Nancy L. Chamberlin

Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS.

We used a recombinant adeno-associated virus vector (AAV) to deliver a foreign gene, green fluorescent protein (GFP), into mature neurons in adult rat CNS in vivo. Microinjections of AAV as small as 50 nl transduced hundreds of neurons at the injection site. There was virtually no retrograde transport as fewer than one neuron per brain was found distant from the injection site that exhibited GFP immunoreactivity. The gene product, GFP, filled the entire neuronal cytoplasmic compartment; GFP immunoreactivity was robust in cell bodies, axons, and nerve terminals. There was no tissue damage at the injection sites or pathogenicity indicated by changes in astrocytic or microglial markers. There was no inflammatory response as judged by leukocytic invasion. Gene expression in transduced cells was robust and apparently permanent: there was no evidence of phenotypic reversion up to 12 weeks following infection. AAV is an excellent vector for introducing foreign genes into mature CNS neurons. Not only might it be an ideal vehicle for gene therapy, but also the GFP-containing AAV presents a new strategy for tracing long axonal pathways in the CNS, which is difficult with current tracers (PHAL, biotinylated dextrans).

Role of endogenous sleep‐wake and analgesic systems in anesthesia

Classical anesthetics of the γ‐aminobutyric acid type A receptor (GABAA)‐enhancing class (e.g., pentobarbital, chloral hydrate, muscimol, and ethanol) produce analgesia and unconsciousness (sedation). Dissociative anesthetics that antagonize the N‐methyl‐D‐aspartate (NMDA) receptor (e.g., ketamine, MK‐801, dextromethorphan, and phencyclidine) produce analgesia but do not induce complete loss of consciousness. To understand the mechanisms underlying loss of consciousness and analgesia induced by general anesthetics, we examined the patterns of expression of c‐Fos protein in the brain and correlated these with physiological effects of systemically administering GABAergic agents and ketamine at dosages used clinically for anesthesia in rats. We found that GABAergic agents produced predominantly delta activity in the electroencephalogram (EEG) and sedation. In contrast, anesthetic doses of ketamine induced sedation, followed by active arousal behaviors, and produced a faster EEG in the theta range. Consistent with its behavioral effects, ketamine induced Fos expression in cholinergic, monoaminergic, and orexinergic arousal systems and completely suppressed Fos immunoreactivity in the sleep‐promoting ventrolateral preoptic nucleus (VLPO). In contrast, GABAergic agents suppressed Fos in the same arousal‐promoting systems but increased the number of Fos‐immunoreactive neurons in the VLPO compared with waking control animals. All anesthetics tested induced Fos in the spinally projecting noradrenergic A5–7 groups. 6‐hydroxydopamine lesions of the A5–7 groups or ibotenic acid lesions of the ventrolateral periaqueductal gray matter (vlPAG) attenuated antinociceptive responses to noxious thermal stimulation (tail‐flick test) by both types of anesthetics. We hypothesize that neural substrates of sleep‐wake behavior are engaged by low‐dose sedative anesthetics and that the mesopontine descending noradrenergic cell groups contribute to the analgesic effects of both NMDA receptor antagonists and GABAA receptor‐enhancing anesthetics. J. Comp. Neurol. 508:648–662, 2008.

Effects of adenosine on gabaergic synaptic inputs to identified ventrolateral preoptic neurons

The ventrolateral preoptic nucleus (VLPO) is a key regulator of behavioral state that promotes sleep by directly inhibiting brain regions that maintain wakefulness. Subarachnoid administration of adenosine (AD) or AD agonists promotes sleep and induces expression of Fos protein in VLPO neurons. Therefore, activation of VLPO neurons may contribute to the somnogenic actions of AD. To define the mechanism through which AD activates VLPO neurons, we prepared hypothalamic slices from 9 to 12-day-old rat pups and recorded from 43 neurons in the galaninergic VLPO cluster; nine neurons contained galanin mRNA by post hoc in situ hybridization. Bath application of AD (20 microM) to seven of these neurons had no direct effect but caused a significant decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents in the presence of tetrodotoxin, indicating a presynaptic site of action. We conclude that AD-mediated disinhibition increases the excitability of VLPO neurons thus contributing to the somnogenic properties of AD.

A Brainstem Network Mediating Apneic Reflexes in the Rat

Apnea is an important protective response to upper airway irritation, but the central mechanisms responsible for eliciting sensory-induced apnea are not well understood. Recent studies have emphasized the Kölliker-Fuse nucleus in producing apnea and proposed a trigeminoparabrachial pathway for mediating these reflexes. However, in our earlier study of apneic responses produced by glutamate stimulation in the dorsolateral pons, we found that apnea was elicited from the area just ventral to the Kölliker-Fuse nucleus, rather than within it. Because this region was not known to be involved in respiratory control, we combined chemical microstimulation with both anterograde and retrograde axonal tracing to characterize the sites in the pons that produce apneic responses. We found that apneic sites were consistently associated with the intertrigeminal region, between the principal sensory and motor trigeminal nuclei. Injections of anterograde tracer at these sites labeled terminals in the ventral respiratory group, in the ventrolateral medulla. Injection of retrograde tracer into this target region in the ventrolateral medulla disclosed a previously unrecognized population of neurons among the trigeminal motor rootlets. Injection of retrograde tracer into this intertrigeminal region demonstrated inputs from portions of the spinal trigeminal nucleus and the nucleus of the solitary tract that have been associated with producing sensory apnea. Our observations suggest that the intertrigeminal region receives a convergence of sensory inputs capable of driving apneic responses and that it may represent a common link between input from different portions of the airway and the respiratory neurons that mediate apneic reflexes.

Unwarranted Administration of Acetylcholinesterase Inhibitors Can Impair Genioglossus and Diaphragm Muscle Function

Background:It is standard practice to administer a cholinesterase inhibitor (e.g., neostigmine) at the end of a surgical case to reverse suspected effects of neuromuscular blocking agents regardless of whether such residual effects are present. The authors hypothesized that cholinesterase inhibition when given the in absence of neuromuscular blockade (NB) would decrease upper airway dilatory muscle activity and consequently upper airway volume. Methods:The authors measured genioglossus and diaphragm electromyograms during spontaneous ventilation in anesthetized, tracheostomized rats before and after administration of neostigmine (0.03, 0.06, or 0.12 mg/kg), after recovery of the train-of-four ratio (quadriceps femoris muscle) to unity after NB (n = 18). For comparison, the authors made the same measurements in rats that had no previous NB (n = 27). In intact anesthetized rats, the authors measured upper airway volume and end-expiratory lung volume by magnetic resonance imaging before and after 0.12 mg/kg neostigmine (n = 9). Results:Neostigmine treatment in rats that had fully recovered from NB based on the train-of-four ratio caused dose-dependent decreases in genioglossus electromyogram (to 70.3 ± 7.6, 49.2 ± 3.2, and 39.7 ± 2.3% of control, respectively), decreases in diaphragm electromyogram (to 103.1 ± 6.5, 83.1 ± 4.7, and 68.7 ± 7.3% of control), and decreases in minute ventilation to a nadir value of 79.6 ± 6% of preneostigmine baseline. Genioglossus electromyogram effects were the same when neostigmine was given with no previous NB. Neostigmine caused a decrease in upper airway volume to 83 ± 3% of control, whereas end-expiratory lung volume remained constant. Conclusions:The cholinesterase inhibitor neostigmine markedly impairs upper airway dilator volume, genioglossus muscle function, diaphragmatic function, and breathing when given after recovery from vecuronium-induced neuromuscular block.

Glutamatergic Signaling from the Parabrachial Nucleus Plays a Critical Role in Hypercapnic Arousal

The mechanisms of arousal from apneas during sleep in patients suffering from obstructive sleep apnea are not well understood. However, we know that respiratory chemosensory pathways converge on the parabrachial nucleus (PB), which sends glutamatergic projections to a variety of forebrain structures critical to arousal, including the basal forebrain, lateral hypothalamus, midline thalamus, and cerebral cortex. We tested the role of glutamatergic signaling in this pathway by developing an animal model for repetitive CO2 arousals (RCAs) and investigating the effect of deleting the gene for the vesicular glutamate transporter 2 (Vglut2) from neurons in the PB. We used mice with lox P sequences flanking exon2 of the Vglut2 gene, in which adeno-associated viral vectors containing genes encoding Cre recombinase and green fluorescent protein were microinjected into the PB to permanently and selectively disrupt Vglut2 expression while labeling the affected neurons. We recorded sleep in these mice and then investigated the arousals during RCA. Vglut2 deletions that included the external lateral and lateral crescent subdivisions of the lateral PB more than doubled the latency to arousal and resulted in failure to arouse by 30 s in >30% of trials. By contrast, deletions that involved the medial PB subdivision had minimal effects on arousal during hypercapnia but instead increased non-rapid eye movement (NREM) sleep by ∼43% during the dark period, and increased delta power in the EEG during NREM sleep by ∼50%. Our results suggest that glutamatergic neurons in the lateral PB are necessary for arousals from sleep in response to CO2, while medial PB glutamatergic neurons play an important role in promoting spontaneous waking.

Functional organization of the parabrachial complex and intertrigeminal region in the control of breathing

Although the medulla oblongata contains the epicenter for respiratory rhythm generation, many other parts of the neuraxis play significant substratal roles in breathing. Accumulating evidence suggests that the pons contains several groups of neurons that may belong to the central respiratory system. This article will review data from microstimulation mapping and tract-tracing studies of the parabrachial complex (PB) and intertrigeminal region (ITR). Chemical activation of neurons in these areas has distinct effects on ventilatory and airway muscle activity. Tract-tracing experiments from functionally identified sites reveal specific respiratory-related sensory inputs and outputs that are likely anatomical substrates for these effects. The data suggest that an important physiological role for the rostral pons may be reflexive respiratory responses to airway stimuli.

Neostigmine but not sugammadex impairs upper airway dilator muscle activity and breathing

BACKGROUND Cholinesterase inhibitor-based reversal agents, given in the absence of neuromuscular block, evoke a partial upper airway obstruction by decreasing skeletal upper airway muscle function. Sugammadex reverses neuromuscular block by encapsulating rocuronium. However, its effects on upper airway integrity and breathing are unknown. METHODS Fifty-one adult male rats were anaesthetized with isoflurane, tracheostomized, and a femoral artery and vein were cannulated. First, we compared the efficacy of sugammadex 15 mg kg(-1) and neostigmine 0.06 mg kg(-1) to reverse respiratory effects of rocuronium-induced partial paralysis [train-of-four ratio (T4/T1)=0.5]. Subsequently, we compared the safety of sugammadex and neostigmine given after recovery of the T4/T1 to 1, by measuring phasic genioglossus activity and breathing. RESULTS During partial paralysis (T4/T1=0.5), time to recovery of minute volume to baseline values was 10.9 (2), 75.8 (18), and 153 (54) s with sugammadex, neostigmine, and placebo, respectively (sugammadex was significantly faster than neostigmine and placebo, P<0.05). Recovery of T4/T1 was also faster for sugammadex than neostigmine and placebo. Neostigmine administration after complete recovery of T4/T1 decreased upper airway dilator muscle activity to 64 (30)% of baseline and decreased tidal volume (P<0.05 for both variables), whereas sugammadex had no effect on either variable. CONCLUSIONS In contrast to neostigmine, which significantly impairs upper airway dilator muscle activity when given after recovery from neuromuscular block, a reversal dose of sugammadex given under the same conditions does not affect genioglossus muscle activity and normal breathing. Human studies will be required to evaluate the clinical relevance of our findings.

Genioglossus premotoneurons and the negative pressure reflex in rats

Reflex increases in genioglossus (GG) muscle activity in response to negative pharyngeal pressure are important for maintenance of upper airway patency in humans. However, little is known of the central circuitry that mediates this negative pressure reflex (NPR). We used two approaches to determine which GG premotoneurons relay negative pressure‐related information to the hypoglossal motor nucleus. First, to identify GG premotoneurons, we injected pseudorabies virus (PRV152) into the GG muscle. We found that medullary GG premotoneurons were concentrated mainly in the reticular formation adjacent to the hypoglossal motor nucleus. Second, in order to determine whether these perihypoglossal neurons were involved in the NPR, we quantified GG EMG responses to negative pressure applied to the isolated upper airway in anaesthetized rats before and after microinjection of muscimol (9 nl; 0.25 mm), a GABA‐A receptor agonist, into the perihypoglossal premotor field. Pressures as low as −4 cmH2O increased inspiratory phase‐related GG activity. The NPR was abolished following bilateral injections of muscimol into the perihypoglossal premotor field at and up to 500 μm rostral to the obex. Muscimol in this location also increased the amplitude of basal, unstimulated phasic GG activity. By contrast, inhibition of neurons caudal to the obex decreased phasic GG activity but had no impact on the NPR. These results suggest that perihypoglossal GG premotoneurons near the obex mediate the NPR and those caudal to the obex are important mediators of respiratory‐related GG activity but are not involved in the NPR.

Sleep disturbance induces neuroinflammation and impairment of learning and memory.

Hospitalized patients can develop cognitive function decline, the mechanisms of which remain largely to be determined. Sleep disturbance often occurs in hospitalized patients, and neuroinflammation can induce learning and memory impairment. We therefore set out to determine whether sleep disturbance can induce neuroinflammation and impairment of learning and memory in rodents. Five to 6-month-old wild-type C57BL/6J male mice were used in the studies. The mice were placed in rocking cages for 24 h, and two rolling balls were present in each cage. The mice were tested for learning and memory function using the Fear Conditioning Test one and 7 days post-sleep disturbance. Neuroinflammation in the mouse brain tissues was also determined. Of the Fear Conditioning studies at one day and 7 days after sleep disturbance, twenty-four hour sleep disturbance decreased freezing time in the context test, which assesses hippocampus-dependent learning and memory; but not the tone test, which assesses hippocampus-independent learning and memory. Sleep disturbance increased pro-inflammatory cytokine IL-6 levels and induced microglia activation in the mouse hippocampus, but not the cortex. These results suggest that sleep disturbance induces neuroinflammation in the mouse hippocampus, and impairs hippocampus-dependent learning and memory in mice. Pending further studies, these findings suggest that sleep disturbance-induced neuroinflammation and impairment of learning and memory may contribute to the development of cognitive function decline in hospitalized patients.