Searching in the wrong place: Might consciousness reside in the brainstem?

Doubtless, the conscious brain integrates masses of information. But declaring that consciousness simply "emerges" when enough has accumulated, doesn't really explain how first person experience is implemented by neurons. Moreover, empirical observations challenge integrated information theory's (IIT) reliance on thalamo-cortical interactions as the information integrator. More likely, the cortex streams processed information to a still-enigmatic consciousness generator, one perhaps located in the brainstem.

Anesthesia in mice activates discrete populations of neurons throughout the brain.

The brain undergoes rapid, dramatic, and reversible transitioning between states of wakefulness and unconsciousness during natural sleep and in pathological conditions such as hypoxia, hypotension, and concussion. Transitioning can also be induced pharmacologically using general anesthetic agents. The effect is selective. Mobility, sensory perception, memory formation, and awareness are lost while numerous housekeeping functions persist. How is selective transitioning accomplished? Classically a handful of brainstem and diencephalic "arousal nuclei" have been implicated in driving brain-state transitions on the grounds that their net activity systematically varies with brain state. Here we used transgenic targeted recombination in active populations mice to label neurons active during wakefulness with one reporter and neurons active during pentobarbital-induced general anesthesia with a second, contrasting reporter. We found 'wake-on' and 'anesthesia-on' neurons in widely distributed regions-of-interest, but rarely encountered neurons labeled with both reporters. Nearly all labeled neurons were either wake-on or anesthesia-on. Thus, anesthesia-on neurons are not unique to the few nuclei discovered to date whose activity appears to increase during anesthesia. Rather neuronal populations selectively active during anesthesia are located throughout the brain where they likely play a causative role in transitioning between wakefulness and anesthesia. The widespread neuronal suppression reported in prior comparisons of the awake and anesthetized brain in animal models and noninvasive imaging in humans reflects only net differences. It misses the ubiquitous presence of neurons whose activity increases during anesthesia. The balance in recruitment of anesthesia-on versus wake-on neuronal populations throughout the brain may be a key driver of regional and global vigilance states. [Correction added on September 22, 2021, after first online publication: Due to a typesetting error, the abstract text was cut off. This has been corrected now.].

A nodal point for brain-state transitions: the mesopontine tegmental anesthesia area (MPTA) in mice.

The mesopontine tegmental anesthesia area (MPTA) was identified in rats as a singular brainstem locus at which microinjection of minute quantities of GABAergic agents rapidly and reversibly induces loss-of-consciousness and a state of general anesthesia, while lesioning renders animals insensitive to anesthetics at normal systemic doses. Obtaining similar results in mice has been challenging, however, slowing research progress on how anesthetics trigger brain-state transitions. We have identified roadblocks that impeded translation from rat to mouse and tentatively located the MPTA equivalent in this second species. We describe here a series of modifications to the rat protocol that allowed us to document pro-anesthetic changes in mice following localized stereotactic delivery of minute quantities (20 nL) of the GABAA-receptor agonist muscimol into the brainstem mesopontine tegmentum. The optimal locus identified proved to be homologous to the MPTA in rats, and local neuronal populations in rats and mice were similar in size and shape. This outcome should facilitate application of the many innovative gene-based methodologies available primarily in mice to the study of how activity in brainstem MPTA neurons brings about anesthetic loss-of-consciousness and permits pain-free surgery.

Reduced Sensitivity to Anesthetic Agents upon Lesioning the Mesopontine Tegmental Anesthesia Area in Rats Depends on Anesthetic Type.

BACKGROUND: The brainstem mesopontine tegmental anesthesia area is a key node in circuitry responsible for anesthetic induction and maintenance. Microinjecting the γ-aminobutyric acid-mediated (GABAergic) anesthetic pentobarbital in this nucleus rapidly and reversibly induces general anesthesia, whereas lesioning it renders the animal relatively insensitive to pentobarbital administered systemically. This study investigated whether effects of lesioning the mesopontine tegmental anesthesia area generalize to other anesthetic agents. METHODS: Cell-selective lesions were made using ibotenic acid, and rats were later tested for changes in the dose-response relation to etomidate, propofol, alfaxalone/alfadolone, ketamine, and medetomidine delivered intravenously using a programmable infusion pump. Anesthetic induction for each agent was tracked using five behavioral endpoints: loss of righting reflex, criterion for anesthesia (score of 11 or higher), criterion for surgical anesthesia (score of 14 or higher), antinociception (loss of pinch response), and deep surgical anesthesia (score of 16). RESULTS: As reported previously for pentobarbital, on-target mesopontine tegmental anesthesia area lesions reduced sensitivity to the GABAergic anesthetics etomidate and propofol. The dose to achieve a score of 16 increased to 147 ± 50% of baseline in control animals ± SD (P = 0.0007; 7 lesioned rats and 18 controls) and 136 ± 58% of baseline (P = 0.010; 6 lesioned rats and 21 controls), respectively. In contrast, responsiveness to the neurosteroids alfaxalone and alfadolone remained unchanged compared with baseline (94 ± 24%; P = 0.519; 6 lesioned rats and 18 controls) and with ketamine increased slightly (90 ± 11%; P = 0.039; 6 lesioned rats and 19 controls). The non-GABAergic anesthetic medetomidine did not induce criterion anesthesia even at the maximal dose tested. The dose to reach the maximal anesthesia score actually obtained was unaffected by the lesion (112 ± 8%; P = 0.063; 5 lesioned rats and 18 controls). CONCLUSIONS: Inability to induce anesthesia in lesioned animals using normally effective doses of etomidate, propofol, and pentobarbital suggests that the mesopontine tegmental anesthesia area is the effective target of these, but not necessarily all, GABAergic anesthetics upon systemic administration. Cortical and spinal functions are likely suppressed by recruitment of dedicated ascending and descending pathways rather than by direct, distributed drug action.

Location of the Mesopontine Neurons Responsible for Maintenance of Anesthetic Loss of Consciousness.

The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinociception). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats by microinjection of small amounts of GABAA-receptor agonists into an upper brainstem region named the mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here, using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state transitions.SIGNIFICANCE STATEMENT General anesthesia permits pain-free surgery. Furthermore, because anesthetic agents have the unique ability to reversibly switch the brain from wakefulness to a state of unconsciousness, knowing how and where they work is a potential route to unraveling the neural mechanisms that underlie awareness itself. Using a novel method, we have located a small, and apparently one of a kind, cluster of neurons in the mesopontine tegmentum that are capable of effecting brain-state switching when exposed to GABAA-receptor agonists. This action appears to be mediated by a network of dedicated axonal pathways that project directly and/or indirectly to nearby arousal nuclei of the brainstem and to more distant targets in the forebrain and spinal cord.

sec-Butylpropylacetamide (SPD), a new amide derivative of valproic acid for the treatment of neuropathic and inflammatory pain.

Chronic pain is a multifactorial disease comprised of both inflammatory and neuropathic components that affect ∼20% of the world's population. sec-Butylpropylacetamide (SPD) is a novel amide analogue of valproic acid (VPA) previously shown to possess a broad spectrum of anticonvulsant activity. In this study, we defined the pharmacokinetic parameters of SPD in rat and mouse, and then evaluated its antinociceptive potential in neuropathic and acute inflammatory pain models. In the sciatic nerve ligation (SNL) model of neuropathic pain, SPD was equipotent to gabapentin and more potent than its parent compound VPA. SPD also showed either higher or equal potency to VPA in the formalin, carrageenan, and writhing tests of inflammatory pain. SPD showed no effects on compound action potential properties in a sciatic nerve preparation, suggesting that its mechanism of action is distinct from local anesthetics and membrane stabilizing drugs. SPD's activity in both neuropathic and inflammatory pain warrants its development as a potential broad-spectrum anti-nociceptive drug.

Model of anaesthetic induction by unilateral intracerebral microinjection of GABAergic agonists.

General anaesthetic agents induce loss of consciousness coupled with suppression of movement, analgesia and amnesia. Although these diverse functions are mediated by neural structures located in wide-ranging parts of the neuraxis, anaesthesia can be induced rapidly and reversibly by bilateral microinjection of minute quantities of γ-aminobutyric acid (GABA)A -R agonists at a small, focal locus in the mesopontine tegmentum (MPTA). State switching under these circumstances is presumably executed by dedicated neural pathways and does not require widespread distribution of the anaesthetic agent itself, the classical assumption regarding anaesthetic induction. Here it was asked whether these pathways serve each hemisphere independently, or whether there is bilateral redundancy such that the MPTA on each side is capable of anaesthetizing the entire brain. Either of two GABAA -R ligands were microinjected unilaterally into the MPTA in awake rats, the barbiturate modulator pentobarbital and the direct receptor agonist muscimol. Both agents, microinjected on either side, induced clinical anaesthesia, including bilateral atonia, bilateral analgesia and bilateral changes in cortical activity. The latter was monitored using c-fos expression and electroencephalography. This action, however, was not simply a consequence of suppressing spike activity in MPTA neurons, as unilateral (or bilateral) microinjection of the local anaesthetic lidocaine at the same locus failed to induce anaesthesia. A model of the state-switching circuitry that accounts for the bilateral action of unilateral microinjection and also for the observation that inactivation with lidocaine is not equivalent to inhibition with GABAA -R agonists was proposed. This is a step in defining the overall switching circuitry that underlies anaesthesia.

Mesopontine Switch for the Induction of General Anesthesia by Dedicated Neural Pathways.

We review evidence that the induction of anesthesia with GABAergic agents is mediated by a network of dedicated axonal pathways, which convey a suppressive signal to remote parts of the central nervous system. The putative signal originates in an anesthetic-sensitive locus in the brainstem that we refer to as the mesopontine tegmental anesthesia area (MPTA). This architecture stands in contrast to the classical notion that anesthetic molecules themselves directly mediate anesthetic induction after global distribution by the vascular circulation. The MPTA came to light in a systematic survey of the rat brain as a singular locus at which microinjection of minute quantities of GABAergic anesthetics is able to reversibly induce a state resembling surgical anesthesia. The rapid onset of anesthesia, the observed target specificity, and the fact that effective doses are far too small to survive dilution during vascular redistribution to distant areas in the central nervous system are all incompatible with the classical global suppression model. Lesioning the MPTA selectively reduces the animal's sensitivity to systemically administered anesthetics. Taken together, the microinjection data show that it is sufficient to deliver γ-aminobutyric acid A receptor (GABAA-R) agonists to the MPTA to induce an anesthesia-like state and the lesion data indicate that MPTA neurons are necessary for anesthetic induction by the systemic route at clinically relevant doses. Known connectivity of the MPTA provides a scaffold for defining the specific projection pathways that mediate each of the functional components of anesthesia. Because MPTA lesions do not induce coma, the MPTA is not a key arousal nucleus essential for maintaining the awake state. Rather, it appears be a "gatekeeper" of arousal function, a major element in a flip-flop switching mechanism that executes rapid and reversible transitions between the awake and the anesthetic state.

Does the Golem Feel Pain? Moral Instincts and Ethical Dilemmas Concerning Suffering and the Brain.

Pain has variously been used as a means of punishment, extracting information, or testing commitment, as a tool for education and social control, as a commodity for sacrifice, and as a draw for sport and entertainment. Attitudes concerning these uses have undergone major changes in the modern era. Normative convictions on what is right and wrong are generally attributed to religious tradition or to secular-humanist reasoning. Here, we elaborate the perspective that ethical choices concerning pain have much earlier roots that are based on instincts and brain-seated empathetic responses. They are fundamentally a function of brain circuitry shaped by processes of Darwinian evolution. Social convention and other environmental influences, with their idiosyncrasies, are a more recent, ever-changing overlay. We close with an example in which details on the neurobiology of pain processing, specifically the question of where in the brain the experience of pain is generated, affect decision making in end-of-life situations. By separating innate biological substrates from culturally imposed attitudes (memes), we may arrive at a more reasoned approach to a morality of pain prevention.

Sources of individual variability: miRNAs that predispose to neuropathic pain identified using genome-wide sequencing.

BACKGROUND: We carried out a genome-wide study, using microRNA sequencing (miRNA-seq), aimed at identifying miRNAs in primary sensory neurons that are associated with neuropathic pain. Such scans usually yield long lists of transcripts regulated by nerve injury, but not necessarily related to pain. To overcome this we tried a novel search strategy: identification of transcripts regulated differentially by nerve injury in rat lines very similar except for a contrasting pain phenotype. Dorsal root ganglia (DRGs) L4 and 5 in the two lines were excised 3 days after spinal nerve ligation surgery (SNL) and small RNAs were extracted and sequenced. RESULTS: We identified 284 mature miRNA species expressed in rat DRGs, including several not previously reported, and 3340 unique small RNA sequences. Baseline expression of miRNA was nearly identical in the two rat lines, consistent with their shared genetic background. In both lines many miRNAs were nominally up- or down-regulated following SNL, but the change was similar across lines. Only 3 miRNAs that were expressed abundantly (rno-miR-30d-5p, rno-miR-125b-5p) or at moderate levels (rno-miR-379-5p) were differentially regulated. This makes them prime candidates as novel PNS determinants of neuropathic pain. The first two are known miRNA regulators of the expression of Tnf, Bdnf and Stat3, gene products intimately associated with neuropathic pain phenotype. A few non-miRNA, small noncoding RNAs (sncRNAs) were also differentially regulated. CONCLUSIONS: Despite its genome-wide coverage, our search strategy yielded a remarkably short list of neuropathic pain-related miRNAs. As 2 of the 3 are validated regulators of important pro-nociceptive compounds, it is likely that they contribute to the orchestration of gene expression changes that determine individual variability in pain phenotype. Further research is required to determine whether some of the other known or predicted gene targets of these miRNAs, or of the differentially regulated non-miRNA sncRNAs, also contribute.

Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2.

Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca(2+) channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.

From molecule to oblivion: dedicated brain circuitry underlies anesthetic loss of consciousness permitting pain-free surgery.

The canonical view of how general anesthetics induce loss-of-consciousness (LOC) permitting pain-free surgery posits that anesthetic molecules, distributed throughout the CNS, suppress neural activity globally to levels at which the cerebral cortex can no longer sustain conscious experience. We support an alternative view that LOC, in the context of GABAergic anesthesia at least, results from anesthetic exposure of a small number of neurons in a focal brainstem nucleus, the mesopontine tegmental anesthesia area (MPTA). The various sub-components of anesthesia, in turn, are effected in distant locations, driven by dedicated axonal pathways. This proposal is based on the observations that microinjection of infinitesimal amounts of GABAergic agents into the MPTA, and only there, rapidly induces LOC, and that lesioning the MPTA renders animals relatively insensitive to these agents delivered systemically. Recently, using chemogenetics, we identified a subpopulation of MPTA "effector-neurons" which, when excited (not inhibited), induce anesthesia. These neurons contribute to well-defined ascending and descending axonal pathways each of which accesses a target region associated with a key anesthetic endpoint: atonia, anti-nociception, amnesia and LOC (by electroencephalographic criteria). Interestingly, the effector-neurons do not themselves express GABAA-receptors. Rather, the target receptors reside on a separate sub-population of presumed inhibitory interneurons. These are thought to excite the effectors by disinhibition, thus triggering anesthetic LOC.

Resurgent neuropathic discharge: an obstacle to the therapeutic use of neuroma resection?

ABSTRACT: Ectopic discharge ("ectopia") in damaged afferent axons is a major contributor to chronic neuropathic pain. Clinical opinion discourages surgical resection of nerves proximal to the original injury site for fear of resurgence of ectopia and exacerbated pain. We tested this concept in a well-established animal neuroma model. Teased-fiber recordings were made of ectopic spontaneous discharge originating in the experimental nerve-end neuroma and associated dorsal root ganglia in rats that underwent either a single transection (with ligation) of the sciatic nerve or 2 consecutive transections separated by 7, 14, 21, or 30 days. Ectopia emerged in afferent A and C fibers after a single cut with kinetics anticipated from previous studies. When resection was performed during the early period of intense A-fiber activity, a brief period of resurgence was observed. However, resection of neuromas of more than 14 days was followed by low levels of activity with no indication of resurgence. This remained the case in trials out to 60 days after the first cut. Similarly, we saw no indication of resurgent ectopia originating in axotomized dorsal root ganglion neuronal somata and no behavioral reflection of resurgence. In summary, we failed to validate the concern that proximal resection of a problematic nerve would lead to intense resurgent ectopic discharge and pain. As the well-entrenched concept of resurgence is based more on case reports and anecdotes than on solid evidence, it may be justified to relax the stricture against resecting neuromas as a therapeutic strategy, at least within the framework of controlled clinical trials.

A putative flip-flop switch for control of REM sleep.

Rapid eye movement (REM) sleep consists of a dreaming state in which there is activation of the cortical and hippocampal electroencephalogram (EEG), rapid eye movements, and loss of muscle tone. Although REM sleep was discovered more than 50 years ago, the neuronal circuits responsible for switching between REM and non-REM (NREM) sleep remain poorly understood. Here we propose a brainstem flip-flop switch, consisting of mutually inhibitory REM-off and REM-on areas in the mesopontine tegmentum. Each side contains GABA (gamma-aminobutyric acid)-ergic neurons that heavily innervate the other. The REM-on area also contains two populations of glutamatergic neurons. One set projects to the basal forebrain and regulates EEG components of REM sleep, whereas the other projects to the medulla and spinal cord and regulates atonia during REM sleep. The mutually inhibitory interactions of the REM-on and REM-off areas may form a flip-flop switch that sharpens state transitions and makes them vulnerable to sudden, unwanted transitions-for example, in narcolepsy.

Might pain be experienced in the brainstem rather than in the cerebral cortex?

It is nearly axiomatic that pain, among other examples of conscious experience, is an outcome of still-uncertain forms of neural processing that occur in the cerebral cortex, and specifically within thalamo-cortical networks. This belief rests largely on the dramatic relative expansion of the cortex in the course of primate evolution, in humans in particular, and on the fact that direct activation of sensory representations in the cortex evokes a corresponding conscious percept. Here we assemble evidence, drawn from a number of sources, suggesting that pain experience is unlike the other senses and may not, in fact, be an expression of cortical processing. These include the virtual inability to evoke pain by cortical stimulation, the rarity of painful auras in epileptic patients and outcomes of cortical lesions. And yet, pain perception is clearly a function of a conscious brain. Indeed, it is perhaps the most archetypical example of conscious experience. This draws us to conclude that conscious experience, at least as realized in the pain system, is seated subcortically, perhaps even in the "primitive" brainstem. Our conjecture is that the massive expansion of the cortex over the course of evolution was not driven by the adaptive value of implementing consciousness. Rather, the cortex evolved because of the adaptive value of providing an already existing subcortical generator of consciousness with a feed of critical information that requires the computationally intensive capability of the cerebral cortex.

Anesthetic loss of consciousness induced by chemogenetic excitation of mesopontine effector neurons.

Although general anesthesia is normally induced by systemic dosing, an anesthetic state can be induced in rodents by microinjecting minute quantities of GABAergic agents into the brainstem mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesions to the MPTA render rats relatively insensitive to standard anesthetic doses delivered systemically. Using a chemogenetic approach we have identified and characterized a small subpopulation of neurons restricted to the MPTA which, when excited, render the animal anesthetic by sensorimotor (immobility) and electroencephalographic (EEG) criteria. These "effector-neurons" do not express GABAAδ-Rs, the likely target of GABAergic anesthetics. Rather, we report a distinct sub-population of nearby MPTA neurons which do. During anesthetic induction these likely excite the effector-neurons by disinhibition. Within the effector population ~ 70% appear to be glutamatergic, ~30% GABAergic and ~ 40% glycinergic. Most are projection neurons that send ascending or descending axons to distant targets associated with the individual functional components of general anesthesia: atonia, analgesia, amnesia, and loss-of-consciousness.

Paradoxical anesthesia: Sleep-like EEG during anesthesia induced by mesopontine microinjection of GABAergic agents.

General anesthetic agents are thought to induce loss-of-consciousness (LOC) and enable pain-free surgery by acting on the endogenous brain circuitry responsible for sleep-wake cycling. In clinical use, the entire CNS is exposed to anesthetic molecules with LOC and amnesia usually attributed to synaptic suppression in the cerebral cortex and immobility and analgesia to agent action in the spinal cord and brainstem. This model of patch-wise suppression has been challenged, however, by the observation that all functional components of anesthesia can be induced by focal delivery of minute quantities of GABAergic agonists to the brainstem mesopontine tegmental anesthesia area (MPTA). We compared spectral features of the cortical electroencephalogram (EEG) in rats during systemic anesthesia and anesthesia induced by MPTA microinjection. Systemic administration of (GABAergic) pentobarbital yielded the sustained, δ-band dominant EEG signature familiar in clinical anesthesia. In contrast, anesthesia induced by MPTA microinjection (pentobarbital or muscimol) featured epochs of δ-band EEG alternating with the wake-like EEG, the pattern typical of natural non-rapid-eye-movement (NREM) and REM sleep. The rats were not sleeping, however, as they remained immobile, atonic and unresponsive to noxious pinch. Recalling the paradoxical wake-like quality the EEG during REM sleep, we refer to this state as "paradoxical anesthesia". GABAergic anesthetics appear to co-opt both cortical and spinal components of the sleep network via dedicated axonal pathways driven by MPTA neurons. Direct drug exposure of cortical and spinal neurons is not necessary, and is probably responsible for off-target side-effects of systemic administration including monotonous δ-band EEG, hypothermia and respiratory depression. SIGNIFICANCE STATEMENT: The concept that GABAergic general anesthetic agents induce loss-of-consciousness by substituting for an endogenous neurotransmitter, thereby co-opting neural circuitry responsible for sleep-wake transitions, has gained considerable traction. However, the electroencephalographic (EEG) signatures of sleep and anesthesia differ fundamentally. We show that when the anesthetic state is generated by focal delivery of GABAergics into the mesopontine tegmental anesthesia area (MPTA) the resulting EEG repeatedly transitions between delta-wave-dominant and wake-like patterns much as in REM-NREM sleep. This suggests that systemic (clinical) anesthetic delivery, which indiscriminately floods the entire cerebrum with powerful inhibitory agents, obscures the sleep-like EEG signature associated with the less adulterated form of anesthesia obtained when the drugs are applied selectively to loci where the effective neurotransmitter substitution actually occurs.

Cerebral activity during the anesthesia-like state induced by mesopontine microinjection of pentobarbital.

Microinjection of pentobarbital into a restricted region of rat brainstem, the mesopontine tegmental anesthesia area (MPTA), induces a reversible anesthesia-like state characterized by loss of the righting reflex, atonia, antinociception, and loss of consciousness as assessed by electroencephalogram synchronization. We examined cerebral activity during this state using FOS expression as a marker. Animals were anesthetized for 50 min with a series of intracerebral microinjections of pentobarbital or with systemic pentobarbital and intracerebral microinjections of vehicle. FOS expression was compared with that in awake animals microinjected with vehicle. Neural activity was suppressed throughout the cortex whether anesthesia was induced by systemic or MPTA routes. Changes were less consistent subcortically. In the zona incerta and the nucleus raphe pallidus, expression was strongly suppressed during systemic anesthesia, but only mildly during MPTA-induced anesthesia. Dissociation was seen in the tuberomammillary nucleus where suppression occurred during systemic-induced anesthesia only, and in the lateral habenular nucleus where activity was markedly increased during systemic-induced anesthesia but not following intracerebral microinjection. Several subcortical nuclei previously associated with cerebral arousal were not affected. In the MPTA itself FOS expression was suppressed during systemic anesthesia. Differences in the pattern of brain activity in the two modes of anesthesia are consistent with the possibility that anesthetic endpoints might be achieved by alternative mechanisms: direct drug action for systemic anesthesia or via ascending pathways for MPTA-induced anesthesia. However, it is also possible that systemically administered agents induce anesthesia, at least in part, by a primary action in the MPTA with cortical inhibition occurring secondarily.

Expression of DRG candidate pain molecules after nerve injury--a comparative study among five inbred mouse strains with contrasting pain phenotypes.

Neuropathic pain that develops after trauma to a nerve may be caused by altered transcription of genes in the damaged neurons. We have previously investigated the effect of nerve injury on the expression of six dorsal root ganglion (DRG) pain candidate molecules in five inbred mouse strains with different pain phenotypes after nerve injury. In this study, we present a detailed morphological examination of mRNA expression in the DRG in the same mouse strains. For Na(v) 1.9, TRPA1, and TRPM8, the size spectra of labeled neurons remained mostly unchanged after injury in all strains. However, in CBA, AKR, and C58 mice, injury caused a preferential downregulation of Na(v) 1.8 in large diameter neurons. In CBA mice there was a shift toward larger neuronal profiles expressing TRPV1 after injury, indicating de novo (or upregulated) expression of TRPV1 in a subpopulation of neurons that normally does not express this gene. Finally, in C58 mice there was a shift toward smaller P2X3-expressing neuronal profiles after injury, suggesting that a loss of P2X3 mRNA transcript occurred preferentially in medium-sized cells. We used a multivariate statistical model to compare the regulation patterns of the six DRG genes. Clustering patterns suggested that genes of similar phylogenetic origin and function are regulated similarly.

Patterns of neural activity in the mouse brain: Wakefulness vs. General anesthesia.

In light of the general shift from rats to mice as the leading rodent model in neuroscience research we used c-Fos expression as a tool to survey brain regions in the mouse in which neural activity differs between the states of wakefulness and pentobarbital-induced general anesthesia. The aim was to complement prior surveys carried out in rats. In addition to a broad qualitative review, 28 specific regions of interest (ROIs) were evaluated quantitatively. Nearly all ROIs in the cerebral cortex showed suppressed activity during anesthesia. Subcortically, however, some ROIs showed suppression, some showed little change, and some showed increased activity. The overall picture was similar to the rat. Special attention was devoted to ROIs significantly activated during anesthesia, as such loci might actively drive the transition to anesthetic unconsciousness rather than responding passively to inhbitory agents distributed globally (the "wet blanket" hypothesis). Twelve such "anesthesia-on" ROIs were identified: the paraventricular hypothalamic nucleus, supraoptic nucleus, tuberomamillary nucleus, lateral habenular nucleus, dentate gyrus, nucleus raphe pallidus, central amygdaloid nucleus, perifornical lateral hypothalamus, ventro-lateral preoptic area, lateral septum, paraventricular thalamic nucleus and zona incerta. The same primary anti-FOS antibody was used in all mice, but two alternative reporter systems were employed: ABC-diaminobenzidine and the currently more popular AlexaFluor488. Fluorescence tagging revealed far fewer FOS-immunoreactive neurons, sounding an alert that the reporter system chosen can have major effects on results obtained.