The Nature of Noradrenergic Volume Transmission From Locus Coeruleus to Brainstem Mesencephalic Trigeminal Sensory Neurons

Noradrenergic neurons in the locus coeruleus (LC) release noradrenaline (NA) that acts via volume transmission to activate extrasynaptic G-protein coupled receptors (GPCRs) in target cells throughout the brain. As the closest projection, the dorsal LC laterally adjoins the mesencephalic trigeminal nucleus (MTN), in which proprioceptive primary sensory neurons innervating muscle spindles of jaw-closing muscles are exceptionally located. MTN neurons express α2-adrenergic receptors (α2-ARs) and display hyperpolarization-activated cyclic nucleotide-gated (HCN) currents (Ihs), which is downregulated by α2-AR activation. To quantify the activity-dependent outcome of volume transmission of NA from LC to MTN, we investigated how direct LC activation inhibits Ih in MTN neurons by performing dual whole-cell recordings from LC and MTN neurons. Repetition of 20 Hz spike-train evoked with 1-s current-pulse in LC neurons every 30 s resulted in a gradual decrease in Ih evoked every 30 s, revealing a Hill-type relationship between the number of spike-trains in LC neurons and the degree of Ih inhibition in MTN neurons. On the other hand, when microstimulation was applied in LC every 30 s, an LC neuron repeatedly displayed a transient higher-frequency firing followed by a tonic firing at 5–10 Hz for 30 s. This subsequently caused a similar Hill-type inhibition of Ih in the simultaneously recorded MTN neuron, but with a smaller Hill coefficient, suggesting a lower signal transduction efficacy. In contrast, 20 Hz activity induced by a 1-s pulse applied every 5–10 s caused only a transient facilitation of Ih inhibition followed by a forced termination of Ih inhibition. Thus, the three modes of LC activities modulated the volume transmission to activate α2-adrenergic GPCR to differentially inhibit Ih in MTN neurons.

Noradrenergic neurons in the locus coeruleus (LC) release noradrenaline (NA) that acts via volume transmission to activate extrasynaptic G-protein coupled receptors (GPCRs) in target cells throughout the brain. As the closest projection, the dorsal LC laterally adjoins the mesencephalic trigeminal nucleus (MTN), in which proprioceptive primary sensory neurons innervating muscle spindles of jaw-closing muscles are exceptionally located. MTN neurons express α 2 -adrenergic receptors (α 2 -ARs) and display hyperpolarization-activated cyclic nucleotide-gated (HCN) currents (Ihs), which is downregulated by α 2 -AR activation. To quantify the activity-dependent outcome of volume transmission of NA from LC to MTN, we investigated how direct LC activation inhibits Ih in MTN neurons by performing dual whole-cell recordings from LC and MTN neurons. Repetition of 20 Hz spike-train evoked with 1-s current-pulse in LC neurons every 30 s resulted in a gradual decrease in Ih evoked every 30 s, revealing a Hill-type relationship between the number of spike-trains in LC neurons and the degree of Ih inhibition in MTN neurons. On the other hand, when microstimulation was applied in LC every 30 s, an LC neuron repeatedly displayed a transient higher-frequency firing followed by a tonic firing at 5-10 Hz for 30 s. This subsequently caused a similar Hilltype inhibition of Ih in the simultaneously recorded MTN neuron, but with a smaller Hill coefficient, suggesting a lower signal transduction efficacy. In contrast, 20 Hz activity induced by a 1-s pulse applied every 5-10 s caused only a transient facilitation of Ih inhibition followed by a forced termination of Ih inhibition. Thus, the three modes of LC activities modulated the volume transmission to activate α 2 -adrenergic GPCR to differentially inhibit Ih in MTN neurons.

INTRODUCTION
It is well known that noradrenaline (NA) released from noradrenergic neurons in the locus coeruleus (LC) acts via volume transmission, which is the diffusion of neurotransmitter released at points that may be remote from the target cells with the resulting activation of non/extra-synaptic receptors with a much longer time course than those in synaptic transmission (Bach-y-Rita, 1993;Agnati et al., 1995;Zoli and Agnati, 1996;Taber and Hurley, 2014). Such a prolonged transmitter action, called tonic transmission, activates nonsynaptic G-protein coupled receptors (GPCRs), in contrast to the phasic transmission that occurs rapidly at narrow synaptic cleft of single synapses (Agnati et al., 1995;Zoli et al., 1999;Farrant and Nusser, 2005;Sarter et al., 2009). LC neurons typically maintain a tonic spontaneous firing at 0.5-2 Hz, while LC neurons respond with phasic or burst firing at ∼20 Hz when the animal is exposed to salient stimuli, resulting in NA release in its target area such as the prefrontal cortex (PFC) (Florin-Lechner et al., 1996;Devoto et al., 2005). Such a differential activity of LC neurons may affect the outcome of volume transmission in target cells.
However, there has been no attempt to quantify or characterize the activity-dependent outcome resulting from GPCR activation and the subsequent signal transduction in target neurons following volume transmission from the source to target neurons. This is largely because the target pyramidal neurons in the PFC that have been most extensively studied in in vitro or in vivo preparations are located too remotely from the source LC neurons. Such a remote and diffuse projection makes it very difficult to find a functionally connected pair of LC and PFC neurons in in vivo preparations, and hence is also not suitable for simultaneously controlling and detecting the activity of LC neurons and the intracellular signal transduction in PFC pyramidal neurons, respectively. The closest projection site from LC may be the mesencephalic trigeminal nucleus (MTN), as MTN neurons receive dense noradrenergic fibers arising from the medially adjoining LC (Takahashi et al., 2010). In addition, nonsynaptic α 2 -adrenergic receptor (AR) has been detected in MTN neurons (Aoki et al., 1994;Scheinin et al., 1994). In the brainstem MTN, there exceptionally exists proprioceptive primary sensory neurons that innervate muscle spindles of jaw-closing muscles, thereby receiving various synaptic inputs from the central nervous system (CNS) and onto diverse neurotransmitter receptors (Copray et al., 1990;Lazarov, 2002). It is known that MTN neurons display prominent hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel currents (Ih) (Khakh and Henderson, 1998;Kang et al., 2004), and that HCN1/2 are robustly expressed in the soma of MTN neurons . Therefore, it may be possible that Abbreviations: ACSF, artificial cerebrospinal fluid; α 2 -AR, α 2 -adrenergic receptor; DBH, dopamine beta hydroxylase; GIRK, G protein coupled inward rectifier K + current; GPCR, G-protein coupled receptors; HCN, hyperpolarization-activated cyclic nucleotide-gated; Ih, hyperpolarization-activated, cyclic nucleotide-gated HCN channel current; LC, locus coeruleus; LDCV, large-dense core vesicle; MTN, mesencephalic trigeminal nucleus; NA, noradrenaline; nH, Hill coefficient; PB, phosphate buffer; PBS, phosphate buffered saline; PFC, prefrontal cortex; RT, room temperature; TH, tyrosine hydroxylase. noradrenergic inputs from the LC participates in the modulation of Ih in MTN neurons by α 2 -AR activation (Copray et al., 1990;Scheinin et al., 1994;Rosin et al., 1996). Thus, the projection from LC to MTN maintained in in vitro slice preparations may be suitable to investigate the activity-dependent outcome of the volume transmission of NA.
In the present study, we addressed whether and how volume transmission of NA occurs from LC neurons to modify Ih in MTN neurons by performing dual whole-cell recordings from LC and MTN neurons. It was found that there was a nonlinear relationship between the cumulative numbers of current pulses repetitively injected in LC neurons every 30 s to induce 20 Hz spike trains and the degree of inhibition of Ih in MTN neurons. This non-linear relationship obtained in response to the periodic phasic firings at 20 Hz was best described by the Hill equation with a Hill coefficient (nH) of approximately 1.5, which reflects the sensitivity to input spikes in LC neurons. However, the nH was variable depending on the mode of activity in LC neurons: smaller for the tonic firings at 5-10 Hz than for the periodic phasic firings at 20 Hz. In contrast, the periodic phasic firing evoked every < 10 s can forcibly terminate the Ih inhibition after transient enhancement of Ih inhibition. Thus, the volume transmission of NA from LC to MTN, accompanied by α 2 -adrenergic GPCR activation, is dynamically and differentially controlled by the three distinct patterns of firing activity of LC neurons.

MATERIALS AND METHODS
All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Osaka University, Seoul National University, and by the institutional Animal Welfare and Ethical Review Body at University of Bristol. All experiments were performed in accordance with the relevant guidelines including the UK Animals in Scientific Procedures Act 1986.

Procedure for Immunohistochemical Examination
In three C57BL6/J mice at postnatal day (PD) 21, noradrenergic projections from LC to MTN neurons were visualized by labeling with anti-tyrosine hydroxylase (TH) antibody and anti-advillin antibody, as advillin is known to be a marker for primary sensory neurons (Hasegawa et al., 2007). The mice were sacrificed by deep anesthesia with sodium pentobarbital (80 mg/kg, i.p.) and perfused transcardially with 5 ml of heparinized normal saline, followed by 10 ml of a freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brainstem, including MTN and LC (-9 to -10 mm from bregma), was dissected out and immersed in 4% paraformaldehyde solution for overnight at 4 • C.

Electrophysiological Recordings and Microstimulation
Using two Axopatch 200B preamplifiers (MDS Analytical Technologies, Sunnyvale, CA, United States), dual whole-cell recordings under voltage-clamp and current-clamp conditions were respectively made from MTN and LC neurons that were viewed under Nomarski optics (BX51WI; Olympus, Tokyo, Japan). The distance between the two tips of patch pipettes for dual recordings from a pair of neurons in MTN and medially adjoining dorsal LC was between 100 and 200 µm. The recording chamber, with a volume of 1.0 ml, was continuously perfused with the extracellular solution (N-aCSF) at a flow rate of 1.0-1.5 ml/min. The internal solution of the patch pipettes had the following ionic composition (in mM): 123 K-gluconate, 18 KCl, 10 NaCl, 2 MgCl 2 , 2 ATP-Na 2 , 0.3 GTP-Na 3 , 10 HEPES, and 0.2 EGTA; with pH 7.3 adjusted with KOH. The membrane potential values given in the text were corrected for the junction potential (10 mV) between the internal solution for the whole-cell recording (negative) and the standard extracellular solution. The pipette resistances were 4-6 M . The series resistance was < 10 M . All recordings were made at RT. Records of currents and voltages were low-pass filtered at 5 kHz (3-pole Bessel filter), digitized at a sampling rate of 10 kHz (Digidata 1322A, MDS Analytical Technologies) and stored on a computer hard disk.
The amplitudes of Ih were measured 10-20 ms before the offset of the negative voltage step (Kang et al., 1994;Tanaka et al., 2003). To isolate Ih or to prevent Ih from being contaminated with other ionic currents, any blocker was not used because of two reasons. First, we did not measure tail current of Ih because low voltage activated currents may contaminate with the tail-Ih at the offset of negative command pulses. Secondly, we measured the maximal amplitude of Ih just before the offset of the negative command pulses because the amplitude of non-Ih currents is less than 6% of the maximal Ih measured just before the offset of Ih (Kawasaki et al., 2018). Furthermore, the small contamination of Ih with inward rectifier and leak K + currents did not cause any problems when the conductance of voltagedependent HCN was measured because inward rectifier and leak K + currents are not voltage-dependent, and their conductances are constant and very small.
With a tungsten microelectrode with an impedance of 1 M at 5 kHz, microstimulation at an intensity of < 2 V (2.0 µA) with a negative pulse (0.2 ms duration) was applied at 100 Hz for 0.1 s (Figure 4) or 20 Hz for 1 s (Figure 5) in LC. The current spread may be less than 30-40 µm for such microstimulation (Stoney et al., 1968;Jankowska and Roberts, 1972;Kang et al., 1994).

Viral Vector Transduction of Locus Coeruleus Neurons With ChannelRhodopsin2
To allow optogenetic activation of LC neurons, a Canine-Adenoviral vector (CAV) was used (CAV-PRS-ChR2-mCherry) (Li et al., 2016). The prolyl tRNA synthetase (PRS) promoter in this vector has been previously demonstrated to achieve LC-specific expression by harnessing the Phox2B transcription factor, which drives endogenous dopamine-β-hydroxylase (the final enzyme in the synthesis pathway for NA).
Stereotaxic injection of the CAV vector was made into the dorsal pons in the region of the LC of PD 19 Wistar rats using an established protocol (Li et al., 2016). Rats were anesthetized (intraperitoneally) with ketamine (5 mg/100 g, Vetalar; Pfizer, New York, NY, United States) and medetomidine (30 µg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. The animal was placed in a stereotaxic frame and core temperature was maintained at 37 • C using a homeothermic blanket (Harvard Apparatus, Holliston, MA, United States). Following a craniotomy (from bregma: -1 mm AP, +1.1 mm ML), a microinjection pipette was advanced into the brain with a 10 • rostral tilt. Two hundred nanoliters of vector was injected at 4.6, 4.9. 5.2, and 5.5 mm depth. The titre of vector used was 2.15 × 10 11 physical particles/ml. Aseptic surgical techniques were used throughout.

Optogenetic Stimulation
Brain slices including the LC were prepared from PD 27-31 Wistar rats (8-12 days after CAV injection). Rats were terminally anesthetised with isoflurane before decapitation. The brain was dissected, and a tissue block containing the medulla and pons was prepared, in ice-cold dissection aCSF (identical to recording aCSF below, except NaCl was reduced to 86 mM, and sucrose added at 58.4 mM). The tissue block was glued, rostral face down, to the stage of a Dosaka LinearSlicePro vibratome (DSK, Japan), and 300 µm thick coronal slices were cut. Slices were incubated in carbogen bubbled recording aCSF at 37 • C for 1 h prior to recordings (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2 MgCl 2 , 2 CaCl 2 and 10 D-glucose, pH 7.3, and osmolality 290 mOsm/l. For recordings, the slices were placed in the perfusion chamber of an upright fluorescence microscope (DMLFSA, Leica Microsystems, Heidelberg Germany) and continuously perfused with recording aCSF at a ∼2 ml/min flow rate at 30 • C. Patch pipettes (4-6 M ) were filled with the internal solution as described above. The membrane potential values given in the text were corrected for the junction potential (10 mV). Channelrhodopsin-2 (ChR2) expression neurons were identified using epifluorescence illumination (Chroma filterset 41034) via membrane-bound mCherry fluorescence. Recordings were made in voltage clamp mode (Axon Multiclamp 700A, Molecular Devices, San Jose, CA, United States). All recordings were low-pass filtered with a 3 kHz cut-off frequency, and digitized at a sampling rate of 10 kHz (power1401, Cambridge Electronic Design, Cambridge, United Kingdom) and stored on a computer hard disk using Spike2 software (CED). For optogenetic stimulation, light was pulsed onto the cells (5-20 mW, 20 pulses of 4 ms duration at 20 Hz) using a focally placed optical fiber (400 µm tip diameter) proximate to the LC pigtailed to a 473 nm LED source (Doric Lenses, Quebec, QC, Canada) using stimulation scripts implemented in Spike2.

Statistical Analysis
Statistical analysis was performed using STATISTICA10J (StatSoft Japan, Tokyo, Japan). Numerical data were expressed as the mean ± SD. The statistical significance was assessed using t-test and/or ANOVA followed by Bonferroni post hoc test. Statistical results were given as a specific p-value, except when p was smaller than 0.001. p < 0.05 was considered statistically significant.

Noradrenergic Projections From Locus Coeruleus Neurons Onto Mesencephalic Trigeminal Nucleus Neurons and Expression of α 2A -Adrenergic Receptors on Mesencephalic Trigeminal Nucleus Neurons
First, the noradrenergic projections from LC neurons to MTN neurons were investigated by immunohistochemistry. THpositive LC neurons project noradrenergic fibers throughout the neuraxis, including MTN which is located dorso-laterally adjacent to the LC nucleus (Paxinos and Watson, 2013). Histological identification of MTN neurons in the mesencephalic region was done by staining with advillin which is a marker for primary sensory neurons (Hasegawa et al., 2007). Advillinpositive MTN neurons were located medially adjoining LC, and occasionally found to be intermingled with the soma of noradrenergic LC neurons ( Figure 1A). MTN neurons received noradrenergic projections from LC, as reported previously (Takahashi et al., 2010). Indeed, many TH-positive beaded axonal projections to an MTN neuron can be seen in a z-stack of eight images ( Figure 1B) of the area enclosed with a square ( Figure 1A) when visualized at 100x magnification. Four terminal-like swellings arising from such TH-positive beaded axons indicated with filled arrow head ( Figure 1C) were found to surround the MTN neuron almost without any direct contacts when examined at respective single plane images. The distance between the closest terminal-swelling and MTN soma membrane was larger than 1 µm. In the single plane images either over the second (Figure 1Ca) or under the fifth plane image (Figure 1Cd), there were no terminal-like swellings in close proximity to the MTN neuron. These results were in good agreement with a previous study (Takahashi et al., 2010) which demonstrated that there were almost no synapse-like contacts between terminal-like swelling arising from LC neurons and somata of MTN neurons.
Volume transmission of NA from LC to MTN neurons would take place from the terminal swellings to the cell surface receptors, the distance between which was larger than 1 µm (see Figure 1C). We further examined if there is any difference in the distribution of TH and DBH immunoreactive terminals of LC neurons. We found that immunoreactivities for TH and DBH were almost the same in the somata of LC neurons, while fine terminal arborizations in the MTN region were not immunepositive to DBH but positive to TH (Supplementary Figure 1). This suggests that only small vesicles containing NA produced in the soma are conveyed through short axons to be released from the terminals surrounding MTN neurons. We next examined whether MTN neurons express α 2A -ARs. In MTN neurons, the intense immunoreactivities for advillin, a sensory neuron-specific protein (Hasegawa et al., 2007), and α 2A -ARs were seen along the plasma membrane and partly in the cytoplasm ( Figure 1D).

Volume Transmission of Noradrenaline From a Single Locus Coeruleus Neuron to an Mesencephalic Trigeminal Nucleus Neuron
To investigate the activity-dependent nature of volume transmission of NA from LC to MTN neurons, we first performed the dual-whole cell recordings from LC and MTN neurons and examined the effects of spiking of the LC neuron on the h-current (Ih) evoked in the simultaneously recorded MTN neuron. Ih was repeatedly evoked by a 1.5-s negative voltage pulse applied from −70 to −100 mV in the MTN neuron every 30 s, while at 4.5 s before the voltage pulse, a 1-s depolarizing current pulse was applied every 30 s to induce a spike train approximately at 20 Hz in the LC neuron after control Ih was evoked six times (Figure 2A). Because the spike train at 20 Hz is usually followed by a long-lasting (∼30 s) afterhyperpolarization, presumably due to autoinhibition (Arima et al., 1998), together with Ca 2+ dependent K + channel activity (Osmanovic and Shefner, 1993; Supplementary Figure 2), we usually applied current pulses at an inter-pulse interval of 30 s. These six responses of Ih in the MTN neuron without preceding spikes in the LC neuron showed an almost constant amplitude of Ih. However, as shown in Figures 2A,B (compare black trace with blue and red traces), Ih gradually decreased in amplitude and reached a minimal level after a 20 Hz spike-train was repeatedly induced in the LC neuron.
After the simultaneous whole-cell recording, the LC and MTN neurons were identified with Lucifer yellow, and the LC neuron was found to be TH-positive among other THpositive neurons ( Figure 2C). This TH-positive LC neuron was electrophysiologically characterized as showing a delayed spiking with or without the immediate-onset spike in response to depolarizing current pulses ( Figure 2B, upper blue and red traces showing LC firings), which is in good agreement with those of LC neurons reported previously (Masuko et al., 1986;van den Pol et al., 2002;Wagner-Altendorf et al., 2019). Thus, following the repetitive release of NA around the recorded MTN neuron, Ih evoked in the MTN neuron was gradually suppressed by repetitive activation of AR every 30 s and reached a plateau level.

Nature of Volume Transmission of Noradrenaline From a Locus Coeruleus Neuron With Periodic Phasic Activity
Considering the long inter-stimulus interval (30 s), the apparent saturation of inhibitory effects of LC activity on Ih in MTN neurons is not likely due to the simple saturation of NA around AR. Because the whole time course of the volume transmission accompanied by GPCR activation may be very slow, the repetitive activation of LC neurons may cause cumulative increases in the signal transduction to gradually decrease Ih in MTN neurons. Therefore, we analyzed this cumulative increase quantitatively. As illustrated in Figure 2D, with an increase in the cumulative spike number in the LC neuron, Ih decreased in amplitude consistent with the outward shift of the baseline current, reflecting the steady state Ih at −70 mV. The normalized inhibition of Ih was plotted against either the cumulative spike number or the number of current pulse injection ( Figure 2E). These relationships were fitted with various functions and were found to be best described by Hill equation with an nH of 1.49 or 1.44 (correlation coefficient = 0.99). The nH would represent the efficacy of signal transduction in response to LC spike inputs, and therefore can quantitatively characterize the volume transmission.
In a total of 55 paired recordings from LC and MTN neurons, 17 pairs of LC and MTN neurons were found to have positive connections, in which Ih in MTN neurons was significantly inhibited by the activity of LC neurons. Of the 17 positive pairs, effects of repetitive activation of LC neurons were successfully examined in 7 MTN neurons, while effects of antagonist for α 2 -AR and of the microstimulation of LC were successfully examined in 4 and 6 paired recordings, respectively. The maximal normalized inhibition of Ih was 0.19 ± 0.07 and of nH was 1.48 ± 0.32 (n = 6) with one exception that showed a smaller nH (0.6), with its maximal inhibition largely diverged from the calculated value ( Figure 2F). The averaged correlation coefficient was 0.98 ± 0.01. Thus, our analysis revealed that the volume transmission of NA from LC neurons to MTN neurons, which was induced following repetitive activation of LC neurons every 30 s, can be best described by Hill equation with an nH of approximately 1.5. This value was in good agreement with the averaged value of nH (1.53 ± 0.10) obtained in the relationship between the dose of NA and normalized inhibition of Ih in respective MTN neurons (n = 8, Supplementary  Figure 3). Because the amplitude of Ih was decreased in a dose-dependent manner with K i = 16 µM and a dynamic range (10-90% inhibition) spanning more than 23-fold range (E) Plotting of voltage and normalized conductance of Ih obtained before 1st spike train (black square), after 20th spike train (red circle), and after 20th spike train in the presence of atipamezole (blue diamond) in one paired recording. Two-way ANOVA: p < 0.001, followed by Bonferroni post hoc test, *p < 0.001, and #p < 0.001. (F) Plotting of voltage and normalized conductance of Ih in a total of four paired recordings. Two-way repeated measures ANOVA: p = 0.006, followed by Bonferroni post hoc test, *p < 0.002 (the largest p-value is 0.0015) and #p < 0.001 (the largest p-value is 0.0002). of concentration (3-70 µM), it is suggested that the basal level of cyclic monophosphate (cAMP) reflecting tonic activity of adenylate cyclase is high enough for NA to inhibit Ih in MTN neurons in a dose-dependent manner.
These results suggest that the Hill-type relationship obtained in response to repetitive activation of spike trains in LC neurons represents the nature of the volume transmission of NA from LC to MTN neurons and can be a measure of volume transmission (see section "Discussion").  (Figure 3A, red trace), Ih was examined at respective voltages. As seen in the superimposed traces ( Figure 3B, bottom traces), Ih responses were invariably suppressed at any voltage steps, consistent with the previous results (Figure 2). Next, in the presence of atipamezole, Ih responses that were evoked after spike trains were repeatedly evoked 20 times every 30 s were larger than those evoked after 20 spike trains in the absence of atipamezole as shown in the superimposed traces in Figure 3D (bottom traces). Thus, the inhibition of Ih by the activity of LC neurons was abolished after applying atipamezole. Voltage-dependent conductance curve of Ih was significantly shifted in the left direction following LC activation [two-way ANOVA: F(2,8) = 77.8, p < 0.001, followed by Bonferroni post hoc test, * p < 0.001], while such leftward shift was reversed and abolished by atipamezole (Bonferroni post hoc test: #p < 0.001; Figure 3E). In a total of four paired recordings (of 17 total positive paired recordings) with multiple step pulse responses, a similar leftward shift of conductance-voltage (G-V) relationship was observed following repetitive LC activation [two-way repeated measures ANOVA: F(8,24) = 3.73, p = 0.006, followed by Bonferroni post hoc test, * p < 0.002 (the largest p value is 0.0015)], while atipamezole reversed and abolished such a leftward shift of the G-V relationship [Bonferroni post hoc test: #p < 0.001 (the largest p-value is 0.0002)] (Figure 3F). Taken together, these results clearly indicate that the inhibition of Ih in MTN neurons was mediated by the activation of α 2 -AR through volume transmission of NA from single LC neurons.

Volume Transmission of Noradrenaline From Locus Coeruleus Neurons That Show Phasic-Tonic Activity
A similar nature of volume transmission was also observed following activation of multiple LC neurons with microstimulation. Dual whole-cell recordings were performed from LC and MTN neurons (Figures 4A-D) with two patch pipettes, and a stimulating electrode was placed in LC ( Figure 4F). In response to microstimulation applied in LC every 30 s, just 0.5 s before the onset of the voltage pulse to evoke Ih in MTN neurons, the LC neuron displayed a spontaneous firing at approximately 0.8 Hz, while Ih was simultaneously induced in the recorded MTN neuron (Figure 4A, * 1). The first microstimulation applied in LC induced a transient increase in spike firing, which did not cause any marked inhibition of Ih ( Figure 4B, * 2), indicating that the latency to the onset of Ih inhibition is longer than ∼2 s. However, the fourth microstimulation induced a persistent firing at 10-20 Hz, and a clear inhibition of Ih was simultaneously observed in the MTN neuron ( Figure 4C,  *  3). Without microstimulation after the fourth one, the LC neuron kept displaying a tonic firing at approximately 5-10 Hz (Figure 4D,  *  4), resulting in a further inhibition of Ih in the MTN neuron ( Figure 4D, bottom trace). Thus, such tonic firing activities in the LC neuron appeared to cause a progressive inhibition of Ih ( Figure 4E). Because microstimulation may have activated other LC neurons and the simultaneously recorded LC neuron around the stimulating electrode, the normalized inhibition of Ih would not necessarily reflect the activity of the simultaneously recorded LC neuron. Therefore, the normalized inhibition of Ih was plotted against the time or the number of episodes (Figure 4G) which yielded a better correlation (r = 0.99) than what was plotted against the number of spikes in this LC neuron (r = 0.96). Similar results were obtained in 6 paired recordings (of 17 positive paired recordings), while in 4 of 6 paired recordings, the nH ranged between 1 and 1.5 with a mean value of 1.24 ± .19. In addition, the mean normalized inhibition of Ih was 0.17 ± 0.10 ( Figure 4H). In two of 6 paired recordings, the relationship between the normalized inhibition of Ih and the episode number displayed a dose-response curve with the nH smaller than unity, in which the calculated normalized inhibition exceeded 1, indicating that the estimation of nH may have probably been underestimated due to the higher background tonic activation of α 2 -AR. Distinct from the cases of single LC activation, microstimulation of LC invariably induced the inhibition of Ih in MTN neurons in all the 6 paired recordings examined. Furthermore, the averaged value of the nH seen during persistent/tonic firings in LC neurons in response to microstimulation of LC (1.24 ± 0.19, n = 4) was significantly [unpaired t-test: t(7) = 2.31, p = 0.049] smaller than what was obtained (1.57 ± 0.24, n = 5) following periodic burst/phasic activations (1-s pulses applied every 30 s) of single LC neurons at ∼20 Hz when cells showing nH ≤ 1 were excluded in the two groups. Persistent/tonic activation of LC neurons at frequencies of 5∼10 Hz induced by microstimulation (Figures 4C,D) would cause an occlusion in the signal transduction to effectively produce Gα-GTP, resulting in a smaller nH.
The inhibitory effects of microstimulation of LC on Ih evoked in MTN neurons were also abolished by atipamezole (Supplementary Figure 4), indicating that the inhibition of Ih in MTN neurons was induced by the activation of α 2 -ARs through volume transmission of NA from LC neurons.

Optogenetic Stimulation of Locus Coeruleus Revealed a Slow Time Course of Ih Inhibition in Mesencephalic Trigeminal Nucleus Neurons
In a ChR2-expressed LC neuron (Figures 5A,B), a train of optogenetic stimulation (4 ms duration at 20 Hz for 1 s) evoked a train of transient depolarizing potentials, from which a train of spikes was triggered ( Figure 5C). After obtaining the control Ih in an MTN neuron, a train of optogenetic stimulation of a population of LC neurons (Figure 5D) was applied 2-5 s before examining the first Ih. Subsequently, Ih that was evoked in an MTN neuron displayed a slow decrease in amplitude and slowly returned to the original amplitude (Figures 5E,F). This change was accompanied by an outward shift of the baseline current with a similar time course (Figure 5G). In 8 MTN neurons examined, the latency, the time-to-peak, and the time-to-termination of Ih inhibition were 2−5 (3.4 ± 1.1; n = 8) s, 30-80 (55 ± 17; n = 8) s, and 65-120 (98 ± 20; n = 7) s, respectively. All these values were approximated as these were obtained by measuring the amplitudes of Ih that were repetitively evoked at intervals greater than 10 s before and after the optogenetic stimulation.
Following optogenetic LC stimulation, the Ih amplitude significantly decreased from −966 ± 213 to −856 ± 177 pA [paired t-test: t(6) = -12.73, * p < .001; n = 7], consistent with a significant shift of the baseline current in the outward direction from −100 ± 55 to −79 ± 46 pA [paired t-test: t(6) = -3.90, * p = 0.008; n = 7]. The normalized inhibition of Ih was 0.11 ± 0.02 (n = 7; Figure 5H) and the outward shift of the baseline current was 20 ± 14 pA (n = 7; Figure 5I). The maximal inhibition of Ih caused by activation of multiple LC neurons with single optogenetic stimulation was not markedly different from those observed in response to the first activation of single LC neurons in dual whole-cell recordings (compare the first response in Figure 2 and the response shown in Figure 5). Axons of such LC neurons that were most strongly activated by optogenetic stimulation due to their location in the top surface of slices might have been largely severed in slice preparations. Hence, optogenetic activation of multiple LC neurons may have induced multiple small NA transients around the target MTN neuron in a less synchronized manner, resulting in a slower timeto-peak compared to those caused by activation of single LC neurons (Figure 2).
Although the recovery from the cumulative inhibition of Ih induced in MTN neurons by the repetitive stimulation of LC neurons was not shown (Figures 2, 4), single optogenetic stimulation of LC neurons only transiently inhibited Ih induced in MTN neurons and the amplitude of Ih was restored almost completely in a few minutes after single optogenetic stimulation ( Figure 5). Furthermore, atipamezole reversed the Ih inhibition induced by LC activity (Figure 3 and Supplementary Figure 4). These observations completely eliminate the possibility that Ih inhibition was artificially caused.

Inter-Burst Intervals in Locus Coeruleus Neurons Determine Whether Ih Inhibition Increases in a Hill-Type Manner or Is Transiently Followed by a Forced Termination
Similar to the repetitive 1-s phasic activation of the LC neuron at 20 Hz every 30 s (Figures 2, 3), repetition of 1-s optogenetic stimulation evoking 20 Hz spike trains ( Figure 5C) in LC neurons with intervals ranging between 20 and 30 s decreased Baseline current level just before the onset of each Ih was compensated by subtracting the amplitude of the preceding tail-I calculated by exponential fitting of the tail-I of the 1st Ih. (H) Normalized inhibition of Ih obtained before (Control) and after optogenetic stimulation (n = 7). Paired t-test: *p < 0.001. (I) Outward shift of baseline-I obtained before (Control) and after optogenetic stimulation (n = 7). Paired t-test: *p = 0.008. (J,K) The repetitive 1 s optogenetic stimulations at short intervals smaller than 10 s induced a cumulative increase in Ih inhibition only transiently and was followed by a forced termination of the Ih inhibition. Ih amplitude was measured for the response evoked approximately 10 s after the respective stimulation. If 10 s after stimulation was just in between the two responses, the average amplitude of the two responses was used as a response for the stimulation. Note that a short interval of the first and second optogenetic stimulations resulted in a much larger Ih inhibition compared to the case with a longer interval > 20 s. (L,M) Significant cumulative increases in normalized inhibition of Ih induced following up to 6-7 repetitive 1 s microstimulations with an interval of 9 s (M; n = 7), after which the forced termination of Ih inhibition began and the maximal inhibition of Ih was significantly decreased after 10 repetitive microstimulation (M; n = 7). Two-way repeated measures ANOVA: p < 0.001, followed by Bonferroni post hoc test, *p < 0.03 (vs. control; the largest p-value is 0.028) and #p < 0.002 (vs. 7th stimulation; the largest p-value is 0.0012). Vertical scale bars; (E,F,J,L) 200 pA. Horizontal scale bars; (F) 500 ms: (J,L), 10 s. the amplitude of Ih in a cumulative manner as reflected in the Hill type relationship (Supplementary Figure 5; mean nH = 1.53, n = 3). In contrast, however, when the repetitive 1-s optogenetic stimulation was applied at short intervals less than 10 s, Ih inhibition was only induced transiently and was followed by a forced termination of Ih inhibition (n = 3, Figures 5J,K). Because it is known that repetitive optogenetic stimulation may cause a more evident rundown of transmitter releases or evoked responses compared to repetitive electrical stimulation (O'Neill et al., 2017), it was examined with microstimulation whether a forced termination can be observed or not. Indeed, a similar forced termination of Ih inhibition following a transient increase in Ih inhibition was observed in 8 LC neurons following microstimulation of LC for 1 s at 20 Hz repeated with an interval of 6 or 9 s (Figures 5L,M). The normalized inhibition of Ih significantly increased and reached a maximal level following 6-7 repetitive microstimulations, after which the forced termination of Ih inhibition began and the maximal inhibition of Ih was significantly decreased after repeating microstimulation ten times [two-way repeated measures ANOVA: F(12,72) = 6.42, p = 0.008, followed by Bonferroni post hoc test, * p < 0.03 vs. control (the largest p-value is 0.028) and #p < 0.002 vs. 7th stimulation (the largest p-value is 0.0012); n = 7, Figure 5M].
Taken together, the Hill-type relationship was largely affected by the activity of LC neurons depending on either periodic burst/phasic firings or persistent/tonic firings, while burst/phasic firing occurring at > 0.1 Hz can forcibly terminate Ih inhibition. Thus, the volume transmission of NA from LC to MTN is differentially and dynamically controlled by the activity of LC neurons.

Noradrenaline-Mediated Ih Inhibition in Mesencephalic Trigeminal Nucleus Neurons Is Caused by Downregulation of cAMP
We next investigated the signaling pathway responsible for the inhibition of Ih by NA in MTN neurons. Control responses of Ih were evoked by the negative voltage pulses stepped from -70 to -100 and -110 mV ( Figure 6A, black trace). Following the application of 100 µM NA for 10 min, the baseline current reflecting the steady state Ih at -70 mV was shifted in the outward direction, and the Ih responses evoked at -100 and -110 mV were markedly suppressed ( Figure 6A, red trace). The subsequent application of 10 µM chelerythrine, a PKC inhibitor, in addition to NA for 10 min hardly reversed the NA-induced inhibition of Ih ( Figure 6A, blue trace). The pooled data analysis revealed that the application of NA significantly shifted the baseline current in the outward direction from -16 ± 36 to 33 ± 44 pA [one-way repeated measures ANOVA: F(2,10) = 14.19, p = 0.001, followed by Bonferroni post hoc test, p = 0.002], but the subsequent application of chelerythrine hardly shifted the baseline current (from 33 ± 44 pA to 27 ± 39 pA; Bonferroni post hoc test, p = 0.920). The application of NA significantly reduced the normalized amplitude of Ih [one-way repeated measures ANOVA: F(2,10) = 46.4, p < 0.001, followed by Bonferroni post hoc test, * p < 0.001], and the subsequent inhibition of PKC activity had no significant effect on the NA-induced Ih inhibition (Bonferroni post hoc test, p = 0.605; n = 6; Figure 6B). Furthermore, the activation kinetics of Ih were compared among the three conditions. Ih was fitted with a double exponential function, and the fast and slow components were subsequently obtained. The application of NA significantly reduced the normalized amplitudes of both fast and slow components [fast component, one-way repeated measures ANOVA: F(2,10) = 21.11, p < 0.001, followed by Bonferroni post hoc test, * p < 0.001; slow component, one-way repeated measures ANOVA: F(2,10) = 23.94, p < 0.001, followed by Bonferroni post hoc test, * p < 0.001], and the subsequent inhibition of PKC activity had no significant effect on both the normalized amplitudes (fast component, Bonferroni post hoc test, p = 0.735; slow component, Bonferroni post hoc test, p = 0.660; n = 6; Figure 6C). The time constant of the slow component was significantly increased following application of NA [one-way repeated measures ANOVA: F(2,10) = 10.54, p = 0.003, followed by Bonferroni post hoc test, * p = 0.004], while such an increase in the slow time constant was not abolished by the subsequent inhibition of PKC activity (Bonferroni post hoc test, p = 0.067; n = 6; Figure 6D). The time constant of the fast component was hardly affected by NA and the subsequent inhibition of PKC activity [One-way repeated measures ANOVA: F(2,10) = 1.83, p = 0.194, n = 6; Figure 6D].
We then examined the effects of 8-Br-cAMP on the NAinduced inhibition of Ih. The application of 100 µM NA for 10 min shifted the baseline current in the outward direction and suppressed the Ih responses evoked at -100 and -110 mV (Figure 6E, compare black and red traces). The subsequent addition of 500 µM 8-Br-cAMP shifted the baseline current in the inward direction and enhanced the Ih responses evoked at -100 mV and -110 mV ( Figure 6E, compare red and blue traces). The pooled data analysis revealed that application of NA significantly shifted the baseline current in the outward direction from -23 ± 62 to 25 ± 64 pA [One-way repeated measures ANOVA: F(2,8) = 9.21, p = 0.008, followed by Bonferroni post hoc test, p = 0.011], and the subsequent application of 8-Br-cAMP significantly shifted the baseline current in the inward direction from 25 ± 64 to -14 ± 73 pA (Bonferroni post hoc test, p = 0.034). The application of NA also significantly reduced the normalized amplitude of Ih [one-way repeated measures ANOVA: F(2,8) = 15.97, p = 0.002, followed by Bonferroni post hoc test, * p = 0.002], but 8-Br-cAMP application, in addition to NA, significantly reversed the inhibition of Ih by NA (Bonferroni post hoc test, * p = 0.023; n = 5; Figure 6F). The double exponential analysis revealed the significant decrease in the normalized amplitudes of both fast and slow components following NA application [fast component, one-way repeated measures ANOVA: F(2,8) = 16.01, p = 0.002, followed by Bonferroni post hoc test, * p < 0.001; slow component, one-way repeated measures ANOVA: F(2,8) = 16.64, p = 0.001, followed by Bonferroni post hoc test, * p < 0.001], and the subsequent addition of 8-Br-cAMP significantly reversed the inhibition of both the fast and slow components (Bonferroni post hoc test, * p = 0.041 and * p = 0.034, respectively; n = 5; Figure 6G). The time constant of the slow component was significantly increased following application of NA [one-way repeated measures ANOVA: F(2,8) = 42.32, p < 0.001, followed by Bonferroni post hoc test, * p < 0.001], while it was significantly decreased after the subsequent activation of cAMP activity . Chelerythrine had no effect on NA-mediated Ih inhibition. (B) Normalized amplitude of Ih at respective conditions in (A) (control, 1.0; NA, 0.55 ± 0.14; NA + chelerythrine, 0.54 ± 0.14; n = 6). One-way repeated measures ANOVA: p < 0.001, followed by Bonferroni post hoc test, *p < 0.001 (control vs. NA), and *p < 0.001 (control vs. NA + chelerythrine). (C) Normalized amplitude of fast and slow components (blue and red squares, respectively) at respective conditions in (A) (n = 6). Normalized amplitude of fast component: control, 1.0; NA, 0.59 ± 0.17; NA + chelerythrine, 0.63 ± 0.22. One-way repeated measures ANOVA: p < 0.001, followed by Bonferroni post hoc test, *p < 0.001 (control vs. NA) and *p = 0.001 (control vs. NA + chelerythrine). Normalized amplitude of slow component: control, 1.0; NA, 0.57 ± 0.23; NA + chelerythrine, 0.56 ± 0.19. One-way repeated measures ANOVA: p < 0.001, followed by Bonferroni post hoc test, *p < 0.001 (control vs. NA) and *p < 0.001 (control vs. NA + chelerythrine). (Bonferroni post hoc test, * p < 0.001; n = 5; Figure 6G). The time constant of the fast component was not significantly changed by NA or the subsequent addition of 8-Br-cAMP [one-way repeated measures ANOVA: F(2,8) = 1.27, p = 0.333; n = 5; Figure 6H]. These results indicate that NA-induced Ih inhibition in MTN neurons is not mediated by PKC, but most likely by downregulation of cAMP through the activation of α 2A -ARs (He et al., 2014).

DISCUSSION
In the present study, we have revealed for the first time the activity-dependent volume transmission of NA from LC neurons to MTN neurons by performing the dual whole-cell recordings from LC and MTN neurons (Figures 2, 3). Furthermore, activities of multiple LC neurons were manipulated by applying microstimulation (Figure 4 and Supplementary Figure 4) and optogenetic stimulation (Figure 5) during whole-cell recordings in MTN neurons. First, in response to repetitive phasic activation (20 Hz, 1 s) of LC neurons every 30 s, Ih inhibition gradually increased in a Hill-type manner with an nH of approximately 1.5 (Figure 2). Secondly, in response to repetition of microstimulations of LC that caused persistent/tonic firings at 5-10 Hz after evoking transient phasic firings at 10-20 Hz (Figure 3), Ih inhibition also gradually increased in a Hill-type manner but with a smaller nH compared to the phasic activity (compare Figure 4 with Figure 2). These findings indicate that the sensitivity to input LC spikes or the efficacy of the signal transduction induced by a single spike is much higher for the phasic bursts than for the tonic/persistent firings because the average frequency (0.66 Hz) in over 30 s for the phasic bursts (20 Hz for 1 s) is much lower than the 5-10 Hz tonic firing (lasting for 30 s). However, the dynamic range of input signals (Simon et al., 2014) is larger for tonic activity than for the periodic bursts in LC neurons. In contrast, the 20 Hz activity, when induced every 5-10 s, caused only a transient facilitation of Ih inhibition followed by a forcible termination of Ih inhibition (Figure 5). Thus, the nature of the volume transmission of NA from LC to MTN neurons accompanied by α 2A -adrenergic GPCR activation can be characterized by these three distinct patterns of Ih inhibition in response to the three different patterns of repetitive activation of LC neurons.

Volume Transmission of Noradrenaline to Activate α 2 -Adrenergic Receptor That Downregulates Ih in Mesencephalic Trigeminal Nucleus Neurons
The processes of NA diffusion from the terminals of LC neurons and subsequent NA accumulation around the target MTN neurons to activate α 2A -ARs are very transient, probably with a latency of less than 200 ms for the terminals separated even by 10 µm from the target receptors, given the diffusion constant of ∼0.5 µm 2 /ms. The subsequent receptor binding duration may be also transient (∼200 ms) (Beyene et al., 2017). In response to NA binding to α 2Aadrenergic GPCR, NA accelerates the GDP dissociation rate to facilitate reaction from NA/α 2A -AR/[G α -GDP/G βγ ] to NA/α 2A -AR/[G α /G βγ ], which subsequently shifts the equilibrium between NA/α 2A -AR/[G α /G βγ ] and NA/α 2A -AR/[G α -GTP/G βγ ] to produce the free G α -GTP (Waelbroeck, 1999;Alhadeff et al., 2018). Thereafter, free G α -GTP complex starts to interact with adenylate cyclase to inhibit its activity, and simultaneously undergoes auto-hydrolysis of G α -GTP complex to slowly terminate such inhibition (0.02/s) (Ross and Wilkie, 2000). Thus, after the binding of NA to α 2Aadrenergic GPCR, there are at least three processes that are involved in determining the rate of production of free G α /GTP complex, subsequently causing a slow Ih inhibition and a slower termination of Ih inhibition. These processes are reflected in the latency (2-4 s), the time-to-peak (30-70 s), and the time to termination (80-120 s) of Ih inhibition in 13 MTN neurons examined (Figure 5). Ih inhibition would occur immediately after the reduction of cAMP concentration because Ih is active under the equilibrium condition between free and bound cAMP to HCN channels. Such slow Ih inhibition following NA transmission was similar to those observed in autonomic ganglionic neurons in PNS (Koketsu and Yamamoto, 1975;Kuba and Koketsu, 1976;Konishi and Otsuka, 1985).
It is known that heteromeric channels of HCN1/HCN2 have voltage and cAMP dependency similar to HCN2 rather than HCN1 (Chen et al., 2001;Ulens and Tytgat, 2001), while HCN1 and HCN2 are modulated mostly by PKC (Reetz and Strauss, 2013) and cAMP (Wainger et al., 2001;Kusch et al., 2011), respectively. Then, it is suggested that Ih in MTN neurons is mediated by heteromeric HCN channels composed of HCN1 and HCN2 , as Ih evoked in MTN neurons is largely modulated by cAMP but not by PKC (Figure 6).

Quantification of Volume Transmission
During repetition of phasic spike trains in LC neurons every 30 s, NA is repetitively released while NA concentration would not be cumulatively increased because of the short latency and duration of NA transient (<0.5 s; see above) as opposed to the long interval of 30 s. Nevertheless, Ih inhibition was cumulatively increased with repetition of spike trains. Then, the concentration of free G α -GTP complex should have been cumulatively increased with repetition of spike trains every 30 s, while free G α -GTP complex very slowly changes into inactive G α -GDP due to the GTPase activity of G α (Ross and Wilkie, 2000), given the absence of regulator of G-protein signaling (RGS). Given that the respective NA transients activate a constant number of α 2A -ARs in MTN neurons, the same amount of NA/α 2A -AR/[G α -GTP/G βγ ], from which free G α -GTP is released, would be repetitively produced in response to repetitive stimulation.
Provided that the production and degradation of G α -GTP complex can be approximated to occur simultaneously with two rate constants of α and β, respectively, the probability [P(t)] of being in a state of free G α -GTP complex is given by the special solution of the following 2nd order linear homogenous differential equation: where α > 0 and β > 0 (usually α > β). Then, the concentration of free G α -GTP complex is proportional to P(t).
Given the simple temporal summation of G α -GTP transient in response to repetitive stimulation, the cumulative increase in G α -GTP concentration can be calculated as explained in the supporting information (Supplementary Figure 6). Using this mathematical model, we could confirm that the nH measured from the relationship between the spike/stimulus number and normalized inhibition of Ih is almost the same or slightly larger than that measured from the dose-response relationship (Supplementary Figures 3,6) and can be a useful measure of "volume transmission." However, when LC neurons displayed persistent/tonic activities, the value of apparent nH became smaller compared to that seen in the slow periodic phasic/burst activity (compare Figure 2 and Figure 4). It is unclear whether tonic/persistent activity served as a background activity that may decrease an apparent nH or caused some occlusion in production of G α -GTP complex. A further study is necessary to clarify this mechanism.
In contrast, when the 20 Hz spike train for 1-s duration was repeated at an interval less than 10 s, Ih inhibition was enhanced only transiently and was followed by a rapid termination of Ih inhibition. In response to such a short interval of repetitive activation of LC neurons, G α -GTP complex and G βγ are rapidly produced and accumulated around the GPCR. Then, Ih inhibition would be rapidly terminated because G βγ can indirectly facilitate the hydrolysis of G α -GTP complex through activation of G αi -selective RGS protein (Huang et al., 2014). In a physiological condition, volume transmission from LC neurons may occur in similar manners following LC activity transitions between tonic and phasic firings. In order to firmly establish the three modes of LC activity-dependent volume transmission to differentially modulate Ih in MTN neurons, a further study to investigate the intracellular mechanisms in MTN neurons is necessary.

Slow Modulation of Ih by Volume Transmission of Noradrenaline Was Necessary to Facilitate Fast Glutamate Synaptic Transmission in Mesencephalic Trigeminal Nucleus Neurons
It is generally considered that noradrenergic fibers arising from LC exert volume transmission onto pyramidal neurons in the PFC. However, 10∼20% of the varicosities arising from noradrenergic fibers are found to form synaptic junctions on the shaft of dendritic spines of pyramidal cells (Seguela et al., 1990;Aoki et al., 1998), while most of α 2 -ARs are found at sites lacking electronmicroscopically identifiable synapses but are invariably near a terminal (Aoki et al., 1998). Therefore, regardless of whether noradrenergic fibers form synaptic junction, activation of extrajunctional α 2 -AR is always in a manner of volume transmission. It should be especially noted that out of 745 terminal-like swellings arising from LC neurons examined in the MTN regions, only 180 swellings (24%) were found in the vicinity of the somata of MTN neurons despite how none of these swellings formed synapse-like contact (Takahashi et al., 2010).
It has been reported that NA was released by exocytosis of a large-dense core vesicle (LDCV) from the soma and dendrites of LC neurons following their phasic firing (Huang et al., 2007), subsequently causing an autoinhibition of LC neurons through activation of G-protein coupled inward rectifier K + currents (GIRKs) (Arima et al., 1998;Torrecilla et al., 2013). This LDCV-mediated transmission is distinct from the classical fast synaptic transmission. However, there was a clear segregation between the two cell groups of LC and MTN neurons ( Figure 1A). In addition, many THimmunopositive fibers project toward and surround MTN neurons (Figures 1B,C). Therefore, it is likely that the majority of MTN neurons receive axonal volume transmission from LC neurons, but rarely the direct LDCV-mediated transmission from the somata/dendrites of LC neurons.
We have previously demonstrated that 8-Br-cAMP application can suppress the burst firing caused by glutamate puff in MTN neurons through an enhancement of Ih (Kawasaki et al., 2018). The present study provides convincing evidence that LC inputs can manipulate the firing pattern of MTN neurons by modulating Ih through α 2 -AR activation. Thus, in MTN neurons, fast glutamatergic synaptic transmission can only function in the presence of slow volume transmission of NA from LC neurons. However, in order to demonstrate the functional significance and dynamics of this volume transmission from LC to MTN neurons, we have to further perform the experiment in in vivo conditions by using GPCR-activation based NE (GRAB_NE) sensor (Feng et al., 2019) and electromyographic (EMG) recording of the jaw closing muscles. GRAB_NE sensor can visualize the spread of the NA and the strength of this effect on a larger scale during jaw-closing movement either in stressful condition or induced by optogenetic stimulation of LC.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Osaka University and Seoul National University and the institutional Animal Welfare and Ethical Review Body at University of Bristol.

AUTHOR CONTRIBUTIONS
YK and SO designed the research. HT, JW, WK, HK, and OD performed the research. HT, JW, WK, HK, OD, MS, TT, and DK analyzed the data. HT, YK, and SO wrote the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea grants (NRF-2018R1A5A2024418 and NRF-2021R1A2C3003334) funded by the Korean government (Ministry of Science and ICT). This research was also funded by the Japan Society for the Promotion of Science (26290006 and 21K06441 to YK, 20K06926 to HT, 16K11482 to MS, and 19K12184 to TT). YK was supported by Brain Pool Program (NRF) funded by the Korean government (Ministry of Science and ICT). OD is a Wellcome Trust funded graduate student on the Neural Dynamics program (203963/Z/16/Z).


s2fieldsofstudy:
Biology
Agricultural And Food Sciences
Medicine

text:
Occurrence, Phenotypic and Molecular Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Healthy Turkeys in Northern Egypt

Poultry is one of the most important reservoirs for zoonotic multidrug-resistant pathogens. The indiscriminate use of antimicrobials in poultry production is a leading factor for development and dissemination of antimicrobial resistance. This study aimed to describe the prevalence and antimicrobial resistance of E. coli isolated from healthy turkey flocks of different ages in Nile delta region, Egypt. In the current investigation, 250 cloacal swabs were collected from 12 turkey farms in five governorates in the northern Egypt. Collected samples were cultivated on BrillianceTM ESBL agar media supplemented with cefotaxime (100 mg/L). The E. coli isolates were identified using MALDI-TOF-MS and confirmed by a conventional PCR assay targeting 16S rRNA-DNA. The phenotypic antibiogram against 14 antimicrobial agents was determined using the broth micro-dilution method. DNA-microarray-based assay was applied for genotyping and determination of both, virulence and resistance-associated gene markers. Multiplex real-time PCR was additionally applied for all isolates for detection of the actual most relevant Carbapenemase genes. The phenotypic identification of colistin resistance was carried out using E-test. A total of 26 E. coli isolates were recovered from the cloacal samples. All isolates were defined as multidrug-resistant. Interestingly, two different E. coli strains were isolated from one sample. Both strains had different phenotypic and genotypic profiles. All isolates were phenotypically susceptible to imipenem, while resistant to penicillin, rifampicin, streptomycin, and erythromycin. None of the examined carbapenem resistance genes was detected among isolates. At least one beta-lactamase gene was identified in most of isolates, where blaTEM was the most commonly identified determinant (80.8%), in addition to blaCTX-M9 (23.1%), blaSHV (19.2%) and blaOXA-10 (15.4%). Genes associated with chloramphenicol resistance were floR (65.4%) and cmlA1 (46.2%). Tetracycline- and quinolone-resistance-associated genes tetA and qnrS were detected in (57.7%) and (50.0%) of isolates, respectively. The aminoglycoside resistance associated genes aadA1 (65.4%), aadA2 (53.8%), aphA (50.0%), strA (69.2%), and strB (65.4%), were detected among isolates. Macrolide resistance associated genes mph and mrx were also detected in (53.8%) and (34.6%). Moreover, colistin resistance associated gene mcr-9 was identified in one isolate (3.8%). The class 1 integron integrase intI1 (84.6%), transposase for the transposon tnpISEcp1 (34.6%) and OqxB -integral membrane and component of RND-type multidrug efflux pump oqxB (7.7%) were identified among the isolates. The existing high incidence of ESBL/colistin-producing E. coli identified in healthy turkeys is a major concern that demands prompt control; otherwise, such strains and their resistance determinants could be transmitted to other bacteria and, eventually, to people via the food chain.

Introduction
The increasing incidence of intestinal colonization with extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae observed in food animals including poultry, emphasizes the possibility that food animals are major sources of ESBL-producing bacteria. This can result in an emerging public health hazard due to the compromised efficacy of the treatment of infectious diseases in humans and animals [1][2][3][4]. ESBL producing bacteria have been recovered from livestock (swine, cattle, poultry and turkey), from companion animals (cats, dogs and horses) and from wild animals [5]. The continuous exposure of bacteria to β-lactams as the most extensively used group of antibiotics in the world at the mean time has led to massive proliferation and extensive evolution of β-lactamases [6]. The ESBLs are those that hydrolyze third generation cephalosporins. Some are derived from mutations in the blaTEM-1 and blaSHV-1 β-lactamase genes, while others originate from horizontal gene transfer from environmental bacteria, such as blaCTX-M enzymes from Kluyvera species. ESBLs are mainly linked to the family of Enterobacteriaceae, including Klebsiella spp., Salmonella enterica, Citrobacter spp., Enterobacter spp., Serratia spp. and Escherichia coli [6]. Many ESBL-producers are also resistant to non-β-lactam antibiotics, including aminoglycosides, fluoroquinolones, tetracyclines, trimethoprim, sulfonamides, and chloramphenicol [7,8].
This concern encouraged the European Union (EU) to forbid the use of antimicrobials as growth promoters in food animals [9]. However, in some countries including Egypt, antimicrobials are still used in the poultry industry for non-therapeutic purposes such as prevention of diseases and promotion of growth [10][11][12]. ESBLs has been described in healthy poultry, in broiler chicken fecal and cecal samples and in turkey flocks [13][14][15]. In Egypt, Escherichia (E.) coli infections are considered one of the most serious infections causing economic losses in poultry production [16]. Despite the huge economic size of the Egyptian poultry industry, turkey production is still considered a growing sector limited to a small scale. Many recent studies have discussed the presence of ESBL producing E. coli in chickens in Egypt, while very few data are available about the same problem in turkeys [17,18].
Carbapenems are considered the last-line agents against ESBL-producing Enterobacteriaceae. The carbapenem resistance is either due to carbapenemase production, decrease in bacterial outer membrane permeability with production of AmpC/ESBL, or due to the efflux pumps [19]. Carbapenemases are beta-lactamases having high affinity for hydrolysis of penicillins, cephalosporins, and carbapenems [20]. Carbapenem-resistant Enterobacteriaceae (CRE) including E. coli, have gained a great attention since their outbreaks in the last two decades [21]. Infections caused by CRE cause high fatality rates in humans due to their resistance against the whole class of beta lactam antibiotics [22,23]. Carbapenemases are usually associated with resistances against other antimicrobial classes such as β-lactamases, aminoglycosides, and fluoroquinolones leaving very limited treatment options. Additionally, carbapenemase-encoding genes are easily transferable through mobile genetic elements as, e.g., plasmids and transposons, and spread easily among Enterobacteriaceae [24]. The CRE were classified as "critical priority pathogens" by World Health Organization [WHO] in 2017 [25] Common carbapenemase genes include oxacillinase-48-like , Klebsiella pneumoniae carbapenemase [KPC], and the New Delhi metallo-β-lactamase [NDM].
Colistin is considered the drug of choice in veterinary medicine for the treatment of recurrent E. coli-related digestive tract infections in animals used for food production. The drug is among the last-resort antimicrobials for the treatment of diseases caused by MDR Gram-negative bacteria. The rising rate of colistin resistance is mainly attributed to the use of the drug in veterinary medicine. The evolution of colistin resistance has recently raised serious concerns and the plasmid-borne mcr gene has been implicated in resistance in Enterobacteriaceae around the globe [17,26].
Many different single PCRs or phenotypic tests are available to obtain information about the different genetic features of E. coli isolates. More extensive information about the bacterial genotype can be obtained by DNA microarrays, which allow the parallel identification of a variety of genes. The microarray includes many oligonucleotide probes in total, covering various genes for clinically relevant features and typing.
The objectives of this study were to estimate the prevalence and antimicrobial resistance (AMR) in E. coli isolates originating from healthy turkey flocks in different districts in northern Egypt and to understand its public health significance. In addition, ESBL, Carbapenemase, and colistin producing E. coli and the possibility of their transmission to humans were investigated.

Isolation and Characterisation of E. coli Strains
A total of 250 cloacal swabs were randomly obtained from healthy turkeys housed in 12 farms spread over five governorates in the Nile Delta of Egypt, namely Dakahlia, Damietta, Kafr El-Sheikh, Sharkiya, and Gharbiya. Table 1 provides information about the studied poultry farms, the number of birds and the number of samples that were collected. Sterile swabs were used to carry out the sampling. The samples were subjected to microbiological analysis in the laboratory directly after being transported at a temperature of 4 • C. The samples were enriched using buffered peptone water. In order to identify ESBL-producing E. coli, after 24 h incubation at 37 • C cultures were streaked on Brilliance™ ESBL agar (Oxoid GmbH, Wesel, Germany), a commercial selective media that contains cefpodoxime (100 mg/L).

Phenotypic Testing for Antimicrobial Susceptibility
All isolates were tested for antimicrobial susceptibility with the MICRONAUT system (Merlin, Bornheim, Germany) using commercial 96-well microtiter plates as per manufacturer's instructions. With the use of this technique, the minimum inhibitory concentrations (MICs) of 14 antimicrobial drugs against E. coli were evaluated (Table 2) using serial antibiotic dilutions. To achieve a turbidity corresponding to a McFarland standard of 0.5, overnight-grown bacteria were suspended in sterile phosphate-buffered saline (PBS, 7.4 pH). The solution was diluted by adding 100 µL of the suspension to 10 mL of Mueller-Hinton broth (Oxoid GmbH), yielding a concentration of roughly 10 6 -10 7 colony forming units (cfu) per ml. A total of 100 µL of the suspension was pipetted in each well of the plate. The plates were then sealed and incubated aerobically for 18 to 24 h at 37 • C. At a wavelength of 620 nm, plates were read using a photometer (MICRONAUT, MERLIN Diagnostika GmbH, Bornheim, Germany). Growth was considered to be indicated by an optical density of >0.1. The German Institute for Standardization's (DIN, Berlin, Germany) criteria were used to interpret MICs using the advanced expert system MCN-6 (Merlin). E. coli ATCC 25922, E. coli ATCC 35218, and K. pneumoniae ATCC 700603 were used as controls.

Identification of Colistin Resistance
The MICs for colistin resistance among isolates were determined using the RUO E-test colistin CO 256 according to the manufacturer's instructions (bioMérieux Deutschland GmbH). To ensure uniform growth, Mueller-Hinton agar plates were equally streaked with an overnight bacterial suspension in Mueller-Hinton broth that had been adjusted to a density of McFarland 0.5. Once the agar surface dried, sterile forceps were used to apply an E-test ® colistin strip to the plate (concentration range: 0.016 to 256 g/mL). After 20 h of aerobic incubation at 37 • C, the MICs were identified as the location where the E-test strip was intersected by the suppression of bacterial growth. According to EUCAST's clinical breakpoints, an isolate was deemed to be colistin-resistant if the MIC value was greater than 2 g/mL [17].

DNA Extraction and Purification
Genomic DNA was extracted from heat-inactivated pure cultures using the HighPure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer's instructions. DNA quantity and purity were determined using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA).

Confirmation of E. coli Isolates Using PCR
The confirmation of an isolate as E. coli was applied using a specific PCR assay targeting 16S rRNA-DNA genes as described in a previous study [29]. The PCR reaction was carried out with the following thermal profile: An initial denaturation step at 96 • C for 60 s was followed by 35 cycles of denaturation (96 • C for 15 s), annealing (58 • C for 60 s), and extension (72 • C at 45 s) with a final extension at 72 • C for 60 s. PCR amplicons of 585 bp were analyzed on a 1.5% non-denaturing agarose gel (PEQGOLD UNIVERSAL-AGAROSE, VWR, Mönchweiler, Germany), stained with ethidium bromide and visualized under UV light (Syngene G: BOX Chemi XT4, VWR, Germany).

Microarray-Prediction of Genoserotypes of E. coli Isolates
The E. coli SeroGenoTyping AS-1 Kit (Abbott Technologies GmbH, Jena, Germany) and the DNA microarray-based assay CarbaResist from INTER-ARRAY (fzmb GmbH, Bad Langensalza, Germany), were used to determine the genotypes of E. coli isolates. Five microliters of extracted RNA-free high-quality DNA (with a concentration of at least 100 ng/µL) were labeled with biotin by a primer extension amplification using E. coli SeroGenoTyping AS-1 Kit according to manufacturer's instructions. The procedures for multiplex labelling, hybridization, and data analysis were carried out as described in previous studies [30].

Detection of Carbapenemase Genes Using Multiplex Real-Time PCR
A multiplex real-time PCR detection of the most common carbapenemase genes blaKPC, blaNDM, blaVIM, and blaOXA-48 was carried out for all E. coli isolates. Primers and probes sequences are included in Table 2. PCR condition was initiated by initial denaturation at 95 • C for 4 min, followed by 50 cycles of denaturation at 95 • C for 30 s, annealing at 50 • C for 30 s and elongation at 72 • C for 60 s and final elongation step at 72 • C for 5 min. Klebsiella (K.) pneumoniae, Citrobacter (C.) freundii, Pseudomonas (P.) aeruginosa, and E. coli were positive controls for the reaction. CTs ≥ 40 were considered negative [31].

Statistical Analysis
A correlation analysis was performed to determine the association of antimicrobial resistance genes belonging to the same antimicrobial class or different classes among the examined isolates. Binary data (0/1) that denote absence/presence of resistance genes were imported into an R software (version 3.6.1; https://www.r-project.org, (accessed on 20 May 2022). The correlations were then calculated with package "corrplot" using the function "cor" and "cor.mtest" at a significance of p < 0.05.

Isolation and Identification of E. coli
Out of 250 samples cultivated on Brilliance TM ESBL agar, 26 suspicious isolates were confirmed as E. coli by MALDI-TOF and conventional PCR.

Antimicrobial Susceptibility Tsting
The results of the phenotypic antimicrobial susceptibility tests of E. coli isolates are illustrated in Table 3. E. coli isolates were all resistant to penicillin, rifampicin, streptomycin, and erythromycin. The resistance rates to tetracycline and trimethoprim/ sulfamethoxazole were 92.3%, each. All isolates were sensitive to imipenem. Concentration ranges provided for each antimicrobial drug, are presented in shadows.
A total of 17 E. coli isolates (65.4%) were confirmed phenotypically as ESBL-producers. Among all isolates, only one E. coli isolate (3.8%) was phenotypically resistant to colistin.

Serogenotyping of E. coli Isolates Using Microarray Analysis
Serogenotyping by microarray revealed that seven out of 26 E. coli isolates (26.9%) belonged to O9, 27, 29, 112, 123, 126, and 141. Other isolates could not be assigned, most likely because several O-serotypes were not covered by the assay.
H-antigen serotypes were identified in all isolates. Eleven different types of H-antigens were detected. H32 was the most common type, it was identified in four isolates, followed by H38 and H05 (two isolates each), while H37, 45,31,25,30,19, 04, and 10 were identified in only single isolates.
Most of resistance and virulence genes against other antimicrobial classes were also identified among these isolates (Tables 4 and S1).

Association of Antimicrobial Resistance Genotypes among the Examined Isolates
The associations between resistance genes either those belonged to the same antimicrobial class or different classes were determined among the examined isolates using correlation analysis. Positive correlations were observed for pairs of resistance genes belonging to the same class, i.e., blaSHV and blaOXA-10 (r = 0.75), sul1 with sul2 (r = 0.57) and sul3 (r = 0.54), mph and mrx (r = 0.67). Positive correlations were also found for the co-occurrence of resistance genes from different antibiotic classes. For instance, blaCTX-M-9 showed positive significant associations with aminoglycosides (aadA2, aphA), macrolide (mph, mrx), sulfonamides (sul1, sul3), and dihydrofolate reductase (dfrA12, dfrA14) resistance genes. On the other hand, negative correlations were observed for genes belonging to the same antimicrobial class (dfrA1 and dfrA14) as well as genes belonging to different antimicrobial classes (blaCTX-M-9 and qnrS; sul1 and qnrS) Figure 1.  Significance was calculated at p < 0.05, and boxes with non-significant correlations were left blank.

Discussion
Commensal E. coli is a normal inhabitant that maintains the normal gut microbiota in poultry. However, avian pathogenic strains (APEC) mostly carry virulence genes and cause extraintestinal infections in birds. Colibacillosis is mostly caused by APEC, including systemic and localized infections such as omphalitis, swollen head syndrome, cellulitis, diarrhea, respiratory colisepticaemia and enteric colisepticemia [17,32]. Furthermore, it could have a zoonotic pathogenic potential such as neonatal meningitis [33]. The infec-

Discussion
Commensal E. coli is a normal inhabitant that maintains the normal gut microbiota in poultry. However, avian pathogenic strains (APEC) mostly carry virulence genes and cause extraintestinal infections in birds. Colibacillosis is mostly caused by APEC, including systemic and localized infections such as omphalitis, swollen head syndrome, cellulitis, diarrhea, respiratory colisepticaemia and enteric colisepticemia [17,32]. Furthermore, it could have a zoonotic pathogenic potential such as neonatal meningitis [33]. The infection of E. coli in poultry farms can spread both vertically and horizontally. It can spread directly by contaminated dust, water, fomites, and feces. E. coli can be inhaled or consumed to cause infection, resulting in illness. High ammonia concentrations, which can harm the skin or respiratory epithelium and make it easier for E. coli to enter the body, are among the most significant non-infectious predisposing variables. The prevalence and severity of E. coli infections are consequently increased by these factors in addition to poor hygiene, short distances between houses, flocks of different ages on a farm, and short service intervals between flocks. E. coli can cause omphalitis and inflammation of the yolk sac when it is vertically transmitted from parent flocks to their offspring. Additionally, it can be transmitted between chicks during hatching and is typically linked to a high mortality rate whenever the yolk sacs become inflamed. Instead of actual vertical transmission within the egg, contamination of the eggshell is mostly to blame for this sequence. Hygiene and management practices are part of the control regimens to stop both the introduction and spread of infection. Longer downtimes between flocks, improved air quality, and thorough cleaning and disinfection are other additional approaches to be considered [34].
Previous studies reported high prevalence of antibiotic resistance rates among APEC in turkeys [15,33,35].
In this study, 26 E. coli were isolated from 250 cloacal swabs collected from 12 turkey farms located in 5 governorates in northern Egypt. The samples were investigated for the presence of ESBL and/or carbapenemase producing E. coli. The E. coli isolates were confirmed either as ESBLs or non ESBLs producers through phenotypic resistance to Ceftazidime. This was a high percentage in healthy turkeys in comparison with a previous result of only 2.2% ESBL producing E. coli isolates from turkeys in other studies [4].
Antimicrobials are usually used in turkey hatcheries to prevent omphalitis in newly hatched birds [36]. Few antimicrobials are available for this purpose, mainly third generation cephalosporins and most are used off-label [37].
In the current investigation, all isolates were MDR, defined as resistant to three or more antimicrobial classes, which is alarming.
Ceftazidime, a third-generation cephalosporin has a high importance in human medicine and it is used to treat severe infections. It is a major concern that the use of ceftazidime in food animals can promote resistance to other cephalosporins, such as ceftriaxone, used in humans and vice versa [38]. Although cephalosporins are not approved for use in poultry, the administration of these drugs in poultry and turkey farms is an emerging problem. Resistance to third and fourth-generation cephalosporins in fecal turkey E. coli has been previously reported [15,35,36,[39][40][41][42]. In this study, the prevalence of ceftazidime resistance among all isolates was 65.4%, while its prevalence in young poults (6-10 days) was 62.5%, which is certainly high.
Gentamicin, an aminoglycoside, is classified as a drug of high importance to humans. In this study, the resistance rate to the drug was found in 65.4% of all isolates and in 62.5% of isolates derived from young poults.
It was reported that the highest rates of resistance among pathogenic and commensal E. coli in turkeys were detected against tetracyclines, penicillins, and sulfonamides [15,40]. The high levels of resistance to these classes may be attributed to a long-term selection pressure, as these antimicrobials are very old and widely used in the poultry industry all over the world [43]. The resistance to tetracycline amongst E. coli is suggested to be selected by a bystander effect on commensal E. coli, during treatment of other Enterobacteriales. Bacterial resistance to tetracycline is most commonly mediated by energy-dependent pumping of tetracycline out of the bacterial cell. The tet(A), -(B), -(C), -(D), -(E), -(Y), and -(I) genes in Gram-negative bacteria encode such efflux systems [44].
In this study, the isolates showed resistance prevalence of 88.5% to chloramphenicol, 92.3% for both tetracycline and trimethoprim/sulfamethoxazole and a full phenotypic resistance prevalence (100%) to penicillin, streptomycin, erythromycin and Rifampicin, respectively. Additionally, all isolates from young poults were fully resistant to penicillin, streptomycin, tetracycline, erythromycin, chloramphenicol, rifampicin, and trimethoprim/ sulfamethoxazole. This result was in accordance with a previous study which reported a high resistance to tetracycline, streptomycin, and sulfisoxazole in E. coli isolated from turkey hatcheries [37]. Moreover, E. coli isolated from turkeys previously were highly resistant to tetracyclines, penicillins, and sulfonamides [45,46].
Erythromycin resistance is intrinsic in E. coli and the members of the erythromycin esterase family known as ereA and ereB, were first discovered in clinical E. coli strains [47].
It has been recognized that fluoroquinolone resistance can be related to the usage of these drugs in a given country, which is in turn influenced by the market dynamics, mainly the cost of commercial products [48]. In this study, most ESBL producing E. coli isolates showed additional high resistance prevalence to fluoroquinolones, aminoglycosides, chloramphenicol, tetracycline, macrolides, and ansamycins. In a previous study, an additional high resistance prevalence to aminoglycosides, sulfonamides, and tetracyclines was recorded among ESBL producing E. coli isolates in turkeys [46].
The resistance to the majority of antimicrobials tested in this study can be attributed to the acquirement of foreign resistance genes through horizontal transfer, which usually involves both pathogenic and commensal bacteria [49,50].
The colistin resistance associated mcr-1 gene was detected first in 2015 in raw meat, livestock and human-originated samples in China. The latest mcr gene variant, mcr-9, was reported first in 2019. This allele shares about 64.5% amino acid identity with mcr-3 [53]. It was previously reported that phenotypic colistin resistance in E. coli is mostly associated with the carriage of mcr-1 gene [54].
In this study, one E. coli isolate (3.8%) was phenotypically colistin-resistant and harbored mcr-9 gene. The mcr-1 gene was previously reported in E. coli isolates from healthy broiler chickens in Egypt [17].
Bacterial efflux pumps (EPs) are proteins that are positioned and embedded in the bacterium's plasma membrane. Their purpose is to recognize toxic substances that have gotten past the organism's protective cell wall and into the periplasm or cytoplasm and extrude them before they reach their target organelles. They are mostly described as a major mechanism of drug resistance [57]. A common form of drug-resistance emergence in Enterobacteriales is mediated by transposons. E. coli is known to harbor a wide range of transposons such as Tn3, Tn5, Tn7, Tn9, and Tn10, that are all related to antimicrobial resistance ampicillin, kanamycin, trimethoprim, spectinomycin, streptomycin, chloramphenicol, and tetracycline [58].
The transmission of resistance genes in bacteria has been linked to a number of acquired resistance mechanisms in recent years, including bacteriophages, transposons, plasmids, and integrons. Integrons are one of the genetic components that may have a role in the widespread occurrence and dissemination of antibiotic resistance [59]. These components have the ability to seize, incorporate, and mobilize antibiotic resistance gene cassettes. Based on the genetic similarity of the integrase intI gene sequence, the integrons were divided into three major groups, namely 1, 2, and 3. Class 1 integrons are more prevalent in Gram-Negative bacteria. Metallo-beta-lactamase (MBL) and class 1 integron are mostly encoded on the same gene cassettes that are circulating in bacterial populations [60]. Furthermore, newly extended spectrum beta lactamases (ESBL)-encoding genes such as blaCTX-M, blaGES, and blaVEB-1 are typically found on integron-like structures [61].
In the current study, the class 1 Integron integrase intI1 gene was detected in 84.6% among E. coli isolates. Moreover, transposase gene for the transposon tnpISEcp1 and OqxB -integral membrane protein, component of RND-type multidrug efflux pump oqxB were identified in 34.6% and 7.7% of isolates.
Similarities between human and avian E. coli virulence-associated factors were previously reported in many studies [62]. Although the E. coli isolates in this study were commensal, they harbored genes encoding virulence factors including fimbrae (ipf A), toxins (astA and cma) that are shared between avian and human-isolated E. coli, in addition to miscellaneous virulence-associated genes (intl1, hemL, iroN, iss). These similarities could emphasize a human transmission origin.

Conclusions
The obtained results strengthen the need to develop surveillance strategies and control procedures to reduce the use of antibiotics and subsequently the development of antimicrobial resistance.
Understanding how an ESBL and/or carbapenemase gene mobilizes through a bacterial population will be critical for detection methods and ultimately inform infection control practices. Additional understanding of gene mobilization and tracking will require novel approaches to surveillance, which will be required to slow the spread of this emerging resistance.
The strategies to contain AMR emergence rely on a robust antibiotic surveillance system, assessing the threats imposed due to the emergence of MDR.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics11081075/s1, Table S1: Phenotypic and serogenotypic characteristics of E. coli isolates. Funding: This work at IPHT was carried out within the framework of the projects RESISTOVAC (Rapid and economical POC tests for determination of immune status and bacterial resistance factors; 13GW0458D) and DRESI (Rapid and accurate diagnosis and resistance testing of sepsis pathogens in the intensive care unit; 13GW0423C). Both projects are funded by the German Federal Ministry for Education and Research and managed by VDI-TZ Phys. Technology.

Conflicts of Interest:
The authors declare no conflict of interest.