Discharge and Role of GABA Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recordings in Mice.

The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-(ChR2)-EYFP in vesicular GABA transporter (VGAT)-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep.SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states.

Discharge profiles across the sleep-waking cycle of identified cholinergic, GABAergic, and glutamatergic neurons in the pontomesencephalic tegmentum of the rat.

Distributed within the laterodorsal tegmental and pedunculopontine tegmental nuclei (LDT and PPT), cholinergic neurons in the pontomesencephalic tegmentum have long been thought to play a critical role in stimulating cortical activation during waking (W) and paradoxical sleep (PS, also called REM sleep), yet also in promoting PS with muscle atonia. However, the discharge profile and thus precise roles of the cholinergic neurons have remained uncertain because they lie intermingled with GABAergic and glutamatergic neurons, which might also assume these roles. By applying juxtacellular recording and labeling in naturally sleeping-waking, head-fixed rats, we investigated the discharge profiles of histochemically identified cholinergic, GABAergic, and glutamatergic neurons in the LDT, SubLDT, and adjoining medial part of the PPT (MPPT) in relation to sleep-wake states, cortical activity, and muscle tone. We found that all cholinergic neurons were maximally active during W and PS in positive correlation with fast (γ) cortical activity, as "W/PS-max active neurons." Like cholinergic neurons, many GABAergic and glutamatergic neurons were also "W/PS-max active." Other GABAergic and glutamatergic neurons were "PS-max active," being minimally active during W and maximally active during PS in negative correlation with muscle tone. Conversely, some glutamatergic neurons were "W-max active," being maximally active during W and minimally active during PS in positive correlation with muscle tone. Through different discharge profiles, the cholinergic, GABAergic, and glutamatergic neurons of the LDT, SubLDT, and MPPT thus appear to play distinct roles in promoting W and PS with cortical activation, PS with muscle atonia, or W with muscle tone.

Chloride and lactate as prognostic indicators of calf diarrhea from eighty-nine cases.

IMPORTANCE: Deaths due to neonatal calf diarrhea are still one of the most critical problems of cattle breeding worldwide. Determining the parameters that can predict diarrhea-related deaths in calves is especially important in terms of prognosis and treatment strategies for the disease. OBJECTIVE: The primary purpose of this study was to determine mortality rates and durations, survival status, and predictive prognosis parameters based on vital signs, hematology, and blood gas analyses in neonatal diarrheic calves. METHODS: The hospital automation system retrospectively obtained data from 89 neonatal diarrheic calves. RESULTS: It was found that 42.7% (38/89) of the calves brought with the complaint of diarrhea died during hospitalization or after discharge. Short-term and long-term fatalities were a median of 9.25 hours and a median of 51.50 hours, respectively. When the data obtained from this study is evaluated, body temperature (°C), pH, base excess (mmol/L), and sodium bicarbonate (mmol/L) parameters were found to be lower, and hemoglobin (g/dL), hematocrit (%), lactate (mmol/L), chloride (mmol/L), sodium (mmol/L) and anion gap (mmol/L) parameters were found to be higher in dead calves compared to survivors. Accordingly, hypothermia, metabolic acidosis, and dehydration findings were seen as clinical conditions that should be considered. Logistic regression analysis showed that lactate (odds ratio, 1.429) and CI- (odds ratio, 1.232) concentration were significant risk factors associated with death in calves with diarrhea. CONCLUSIONS AND RELEVANCE: According to the findings obtained from this study, the determination of lactate and Cl- levels can be used as an adjunctive supplementary test in distinguishing calves with diarrhea with a good prognosis.

Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza.

BACKGROUND: Severe influenza is characterized by cytokine storm and multiorgan failure with edema. The aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. METHODS: Weanling mice were infected with influenza A WSN/33(H1N1) virus. The levels of proinflammatory cytokines, tumor necrosis factor (TNF) alpha, interleukin (IL) 6, IL-1beta, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. The effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. RESULTS: Influenza A virus infection resulted in significant increases in TNF-alpha, IL-6, IL-1beta, viral hemagglutinin-processing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. Trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. Calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokine- and trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. CONCLUSIONS: The influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza.

Discharge and Role of Acetylcholine Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recording in Mice.

Acetylcholine (ACh) neurons in the pontomesencephalic tegmentum (PMT) are thought to play an important role in promoting cortical activation with waking (W) and paradoxical sleep [PS; or rapid eye movement (REM)], but have yet to be proven to do so by selective stimulation and simultaneous recording of identified ACh neurons. Here, we employed optogenetics combined with juxtacellular recording and labeling of neurons in transgenic (TG) mice expressing ChR2 in choline acetyltransferase (ChAT)-synthesizing neurons. We established in vitro then in vivo in anesthetized (A) and unanesthetized (UA), head-fixed mice that photostimulation elicited a spike with short latency in neurons which could be identified by immunohistochemical staining as ACh neurons within the laterodorsal (LDT)/sublaterodorsal (SubLDT) and pedunculopontine tegmental (PPT) nuclei. Continuous light pulse stimulation during sleep evoked tonic spiking by ACh neurons that elicited a shift from irregular slow wave activity to rhythmic θ and enhanced γ activity on the cortex without behavioral arousal. With θ frequency rhythmic light pulse stimulation, ACh neurons discharged in bursts that occurred in synchrony with evoked cortical θ. During natural sleep-wake states, they were virtually silent during slow wave sleep (SWS), discharged in bursts during PS and discharged tonically during W. Yet, their bursting during PS was not rhythmic or synchronized with cortical θ but associated with phasic whisker movements. We conclude that ACh PMT neurons promote θ and γ cortical activity during W and PS by their tonic or phasic discharge through release of ACh onto local neurons within the PMT and/or more distant targets in the hypothalamus and thalamus.

Oseltamivir reduces hippocampal abnormal EEG activities after a virus infection (influenza) in isoflurane-anesthetized rats.

BACKGROUND: Oseltamivir phosphate (OP, Tamiflu(®)) is a widely used drug in the treatment of influenza with fever. However, case reports have associated OP intake with sudden abnormal behaviors. In rats infected by the influenza A virus (IAV), the electroencephalogram (EEG) displayed abnormal high-voltage amplitudes with spikes and theta oscillations at a core temperature of 39.9°C to 41°C. Until now, there has been no information describing the effect of OP on intact brain hippocampal activity of IAV-infected animals during hyperthermia. OBJECTIVE: The aim of the present study was to examine the effect of OP on abnormal EEG activities in the hippocampus using the rat model of influenza-associated encephalopathy. METHODS: Male Wistar rats aged 3 to 4 weeks were used for the study. Influenza A/WSN/33 strain (1 × 10(5) plaque forming unit in PBS, 60 µL) was applied intranasally to the rats. To characterize OP effects on the IAV-infected rats, EEG activity was studied more particularly in isoflurane-anesthetized IAV-infected rats during hyperthermia. RESULTS: We found that the hippocampal EEG of the OP-administered (10 mg/kg) IAV-infected rats showed significant reduction of the high-voltage amplitudes and spikes, but the theta oscillations, which had been observed only at >40°C in OP non-administered rats, appeared at 38°C core temperature. Atropine (30 mg/kg) blocked the theta oscillations. CONCLUSION: Our data suggest that OP efficiently reduces the abnormal EEG activities after IAV infection during hyperthermia. However, OP administration may stimulate ACh release in rats at normal core temperature.

Mechanisms of matrix metalloproteinase-9 upregulation and tissue destruction in various organs in influenza A virus infection.

Severe influenza is characterized clinicopathologically by multiple organ failure, although the relationship amongst virus and host factors that influence this morbid outcome and the underlying mechanisms of action remain unclear. The present study identified marked upregulation of matrix metalloproteinase (MMP)-9 and pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in various organs after intranasal infection of influenza A WSN virus. MMP-9 and TNF-alpha were upregulated in the lung, the site of initial infection, as well as in the brain and heart. The infection-induced MMP-9 upregulation was inhibited by anti-TNF-alpha antibodies and by anti-oxidative reagents pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, which inhibit activation of nuclear factor kappa B (NF-kappaB), as well as by nordihydroguaiaretic acid, which inhibits activation of activator protein 1 (AP-1). In addition, MMP-9 upregulation via TNF-alpha was also suppressed by inhibitors of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 and p38, and partly by a c-Jun N-terminal kinase inhibitor. These results indicated that the influenza-induced MMP-9 upregulation in various organs is mediated through MAPK-NF-kappaB- and/or AP-1-dependent mechanisms. Strategies that neutralize TNF-alpha as well as inhibitors of MAPK-NF-kappa B- and/or AP-1-dependent pathways may be useful for suppressing the MMP-9 effect and thus preventing multiple organ failure in severe influenza.

EPSP depression following neocortical seizures in cat.

To study the possible mechanism(s) underlying unresponsiveness following neocortical seizures, we recorded excitatory postsynaptic potentials (EPSPs) of cortical neurons evoked by ipsilateral cortical stimulation before and after spontaneous or elicited seizures. Regular-spiking neurons (n = 32) were intracellularly recorded in association area five of cats under ketamine-xylazine or barbiturate anesthesia. Compared with control responses, cortically evoked EPSPs were characterized by decreased amplitude after electrographic seizures. Synaptic responses and intrinsic properties were measured by applying extracellular electrical stimuli followed by intracellular hyperpolarizing current pulses. The input resistance decreased during seizures but quickly recovered to control level after the paroxysms, whereas the amplitude of evoked EPSPs remained lower following seizures, generally for 2-12 min, suggesting that the decreased EPSPs were not due to an alteration of intrinsic response. Data demonstrate a long-lasting decreased synaptic responsiveness following generalized spike-wave seizures slowly recovering in time.

State-dependent slow outlasting activities following neocortical kindling in cats.

Some forms of electrographic seizures are generated at the level of the cortical network. Neocortical kindling exhibits a resistance to produce generalized convulsive seizures, and therefore, it was rather difficult to use it to study the cortical epileptogenesis. Here, using supra-threshold cortical kindling, we report electrophysiological patterns of field-potential synchronization and intracellular activities in chronically implanted non-anesthetized cats, during different states of vigilance, and during acute seizures elicited by prolonged (20-60 s) electrical stimulation. Acute seizures were easily elicited during transition from slow-wave sleep (SWS) to waking state. The seizures were mainly clonic accompanied with tonic components followed by prolonged postictal depression. Delayed rhythmic outlasting activities (OA) at approximately 1.5 Hz, first time reported here, followed the postictal depression, and lasted up to 2 h. These activities were clear during waking state, slightly reduced during SWS and completely absent during rapid-eye movement sleep. They started focally and following daily stimulations generalized over the entire cortical surface. Extra- and intracellular neuronal recordings during OA displayed spike-doublets, built on the summation of successive excitatory postsynaptic potentials and fast-prepotentials, entailing an increased dendritic excitation. Our results suggest that such rhythmic long-lasting oscillatory activity outlasting seizures are the key factor of epileptogenesis, leading to epilepsy.

Cortical and thalamic components of neocortical kindling-induced epileptogenesis in behaving cats.

Kindling is an essential operating paradigm of the nervous system extensively used both as a model of epileptogenesis and neuroplasticity. In a parallel study conducted on chronically implanted non-anesthetized kindled cats, we report the occurrence of long-lasting slow oscillatory patterns (1.5-2 Hz) called outlasting activities (OA) following the acute seizures (AS) induced by cortical stimulation. Here, we asked if OA observed in the neocortex of kindled animals are generated exclusively by the cortical networks or if they also rely on the burst firing of thalamic neurons. We analyzed the electrophysiological patterns of synchronization of cortical EEG (areas 4, 5, 7, 21, 17, 18, 22) and thalamic field (EThG) (ventral posterior lateral nucleus-VPL), and the influence of modulatory systems originating in the pedunculo-pontine tegmentum (PPT) and locus coeruleus (LC) on the discharge pattern of thalamic neurons during OA. Synchrony analysis of field recordings showed that during AS cortical paroxysmal activities preceded thalamic ones, while during OA this sequential order was reversed. During OA thalamic neurons regularly discharged bursts with the frequency of OA. Electrical stimulation of either PPT or LC during OA decreased both the probability of bursts in thalamocortical neurons and the amplitude of OA. Yet, neither of them was able to block completely the expression of OA. Following PPT/LC stimulation the burst firing of thalamocortical neurons was replaced by tonic firing. We conclude that the thalamus is involved in the mechanism of generation of OA but that it does not play an exclusive role.

Waking-sleep modulation of paroxysmal activities induced by partial cortical deafferentation.

We investigated the dependency of electrical seizures produced by cortical undercut upon behavioral states of vigilance in chronically implanted cats. Experiments were performed 1-12 weeks after white matter transection. Multisite field potentials and intracellular activity were recorded from suprasylvian and marginal gyri. Paroxysmal activity developed within days and consisted of spike-wave complexes at 3-4 Hz occurring during the waking state (correlated with eye movements), being enhanced during slow-wave sleep (SWS) and blocked during rapid eye movement (REM) sleep. Prolonged hyperpolarizing events were seen not only during SWS (which is the case in normal animals) but also during both waking and REM, thus resulting in bimodal distribution of the membrane potential in all 3 natural states of vigilance. The increased synchrony of field potential activity expressed by shorter time of propagation over the cortical surface and the tendency toward generalization are ascribed to changes in intrinsic neuronal properties and potential disinhibition following cortical undercut.

Increased propensity to seizures after chronic cortical deafferentation in vivo.

Cortical injury may lead to clinical seizures. We investigated the changing patterns of the sleeplike slow oscillation and its tendency to develop into paroxysmal activity consisting of spike-wave (SW) complexes at 2-4 Hz after partial deafferentation of the suprasylvian gyrus. Experiments were carried out in anesthetized cats, at different time intervals (wk 1 to wk 5, W1-W5) after cortical undercut. Multisite field potentials and single or dual intracellular recordings from the whole extent of the deafferented gyrus were used. The field components of the slow oscillation increased in amplitudes and were transformed into paroxysmal patterns, expressed by increased firing rates and tendency to neuronal bursting. The incidence of SW seizures was higher with transition from semiacute (W1) to chronic (W2-W5) stages after cortical undercut. The propagation delay of low-frequency activities decreased from W1 to W5, during both the slow oscillation and seizures. The initiation of seizures took place in territories contiguous to the relatively intact cortex (area 5 in the anterior part of the gyrus), as shown by cross-correlations of field potentials from different sites and simultaneous intracellular recordings from the anterior and posterior parts of the gyrus. The increased amplitudes of both slow oscillation and SW seizures, and their enhanced synchrony expressed by shorter time of propagation, are ascribed to increased neuronal and network excitability after cortical undercut.

Synaptic plasticity in local cortical network in vivo and its modulation by the level of neuronal activity.

Neocortical neurons maintain high firing rates across all behavioral states of vigilance but the discharge patterns vary during different types of brain oscillations, which are assumed to play an important role in information processing and memory consolidation. In the present study, we report that trains of stimuli applied to local neocortical networks of cats, at frequencies that mimic endogenous brain rhythms, produced depression or potentiation of postsynaptic potentials, which lasted for several minutes. This form of synaptic plasticity was not mediated through NMDA receptors since it persisted after blockade of these receptors, but was strongly modulated by the level of background neuronal activity. Using different preparations in vivo, we found that increased background neuronal activity decreased the probability of plastic changes but enhanced the probability of potentiation over depression. Conversely, when the level of background neuronal activity was low, plasticity was observed in all neurons, but mainly depression was induced. Our results demonstrate that high levels of neuronal activity in the cortical network promote potentiation and insure the stability of synaptic connections.

Synaptic enhancement induced through callosal pathways in cat association cortex.

The corpus callosum plays a major role in synchronizing neocortical activities in the two hemispheres. We investigated the changes in callosally elicited excitatory postsynaptic potentials (EPSPs) of neurons from cortical association areas 5 and 7 of cats under barbiturate or ketamine-xylazine anesthesia. Single pulses to callosal pathway evoked control EPSPs; pulse-trains were subsequently applied at different frequencies to homotopic sites in the contralateral cortex, as conditioning stimulation; thereafter, the single pulses were applied again to test changes in synaptic responsiveness by comparing the amplitudes of control and conditioned EPSPs. In 41 of 42 neurons recorded under barbiturate anesthesia, all frequencies of conditioning callosal stimuli induced short-term (5-30 min) enhancement of test EPSPs elicited by single stimuli. Neurons tested with successive conditioning pulse-trains at different frequencies displayed stronger enhancement with high-frequency (40-100 Hz) than with low-frequency (10-20 Hz) rhythmic pulse-trains; >100 Hz, the potentiation saturated. In a neuronal sample, microdialysis of an N-methyl-D-aspartate (NMDA) receptor blocker in barbiturate-treated cats suppressed this potentiation, and potentiation of callosally evoked EPSPs was not detected in neurons recorded under ketamine-xylazine anesthesia, thus indicating that EPSPs' potentiation implicates, at least partially, NMDA receptors. These data suggest that callosal activities occurring within low-frequency and fast-frequency oscillations play a role in cortical synaptic plasticity.

Electrophysiological properties and input-output organization of callosal neurons in cat association cortex.

Intracellular recordings from association cortical areas 5 and 7 were performed in cats under barbiturate or ketamine-xylazine anesthesia to investigate the activities of different classes of neurons involved in callosal pathways, which were electrophysiologically characterized by depolarizing current steps. Excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), and/or antidromic responses were elicited by stimulating homotopic sites in the contralateral cortical areas. Differential features of EPSPs related to latencies, amplitudes, and slopes were detected in closely located (50 microm or less) neurons recorded in succession along the same electrode track. In contrast to synchronous thalamocortical volleys that excited most neurons within a cortical column, stimuli applied to homotopic sites in the contralateral cortex activated neurons at restricted cortical depths. Median latencies of callosally evoked EPSPs were 1.5 to 4 ms in various cortical cell-classes. Fast-rhythmic-bursting neurons displayed EPSPs whose amplitudes were threefold larger, and latencies two- or threefold shorter, than those found in the three other cellular classes. Converging callosal and thalamic inputs were recorded in the same cortical neuron. EPSPs or IPSPs were elicited by stimulating foci spaced by <1 mm in the contralateral cortex. In the overwhelming majority of neurons, latencies of antidromic responses were between 1.2 and 3.1 ms; however, some callosal neurons had much longer latencies,