, GABAergic, and glutamatergic-containing projections to DA-containing neurons of the VTA, are critical to gating normal functioning of this nucleus. The actions of nicotine on LDT neurons are unknown. We addressed this issue by examining the effects of nicotine on identified cholinergic and non-cholinergic LDT neurons using whole-cell patch clamp and Ca2+-imaging methods in brain slices from mice (P12–P45). Nicotine applied by puffer pipette or bath superfusion elicited membrane depolarization that often induced firing and TTX-resistant inward currents. Nicotine also enhanced sensitivity to injected current; and, baseline changes in intracellular calcium were elicited in the dendrites of some cholinergic LDT cells. In addition, activity-dependent calcium transients were increased, suggesting that nicotine exposure sufficient to induce firing may lead to enhancement of levels of intracellular calcium. Nicotine also had strong actions on glutamate and GABA-releasing presynaptic terminals, as it greatly increased the frequency of miniature EPSCs and IPSCs to both cholinergic and non-cholinergic neurons. Utilization of nicotinic acetylcholine receptors (nAChR) subunit antagonists revealed that presynaptic, inhibitory terminals on cholinergic neurons were activated by receptors containing α7, β2, and non-α7 subunits, whereas, presynaptic glutamatergic terminals were activated by nAChRs that comprised non-α7 subunits. We also found that direct nicotinic actions on cholinergic LDT neurons were mediated by receptors containing α7, β2, and non-α7 subunits. These findings led us to suggest that nicotine exposure from smoking will enhance both the excitability and synaptic modulation of cholinergic and non-cholinergic LDT neurons, and increase their signature neurotransmitter outflow to target regions, including the VTA. This may reinforce the direct actions of this drug within reward circuitry and contribute to encoding stimulus saliency.

Differential actions of orexin receptors in brainstem cholinergic and monoaminergic neurons revealed by receptor knockouts: implications for orexinergic signaling in arousal and narcolepsy

Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2) are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca2+ imaging methods to delineate the cellular actions of each receptor within cholinergic [laterodorsal tegmental nucleus (LDT)] and monoaminergic [dorsal raphe (DR) and locus coeruleus (LC)] brainstem nuclei—where orexins promote arousal and suppress REM sleep. In slices from OX−/−2 mice, orexin-A (300 nM) elicited wild-type responses in LDT, DR, and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca2+ transients. In slices from OX−/−1 mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca2+-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX−/−1 mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca2+ transients produced by both receptors appeared mediated by influx via L-type Ca2+ channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor knockout mice.

Noradrenaline excites non-cholinergic laterodorsal tegmental neurons via two distinct mechanisms.

Cholinergic neurons of the laterodorsal tegmental nucleus have been hypothesized to play a critical role in the-generation and maintenance of rapid eye movement sleep. Less is known about the function of non-cholinergic laterodorsal tegmental nucleus neurons. As part of our ongoing studies of the brainstem circuitry controlling behavioral state, we have begun to investigate the functional properties of these neurons. In the course of these experiments, we have observed a novel response to the neurotransmitter noradrenaline. Whole-cell patch-clamp recordings of laterodorsal tegmental nucleus neurons were carried out in 21- to 35-day-old rat brain slices. A subpopulation of laterodorsal tegmental nucleus cells responded to a 30-s application of 50 microM noradrenaline with depolarization and a decrease in input resistance which lasted several minutes. Following return to resting membrane potential, these cells invariably exhibited barrages of excitatory postsynaptic potentials which lasted at least 12 min. These excitatory postsynaptic potentials were reversibly abolished by bath application of tetrodotoxin, as well as by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, but were insensitive to application of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. To examine whether these neurons were cholinergic, the recorded cells were labeled with biocytin and tested for co-localization with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a marker for laterodorsal tegmental nucleus cholinergic neurons. In every instance, neurons with these properties were non-cholinergic. However, they were always located in close proximity to reduced nicotinamide adenine dinucleotide phosphate-diaphorase-positive laterodorsal tegmental nucleus cells. The present data indicate that noradrenaline, in addition to directly inhibiting cholinergic cells of the laterodorsal tegmental nucleus, also results in the direct and indirect excitation of non-cholinergic cells of the laterodorsal tegmental nucleus. The indirect excitation is long lasting and mediated by glutamatergic mechanisms. Our working hypothesis is that these non-cholinergic cells are local circuit inhibitory interneurons and that prolonged excitation of these neurons by noradrenaline may serve as a mechanism for inhibition of cholinergic laterodorsal tegmental nucleus cells during wakefulness, when noradrenaline tone is high.

Muscle atonia can be induced by carbachol injections into the nucleus pontis oralis in cats anesthetized with α-chloralose

Cholinergic excitation of structures in the pontine reticular formation appears to be a key step in the generation of active sleep. For example, muscle atonia which occurs as a result of the postsynaptic inhibition of motoneurons during active sleep is also present after carbachol, a cholinergic agonist, is injected into the nucleus pontis oralis. In the present study, in order to obtain information regarding the mechanisms that generate atonia during active sleep and to provide a paradigm for studying atonia in anesthetized cats, we determined whether cholinergically induced atonia could be generated in an animal that was anesthetized with alpha-chloralose. Cats which were initially anesthetized with alpha-chloralose (40 mg/kg, I.V.) exhibited spikes in the EEG, hippocampus and lateral geniculate nuclei. Muscle atonia occurred after carbachol (200 mM) was injected by microiontophoresis (300-500 nA) into the nucleus pontis oralis; the spikes in the EEG, hippocampus and lateral geniculate nuclei were still present. We believe that the atonia induced by carbachol in alpha-chloralose-anesthetized cats is mediated by the same mechanisms that operate during active sleep in the unanesthetized animal for the following reasons. First, in the same cats when they were not anesthetized with alpha-chloralose, carbachol injections in the identical brainstem sites induced active sleep with its accompanying pattern of muscle atonia. Second, after carbachol was injected into the same sites in alpha-chloralose-anesthetized cats, intracellular recordings from lumbar motoneurons revealed that inhibitory postsynaptic potentials were bombarding motoneurons; these inhibitory potentials were similar to those which are present during naturally occurring active sleep. In addition, stimulation of the nucleus reticularis gigantocellularis (NRGc) was found to induce large amplitude depolarizing potentials in lumbar motoneurons in alpha-chloralose-anesthetized cats prior to the administration of carbachol, whereas after its administration, accompanying muscle atonia there were large amplitude hyperpolarizing potentials and a reduction in the amplitude of depolarizing potentials. We therefore conclude that the cholinergically induced processes that initiate and maintain muscle atonia are not blocked by the actions of alpha-chloralose.

State dependency of the effects of microinjection of cholinergic drugs into the nucleus pontis oralis.

The microinjection of cholinergic drugs into the pontine reticular formation elicits active sleep-like states that are comprised of the principal physiological patterns of activity that characterize naturally-occurring active sleep, i.e., EEG desynchronization, PGO waves, rapid eye movements and atonia. We have reported that other behavioral states arise even when cholinergic drugs are injected into the exact same reticular location. The present study was conducted to explore the basis for the differences in the drug effect. A combination of acetylcholine and neostigmine was injected by microiontophoresis into the dorsal region of the nucleus pontis oralis in four chronic, unanesthetized cats. The states that were induced by cholinergic drug injection depended on the state of the animal at the time of the injection. When the animal was awake, cholinergic injections resulted in a waking-dissociated state, which was characterized by EEG desynchronization and muscle atonia in a cat that appeared to be awake and was able to track objects in its visual field. If the cat was in quiet sleep at the time of the injection, an active sleep-like state followed that was indistinguishable from naturally-occurring active sleep; on a few occasions following cholinergic injections during quiet sleep there was a quiet sleep-dissociated state, which was characterized by PGO waves and muscle atonia in the cat that by other indices appeared to be in quiet sleep. The results of this study indicate that the state of the animal at the time of drug injection is a critical variable that influences the responses which are induced by cholinergic stimulation of the pontine reticular formation.

Substance P in the descending cholinergic projection to REM sleep-induction regions of the rat pontine reticular formation: anatomical and electrophysiological analyses

Release of acetylcholine within the pontine reticular formation (PRF) from the axon terminals of mesopontine cholinergic neurons has long been hypothesized to play an important role in rapid eye movement (REM) sleep generation. As some of these cholinergic neurons are known to contain substance P (SP), we used anatomical, electrophysiological and pharmacological techniques to characterize this projection in the rat. Double immunofluorescence demonstrated that 16% of all cholinergic neurons within the mesopontine tegmentum contained SP; this percentage increased to 27% in its caudal regions. When double immunofluorescence was combined with retrograde tracing techniques, it was observed that up to 11% of all SP‐containing cholinergic neurons project to the PRF. Whole‐cell patch‐clamp recordings from in vitro brainstem slices revealed that SP administration depolarized or evoked an inward current in a dose‐dependent manner in all PRF neurons examined, and that these effects were antagonized by a SP antagonist. The amplitude of the SP‐induced inward current varied with changes in the Na+ concentration, did not reverse at the calculated K+ or Cl– equilibrium potentials, and was not attenuated in the presence of tetrodotoxin, low Ca2+ concentration or caesium ions. These data suggest that activation of a tetrodotoxin‐insensitive cation channel(s) permeable to Na+ is responsible for a SP‐induced inward current at resting membrane potentials. The depolarizing actions of SP appeared to be primarily due to activation of the adenylate cyclase pathway, and were additive with cholinergic receptor activation even at maximal concentrations. These data indicate that SP is colocalized in a subpopulation of mesopontine tegmental cholinergic neurons projecting to REM sleep‐induction regions of the PRF, and that actions of these two neuroactive substances on PRF neurons are additive. If SP is coreleased with acetylcholine, the additive actions of the two neurotransmitters might heighten the excitability of postsynaptic PRF neurons and ensure the initiation and maintenance of REM sleep.

Transmitter modulation of spike‐evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX‐482‐sensitive calcium influx

Nitric oxide synthase (NOS)‐containing cholinergic neurons in the laterodorsal tegmentum (LDT) influence behavioral and motivational states through their projections to the thalamus, ventral tegmental area and a brainstem ‘rapid eye movement (REM)‐induction’ site. Action potential‐evoked intracellular calcium transients dampen excitability and stimulate NO production in these neurons. In this study, we investigated the action of several arousal‐related neurotransmitters and the role of specific calcium channels in these LDT Ca2+‐transients by simultaneous whole‐cell recording and calcium imaging in mouse (P14–P30) brain slices. Carbachol, noradrenaline and adenosine inhibited spike‐evoked Ca2+‐transients, while histamine, t‐ACPD, a metabotropic glutamate receptor agonist, and orexin‐A did not. Carbachol inhibition was blocked by atropine, was insensitive to blockade of G‐protein‐coupled inward rectifier (GIRK) channels and was not inhibited by nifedipine, ω‐conotoxin GVIA or ω‐agatoxin IVA, which block L‐, N‐ and P/Q‐type calcium channels, respectively. In contrast, SNX‐482 (100 nm), a selective antagonist of R‐type calcium channels containing the alpha1E (Cav2.3) subunit, attenuated carbachol inhibition of the somatic spike‐evoked calcium transient. To our knowledge, this is the first demonstration of muscarinic inhibition of native SNX‐482‐sensitive R‐channels. Our findings indicate that muscarinic modulation of these channels plays an important role in the feedback control of cholinergic LDT neurons and that inhibition of spike‐evoked Ca2+‐transients is a common action of neurotransmitters that also activate GIRK channels in these neurons. Because spike‐evoked calcium influx dampens excitability, our findings suggest that these ‘inhibitory’ transmitters could boost firing rate and enhance responsiveness to excitatory inputs during states of high firing, such as waking and REM sleep.

Strychnine blocks inhibitory postsynaptic potentials elicited in masseter motoneurons by sensory stimuli during carbachol-induced motor atonia

In previous studies we reported that large-amplitude inhibitory potentials were elicited in masseter motoneurons by auditory stimuli (95-dB clicks) and stimulation of the sciatic nerve in alpha-chloralose-anesthetized cats [Kohlmeier K. A. et al. (1994) Soc. Neurosci. Abstr. 20, 1218; Kohlmeier K. A. et al. (1995) Sleep Res. 24, 9]. These potentials were always elicited during motor atonia induced by the pontine injection of carbachol into the nucleus pontis oralis and were never elicited prior to atonia. In the present report, the hyperpolarizing potentials that arose in response to clicks and stimulation of the sciatic nerve were blocked following the juxtacellular application of strychnine, a glycinergic antagonist. In contrast, bicuculline, a GABA(A) receptor antagonist, did not suppress the carbachol-dependent hyperpolarizing potentials elicited by these stimuli. In some motoneurons, blockade of the inhibitory potential by strychnine revealed a depolarizing potential. These data suggest that clicks and stimulation of the sciatic nerve not only elicit inhibition of motoneurons but also activate an excitatory drive which is masked by elicited inhibitory postsynaptic potentials. These findings suggest that glycine is likely to be the neurotransmitter that is responsible for the inhibitory postsynaptic potentials elicited in masseter motoneurons following the presentation of auditory and somatosensory stimuli during carbachol-induced motor atonia. We suggest that the same system that mediates glycinergically-dependent motor atonia during naturally occurring active sleep [Chase M. H. et al. (1989) J. Neurosci. 9, 743-751] also mediates the carbachol-dependent response of motoneurons to sensory stimuli.

Age-related changes in nicotine response of cholinergic and non-cholinergic laterodorsal tegmental neurons: implications for the heightened adolescent susceptibility to nicotine addiction

The younger an individual starts smoking, the greater the likelihood that addiction to nicotine will develop, suggesting that neurobiological responses vary across age to the addictive component of cigarettes. Cholinergic neurons of the laterodorsal tegmental nucleus (LDT) are importantly involved in the development of addiction, however, the effects of nicotine on LDT neuronal excitability across ontogeny are unknown. Nicotinic effects on LDT cells across different age groups were examined using calcium imaging and whole-cell patch clamping. Within the youngest age group (P7-P15), nicotine induced larger intracellular calcium transients and inward currents. Nicotine induced a greater number of excitatory synaptic currents in the youngest animals, whereas larger amplitude inhibitory synaptic events were induced in cells from the oldest animals (P15-P34). Nicotine increased neuronal firing of cholinergic cells to a greater degree in younger animals, possibly linked to development associated differences found in nicotinic effects on action potential shape and afterhyperpolarization. We conclude that in addition to age-associated alterations of several properties expected to affect resting cell excitability, parameters affecting cell excitability are altered by nicotine differentially across ontogeny. Taken together, our data suggest that nicotine induces a larger excitatory response in cholinergic LDT neurons from the youngest animals, which could result in a greater excitatory output from these cells to target regions involved in development of addiction. Such output would be expected to be promotive of addiction; therefore, ontogenetic differences in nicotine-mediated increases in the excitability of the LDT could contribute to the differential susceptibility to nicotine addiction seen across age.