Selective pharmacogenetic activation of catecholamine subgroups in the ventrolateral medulla elicits key glucoregulatory responses
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Catecholamine (CA) neurons in the ventrolateral medulla (VLM) contribute importantly to glucoregulation during glucose deficit. However, it is not known which CA neurons elicit different glucoregulatory responses or whether selective activation of CA neurons is sufficient to elicit these responses. Therefore, to selectively activate CA subpopulations, we injected male or female Th-Cre+ transgenic rats with the Cre-dependent DREADD construct, AAV2-DIO-hSyn-hM3D(Gq)-mCherry, at one of 4 rostrocaudal levels of the VLM: rostral C1 (C1r), middle C1 (C1m), the area of A1 and C1 overlap (A1/C1) or A1. Transfection was highly selective for CA neurons at each site. Systemic injection of the DREADD receptor agonist, clozapine-N-oxide (CNO), stimulated feeding in rats transfected at C1r, C1m or A1/C1, but not A1. CNO increased corticosterone secretion in rats transfected at C1m or A1/C1, but not A1. In contrast, CNO did not increase blood glucose or induce c-Fos expression in the spinal cord or adrenal medulla after transfection of any single VLM site, but required dual transfection of both C1m and C1r, possibly indicating that CA neurons mediating blood glucose responses are more sparsely distributed in C1r and C1m than those mediating feeding and corticosterone secretion. Results show that selective activation of C1 CA neurons is sufficient to increase feeding, blood glucose and corticosterone secretion and suggest that each of these responses is mediated by CA neurons concentrated at different levels of the C1 cell group.Selective activation of C1 catecholamine neurons in the ventrolateral medulla is sufficient to increase blood glucose, corticosterone levels and feeding in the absence of glucoprivation.
INTRODUCTION
Glucose is the obligatory substrate for brain energy metabolism, but the brain itself stores very little glucose and depends upon delivery of glucose from the blood. Hence, the brain is equipped with mechanisms that detect glucose deficit and trigger coordinated systemic responses required to defend and replenish its glucose supply. These responses, referred to as counter-regulatory responses (CRRs), activate behavioral, physiological and endocrine systems that counter rapidly developing and profound glucose deficit and facilitate delivery of glucose to the brain (1). These protective responses include, among others, mobilization of stored glucose and fatty acids by adrenal medullary activation and glucagon secretion (2), replenishment of glucose by stimulation of feeding (3), increased gastric motility that facilitates digestion and absorption of ingested food(4), and increased corticosterone (Cort) secretion that increases utilization of fatty acids, thereby decreasing peripheral glucose utilization (5-9). CRRs are essential for survival. Pathological conditions in which they are impaired result in rapid neurological deficit and can be lethal. Hypoglycemia associated autonomic failure (HAAF), a potential side effect of insulin treatment in diabetic individuals (1,10), is an example of such a condition.Several lines of investigation have established definitively that hindbrain catecholamine (CA) neurons are necessary for elicitation of multiple CRRs to acute glucose deficit producedexperimentally by insulin-induced hypoglycemia or by systemic or central administration of the antiglycolytic drugs, 2-deoxy-D-glucose (2DG) or 5-thioglucose (5TG) (11-16). For example, selective ablation of CA neurons using the retrogradely transported immunotoxin, anti-dopamine beta hydroxylase (DBH) conjugated to the ribosomal toxin, saporin (DSAP) (17,18), revealed that lesion of hindbrain CA neurons with projections to the hypothalamus eliminate glucoprivation-induced feeding (19) and elevation of Cort levels (13), modulation of growth hormone secretion (20) and suppression of estrous cycles (21).
Retrograde lesion of CA neurons resulting from DSAP injections into the intermediolateral column (IML) of the spinal cord also established that spinally-projecting CA neurons are necessary for glucoprivic control of adrenal medullary secretion (14, 19), a result later confirmed in rats in which DSAP was injected directly into the rostral subregion of the ventrolateral medulla (VLM), which houses cell bodies of spinally-projecting CA neurons (22).Despite strong evidence from various approaches that hindbrain CA neurons are necessary for elicitation of key CRRs, the specific phenotypes and characteristics of the CA neurons that mediate specific glucoregulatory responses are not known. Furthermore, it is not yet clear whether selective activation of distinct subpopulations of CA neurons in the VLM is sufficient to evoke specific CRRs. These questions have not been answered, in part because of the anatomical complexity of the CA neurons themselves, including their collateralized projections, the apparent co-mingling of their functional subtypes, and the lack of technical approaches to overcome these obstacles. Therefore, in the present experiments, we used viral transfection of a Cre-dependent Designer Receptor Exclusively Activated by Designer Drugs (DREADD), a new technology that would enable selective activation of VLM CA neurons transfected at different rostrocaudal levels in Th-Cre+ transgenic rats by systemic injection of clozapine-n-oxide (CNO), the synthetic agonist for the DREADD receptor (23). We tested the effects of selective CNO activation of CA neurons on Cort secretion, blood glucose and food intake and on c-Fos expression in the VLM, dorsomedial medulla (DMM), hypothalamus, spinal cord and adrenal medulla.
Our results show for the first time that selective pharmacogenetic activation of C1 neurons is sufficient to stimulate robust feeding, Cort and blood glucose responses in the absence of glucoprivation and suggest a differential distribution of neurons mediating these specific responses within the C1 cell column. These results will provide a foundation for further work to identify the specific neurons that mediate different CA responses to glucose deficit, how these responses are integrated to control emergency counter-regulation and how they participate in the overall control of metabolism.Male and female rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (Long Evans-Tg(Th-Cre)3.1, or Th-Cre+) and their wild-type non-Th-Cre (Th-Cre-) littermates were used in these experiments (23). Transgenic rats were bred and raised in the VBRB Vivarium at Washington State University from breeding stock generously provided by Dr. Karl Deisseroth (Stanford University, Stanford, California).Rats were maintained on a 12-hr light/12-hr dark cycle (lights on at 7 a.m.) with ad libitum access to pelleted rodent food (#5001; LabDiet) and tap water. All experimental procedures were approved by Washington State University Institutional Animal Care and Use Committee, which conforms to NIH guidelines.Genotyping was performed at 3 weeks of age using PCR. One ear punch (1.0-mm diameter) was heated at 95 ˚C (1 hr) in 100 µl alkaline lysis buf fer (15 mM NaOH/0.2 mM Na2-EDTA.2H2O; pH, ~12). After mixing with 100µl neutralization bu ffer (40 mM Tris-HCl; pH, ~5), and a 3-min centrifugation, 4 µl of supernatant was used for PC R. PCR was performed in SYBR green buffer with a forward primer (5’-GCGGCATGGTGCAAGTTGAAT-3’) and a reverse primer (5’-CGTTCACCGGCATCAACGTTT-3’). PCR cycles were 94˚C for 3 min, then 94˚C (20 sec), 61˚C (20 sec), and 72˚C (20 sec) for 35 cycles, fol lowed by a melting curve reaction.
A single production band was ctonfirmed by gel electrophoresis, and by a single melting curve peak at 86.5 ˚C.CA cell groups in the hindbrain are defined as in The Rat Brain in Stereotaxic Coordinates (24). Cell groups C1 and A1 are continuously distributed along the rostrocaudal extent of the VLM. Therefore, we have adopted additional terms to subdivide these groups. We refer to A1 as the portion of A1 caudal to overlap with C1 (14.9-14.2 mm caudal to bregma). We refer to caudal portion of C1 overlapping the rostral portion of A1 as A1/C1 (14.1-13.4 mm caudal to bregma). The middle portion of C1 is referred to as C1m (13.3-12.6 mm caudal to bregma). The rostral portion of C1 is designated as C1r (12.5-11.8 mm caudal to bregma). These areas were separately targeted for viral transfection in different groups of TH-Cre+ and TH-Cre- rats. Parameters for accurate targeting of viral injections to each of these sites, including stereotaxic co-ordinates, injection volume, and the effectiveness and selectivity of the viral transfections for targeted CA neurons was determined prior to the main experiments (See Results).After completion of preliminary studies, viral constructs were injected into 12 – 14 week old male and 16 – 18 week old female TH-Cre + rats and TH-Cre- male and female controls of the same age, as follows. A recombinant adeno-associated virus (AAV) containing a Cre-dependent doubly-floxed inverted open reading frame encoding and a reporter gene (mCherry) under a human synapasin I promotor, AAV2-DIO-hSyn-hM3D(Gq)-mCherry (AAV-hM3D), was injected into targeted sites along the rostrocaudal extent of the VLM CA cell column. Rats were anesthetized using 1.0 ml/kg of ketamine/xylazine/acepromazine cocktail (50 mg/kg ketamine HCl, Fort Dodge Animal Health; 5.0 mg/kg xylazine, Vedco; 1.0 mg/kg acepromazine, Vedco) and placed in a stereotaxic device. Two batches of virus (titers were 2 and 4 x 1012 particles/mL; University of North Carolina Vector Core) were used during the experiment. No differences in transfection were found between these two batches in tests conducted prior to use in the main experiments.
The virus was injected as provided (without dilution) using a Picospritzer through a pulled glass capillary pipette (30-µm tip diameter), bilaterally (200 nL/spot) int o A1, A1/C1, C1m or C1r in male rats. Pipettes were positioned dorsolateral to the targeted site and directed medially at a 14-degree angle to avoid damage to or transfection of CA neurons in the nucleus of the solitary tract (NTS) by potential diffusion of the viral construct along the pipette tract (Fig.1). The following coordinates were used: 14.45 mm (for A1), 13.75 mm (for A1/C1), 12.95 mm (for C1m), or 12.25 mm (for C1r) caudal to bregma; 4.0 – 4.1 mm lateral to midline, and 8.4 – 8.9 mm ventral to the skull surface (24).In a separate experiment using female Th-Cre+ rats, AAV-hM3D was unilaterally injected into both C1m and C1r (C1m+C1r AAV) in each rat in order to increase the distribution of the DREADD transcript in the VLM. Injections were made unilaterally, rather than bilaterally as in the other groups, so that the total amount of virus injected into the VLM would be consistent across groups.Measurement of food intake and blood glucose responses to systemic CNO and glucoprivation in VLM hM3D(Gq)-transfected ratsResponses to CNO were tested beginning 5 weeks after DREADD transfection. To activate hM3D(Gq) receptors, CNO (Tocris) was dissolved in sterile saline (0.9 %) and injected at a dose of 1 mg/kg (i.p.). The antiglycolytic drug, 2DG (Sigma-Aldrich), was dissolved in sterile saline, and injected at a dose of 250 mg/kg (s.c.) to induce glucoprivation (19). Food intake was measured in a 4-hr test after injection of CNO, 2-DG or the saline (0.9%) vehicle at 9:30 a.m. in 1 hr-fasted rats. Blood glucose concentrations were measured in the absence of food before and during a 4 or 6 hr period after drug injections at 10:30 a.m. in 2 hr-fasted rats.
Glucose was measured from tail blood using a GlucoCard Vital glucose meter (Arkray). All tests were performed between 5 and 9 weeks after AAV injections, when transfection was stably expressed. Individual tests were separated by at least 3 days.Separate groups of male rats, transfected at the same sites described above for feeding and blood glucose testing (A1AAV-hM3D, A1/C1AAV-hM3D, C1mAAV-hM3D, C1rAAV-hM3D)) were prepared foranalysis of CNO- and 2DG-induced Cort secretion. To minimize handling stress during sample collection for Cort measurement, these rats were implanted with chronic jugular catheters for remote blood collection. The catheter, made of silastic tubing (inside diameter, 0.64 mm; outside diameter, 1.19 mm; Dow Corning), was implanted into the atrium through the right jugular vein under anesthesia induced by ketamine/xylazine/acepromazine cocktail (13). As in other groups, tests were performed between 5 and 9 weeks after stereotaxic DREADD transfection. Catheters were implanted approximately 1 week prior to collection of blood for Cort determination. After recovery, rats were habituated to 30 cm × 10 cm opaque Plexiglas chambers used for remote blood sampling. On test days, 2-hr fasted rats were placed in the chambers, and blood samples (~300 ul) were collected remotely at several time points before and after each treatment. After clotting for 1-2 hr at 4 ˚C, and centrifugation, serum samples were stored at −80 °C (25). Plasma Cort levels were meas ured with a mouse/rat Corticosterone ELISA kit (#IB79175, IBL America), according to the provided procedure.After completion of behavioral tests (8 – 10 weeks after AAV-hM3D injections), rats were food deprived for 2 hr and injected with CNO (1 mg/kg; i.p.), or saline. Two hours later, they were euthanized by deep isoflurane-induced anesthesia (Halocarbon Products). Just prior to cessation of the heartbeat, rats were perfused transcardially with PBS (pH 7.4) followed by freshly made 4% formaldehyde/PBS solution.
Brain, adrenal medulla and spinal cord were rapidly removed and placed in 4% formaldehyde/PBS for 5 hr followed by immersion in 25% sucrose in PBS overnight (16 hr) and then sectioned with a cryostat at 40 µm thickness. Coronal brain sections for hindbrain and hypothalamus (four serial sets), sagittal spinal cord sections (one set) and adrenal medullary sections (two sets) were collected for analysis of the distribution and selectivity of DREADD transfection and for c-Fos expression induced by CNO.For double or triple immunofluorescence (IF) staining, sections were incubated with primary antibodies at 4˚C (2 days) in 10% normal horse serum (NHS)/PBS, washed, and then incubated in secondary antibodies (4 hr) (26, 27). Following antibodies were used: mouse anti-mCherry (ClonTech Labs), rabbit anti-DsRed (ClonTech Labs; for detecting mCherry); rabbit anti-TH (Millipore); mouse anti-DBH (Millipore); goat anti-c-Fos (SantaCruz Biotechnology) antibody, and donkey anti-mouse, donkey anti-rabbit, or donkey anti-goat antibody, conjugated with Alexa 488, Cy3, or Alexa 647 (all 1:500 dilution in 1 % NHS/TPBS; Jackson ImmunoResearch).Sections were mounted and coverslipped with ProLong Gold medium (ThermoFisher Scientific), and observed and photographed using a Zeiss epifluorescent or a Leica confocal microscope.For c-Fos staining in adrenal medulla and spinal cord, standard avidin-biotin-peroxidase methods were used. Sections were incubated with goat anti-c-Fos antibody (1:5,000) for 2 days, followed by overnight incubation in biotinylated donkey anti-goat IgG (1:500; Jackson ImmunoResearch) and a 4-hr incubation in ExtrAvidin-peroxidase (1:1,500; Sigma-Aldrich).
Nickel-intensified diaminobezidine was used to produce a gray/black reaction product by peroxidase reaction (27,28).A virus injection was considered to be targeted accurately if mCherry-expression was contained within one of our defined VLM subdivisions (see Methods). Typically, each defined target zone was comprised of 4 – 5 tissue coronal sections. For quantification in rats with bilateral single site injections, DBH-, TH-, mCherry and c-Fos-positive cells were counted bilaterally in each rat from the three coronal sections within the targeted zone containing the most abundant mCherry signal. In rats with injections at two adjacent sites (C1r+C1m) labeled cells were counted from seven coronal sections containing the most abundant mCherry signal.The DMM also contains CA neurons. These cells are distributed in cell groups A2 in the caudal DMM and C2 in the rostral DMM. Although our pipettes were angled to avoid traversing these areas, we nevertheless examined the DMM in the experimental animals in order to identify any cells that may have been inadvertently transfected by the DREADD construct and to identify areas of c-Fos expression. Cells in A2, subpostrema (Sub-P) and dorsolateral solitary nucleus (Sol-DL) were examined and counted (two sections/rat) at 13.9-13.7 or 14.4-14.2 mm (for Sub-P) caudal to bregma.Two sites innervated by VLM CA neurons, the paraventricular hypothalamus (PVH) and the IML of the thoracic spinal cord were also examined for c-Fos expression in response to CNO in the VLM transfected rats. This was done to verify that CNO activation of VLM sites produced expected activation of VLM CA projection sites. In addition, c-Fos expression in the adrenal medulla, which is innervated by preganglionic neurons in the IML, was assessed. Positive cells in PVH were counted bilaterally from three sections per rat (at 1.5 – 1.9 mm caudal to bregma). IML and adrenal medullary c-Fos expression were not quantified.All results are presented as mean ± SEM. For statistical analysis of data, we used unpaired t-test, one-way or two-way repeated measures ANOVA, as appropriate. After significance was determined by ANOVA, multiple comparisons between individual groups were tested using a post hoc Student-Newman-Keuls test. P < 0.05 was considered to be statistically significant. RESULTS Preliminary experiments were conducted to determine the final protocols for the main experiments. The efficacy, specificity and time course of hM3D(Gq) expression in CA neurons were investigated using double IF staining of the reporter gene mCherry plus DBH or TH in female Th-Cre+ rats after bilateral AAV-hM3D injection into A1/C1. Both CA-directed antibodies produced similar results. Using standard microscopy, as shown Fig. 1, 24 - 26% of DBH- or TH-immunoreactive (ir) neurons in A1/C1 were mCherry-positive 3 weeks after AAV injection. At 6 and 9 weeks, respectively, 41 - 49% of CA neurons were mCherry-positive, 84 and 89% of mCherry-positive cells were DBH-ir and 82 and 89 % were TH-ir. More sensitive analysis using confocal images at 6 weeks revealed that almost all (93%) mCherry-ir cells were CA neurons. The rostrocaudal extent of mCherry expression at each injection site was 0.50 ± 0.02 mm in these rats. In contrast, female control Th-Cre- rats with AAV-hM3D injected unilaterally into A1/C1 showed no mCherry-ir cells at 8 weeks after virus injection in either the injected or non-injected side of VLM. Similar transfection rates were confirmed in males. Male Th-Cre+ rats with AAV-hM3D injection in A1/C1 showed a similar mCherry transfection rates at both 5 and 10 weeks (~ 50% and also see below for details) that were similar to those of female rats at 6 or 9 weeks (shown above). Together, these preliminary results indicate effective and selective mCherry expression in CA neurons. No mCherry-positive cell bodies were found in any area outside of VLM in either male or female rats (n = 3 – 5 rats per sex) . There were no gender-related differences in the extent of virus transfection in female and male Th-Cre+ rats in these studies. Based on these preliminary results, responses to systemic CNO injection were tested in the main experiments between 5 and 9 weeks after AAV injection when m-Cherry expression was stable. Effects of pharmacogenetic activation of CA neurons on feeding, blood glucose and Cort secretion Effects of selective CNO-induced activation of CA neurons on feeding and blood glucose were investigated in male Th-Cre+ rats after bilateral AAV-hM3D injection into A1, A1/C1, C1m or C1r (Fig. 2). CNO (1 mg/kg) injection significantly increased 2 hr and 4 hr food intake compared to intake following saline injection in A1/C1AAV-hM3D, C1mAAV-hM3D, and C1rAAV-hM3D rats, but not in A1AAV-hM3D rats. The magnitude and time course of the feeding response was comparable at all three positive sites to the response induced by 2DG (250 mg/kg) in the same rats. Surprisingly, injection of the same dose of CNO that stimulated food intake did not change blood glucose levels in these same rats, although blood glucose levels were significantly increased in these rats by 2DG (Fig. 2). Changes in plasma Cort levels in rats transfected with AAV-hM3D in A1 , after saline, 2DG (250 mg/kg) and CNO (1 mg/kg) (Fig. 2). CNO increased Cort levels most effectively in A1/C1 and C1m transfected rats, increased Cort only slightly in C1r transfected rats, and did not increase Cort in A1 transfected rats. The effects of 2DG and CNO on food intake and blood glucose at the four injection sites in this group of rats were similar to those shown for the previous group and are not presented here. Between 8 and 10 weeks after injection of AAV-hM3D into VLM subregions (about one week after testing was complete), rats were treated with CNO (1 mg/kg) or saline and prepared for anatomical evaluation of viral transfection selectivity and distribution and CNO-induced c-Fos expression (Fig. 3). The specificity of mCherry expression in CA neurons was investigated by double IF staining of DBH and mCherry. The expression of mCherry was limited to the targeted site. Figure 3A shows the extent of the viral transfection along the rostrocaudal axis of the VLM for each targeted site. Labeling extended 0.62 ± 0. 05, 0.66 ± 0.06, 0.77 ± 0.08 and 0.64 ± 0.06 mm, rostro-caudally along the VLM for A1AAV-hM3D, A1/C1AAV-hM3D, C1mAAV-hM3D, or C1rAAV- hM3D rats, respectively. Supplementary Fig. S1-S4 show distribution of mCherry labeled neurons for each of the 4 targeted VLM levels from 2 representative rats from each target group. Cell counting revealed that 53.9 ± 1.9, 49.9 ± 1.9, 53.5 ± 1.9 and 48.6 ± 2.9% of DBH-ir neurons were mCherry-positive in A1AAV-hM3D, A1/C1AAV-hM3D, C1mAAV-hM3D, or C1rAAV-hM3D rats, respectively (Fig 3D). CNO injection dramatically increased c-Fos expression in virus- transfected CA neurons (Fig. 3B - 3E). Double IF staining with c-Fos/mCherry indicated that 78.2 ± 2.9%, 84.9 ± 1.6%, 90.5 ± 1.8%, or 81.6 ± 1. 8% of mCherry labeled neurons were activated in the injection site in A1AAV-hM3D, A1/C1AAV-hM3D, C1mAAV-hM3D, and C1rAAV-hM3D rats, respectively (Fig. 3B and 3D). Triple IF staining for DBH/c-Fos/mCherry demonstrated that most c-Fos-positive cells (86.5%, 90.2%, 84.7% and 90%, respectively) were triple labeled (Fig. 3C and 3E). Histological analysis revealed 4 rats (from total of 65 rats) in which injections of the AAV-hM3D construct were apparently “off-target”. As the y produced no evidence of transfection at any site, these injections were undoubtedly delivered into non-CA areas, most likely dorsal to the target site. None of these rats responded to CNO, but did show typical responses to 2DG, as shown for control rats. Data were not included in the statistical analyses, as the injection site could not be determined. Activation of neurons in the DMM by CNO-induced stimulation of A1 and C1 neurons was also examined. As noted, mCherry-positive fibers, but no mCherry positive cell bodies, were found in the DMM (Fig. 4A-F and H) in any of the AAV-transfected rats, confirming the selectivity of transfection for VLM-targeted sites. In A1AAV-hM3D, A1/C1AAV-hM3D, C1mAAV-hM3D, C1rAAV-hM3D and C1m+C1rAAV-hM3D transfected rats, c-Fos was found in limited regions of the NTS, especially in areas just ventral to the fourth ventricle, namely in the Sub-P and Sol-DL after CNO treatment. In these two regions A1/C1AAV -hM3D transfected rats expressed higher numbers of c-Fos-ir neurons than the rats transfected at any of the other C1 sites. Double IF staining with DBH revealed that these CNO-activated neurons were non-CA neurons (Fig. 4G). CNO increased c-Fos expression in PVH in A1/C1 AAV-hM3D and C1mAAV-hM3D rats (Fig. 5), the only rats in which PVH c-Fos was examined. This increased activation of PVH is consistent with our previous report (27), with CNO-induced elevation of plasma Cort levels and strong expression of mCherry fibers in PVH in the present study, and with activation of CA neurons known to innervate the PVH (29, 30). Effects of pharmacogenetic activation of C1m plus C1r neurons on feeding, blood glucose and Fos expression in IML and adrenal medulla In this experiment, AAV-hM3D was unilaterally injected into both C1m and C1r in female Th-Cre+ rats. As shown in Fig. 6A and 6B, CNO (1 mg/kg) injection increased both food intake and blood glucose in dual transfected rats to levels roughly equivalent to those produced in the same rats by systemic 2DG (250 mg/kg). Female rats transfected at C1m+C1r did not differ from males with respect to food intake when intake was corrected for differences in body weight. At the time of feeding tests, body weight of females was 61.0 ± 1.3 % of males’ weight, and food intake of females was 60.6 ± 9.2 % of males’ intake (data includes saline, 2DG and CNO tests for A1, A1/C1, C1m and C1r transfected male rats). Dual transfection of both C1m and C1r produced more extensive mCherry expression in rostral and middle C1 than when separate injections were made into each single site. The rostrocaudal extent of mCherry expression was 1.32 ± 0.08 mm in these rats (Fig. 6E and Supplementary Fig. S5). Similarly, 58.4 ± 2.4 % of DBH-ir neurons near the injection sites were mCherry-positive. Expression of c-Fos in C1m+C1r regions was significantly increased by CNO injection compared to control saline injection (Fig. 6C and 6D). Double staining revealed that most (86.5 ± 1.0 %) mCherry-ir cells were c-Fos-positive. Triple IF staining of c-Fos/mCherry/DBH confirmed that 84.6 % of mCherry/DBH cells were activated by CNO treatment. In contrast to rats with single transfection sites, CNO increased c-Fos expression in the IML cell column at thoracic segments T5-T10 and in the adrenal medulla in rats with dual transfection of C1m+C1r (Fig. 7), consistent with the stimulation of a hyperglycemic response in these rats. DISCUSSION Injection of Cre-dependent AAV-hM3D-mCherry into the C1 or A1 cell group in Th-Cre+ transgenic rats produced a selective and effective transfection of CA neurons at each injection site. In Th-Cre+ rats virtually all of the mCherry labeled cells at the injection sites were CA neurons, while no mCherry labeled cells were present in non-Th-Cre (Th-Cre-) rats. Furthermore, we found no evidence of transfection of cell bodies in CA cell groups outside the VLM (A2, A5, C2, C3) following C1 or A1 injections in TH-Cre+ rats. These observations support our conclusion that responses to CNO resulted from activation of DREADD constructs expressed in A1 and C1 and not from DREADD activation of other CA cell groups.Activation of the transfected A1 and C1 neurons following systemic CNO injections evoked robust behavioral, autonomic and endocrine responses that were similar in magnitude and time course to those elicited by systemic 2DG. Double staining for c-Fos and mCherry immunoreactivity revealed that the majority (78-91%) of c-Fos-ir neurons were also mCherry+ immunopositive, while triple IF staining (c-Fos/mCherry/DBH) confirmed that 85-90% of mCherry/DBH cells were activated by CNO treatment. These anatomical results indicate that the CNO-induced feeding, Cort and blood glucose responses we observed in this experiment result from activation of CA neurons in the VLM (and most likely in C1), indicating that selective activation of these CA neurons is sufficient to elicit critical glucoregulatory responses even in normoglycemic rats.As the sole mechanism for replenishment of depleted glucose reserves, increased food intake is of obvious importance as a CRR. Prior results indicate that hindbrain CA neurons that project to the hypothalamus are necessary for increased food intake in response to glucoprivation. Selective retrograde ablation of hypothalamically projecting CA neurons, resulting from PVH or lateral hypothalamic immunotoxin injection, permanently abolish glucoprivic feeding (19, 26), without apparent disruption of other controls of food intake or the ability of lesioned rats to maintain body weight under ad libitum feeding conditions (19). Interestingly, most of the rostral projecting A1/C1 neurons co-express neuropeptide Y (NPY) and selective co-silencing of DBH and NPY genes in A1/C1 also attenuates feeding in response to systemic glucoprivation (2DG) (28). Nevertheless, because injection of DSAP into PVH damages CA neurons in the dorsal as well as ventral medulla (13,19), the sufficiency of ventral medullary CA neurons to increase food intake has remained uncertain. However, the results reported here clearly show that selective CNO activation is sufficient to increase food intake at each of the DREADD transfection sites along the entire longitudinal extent of the C1 cell group. Importantly, although many CA neurons throughout the A1 as well as the C1 cell group are activated by glucose deficit (11, 31), food intake was not evoked by DREADD activation of A1 caudal to its overlap with C1. Hence, our results strongly suggest that the neurons that mediate feeding in response to glucose deficit are C1 CA neurons.Adrenal medullary mediated hyperglycemia is of major importance for rapid restoration of blood glucose in response to acute glucose deficit. Adrenal medullary epinephrine promotes glucagon secretion, inhibits insulin secretion and acts directly on the liver and white adipose tissue to mobilize stored glucose, and also fatty acids. The presence in the VLM of spinally-projecting CA neurons that are necessary for the adrenal medullary hyperglycemic response toglucoprivation has been recognized for some time (19, 22, 32-37). The present results indicate that selective CNO-induced activation of these spinally-projecting CA neurons is sufficient to produce adrenal medullary c-Fos expression and hyperglycemia in the absence of glucoprivation, thus confirming their fundamental role in adrenal medullary activation. Our results may also indicate that the C1 neurons controlling adrenal medullary secretion are more widely distributed along the longitudinal extent of C1r and C1m than previously thought, overlapping considerably with neurons controlling food intake and, to a lesser extent, those controlling Cort secretion. The fact that stimulation of feeding and Cort required transfection of only one C1 site, whereas stimulation of blood glucose responses required transfection of two adjacent sites, may be due to a more diffuse distribution of blood glucose control within the VLM.It has become clear that, in addition to the differences in their projection targets, the functionally distinct populations of the C1 neurons mediating feeding and blood glucose responses differ in other ways as well, including the signaling mechanisms that activate them. Specifically, glucosamine and alloxan (both glucokinase inhibitors), phloridzin (a sodium-linked glucose transporter antagonist) and 5TG (an antiglycolytic agent) all increase feeding when injected intraventricularly (38-42), but only 5TG, injected into these same sites, increases blood glucose (43). Moreover, we have shown that these agents increase feeding in a CA-dependent manner regardless of whether they are injected into the lateral or fourth ventricle (43). Therefore, future investigations are expected to reveal additional phenotypic differences between these CA neuronal subgroups that reflect their distinct physiological roles in glucoregulation.Corticosterone secretion contributes importantly to glucose counter-regulation. Among its other actions, Cort promotes peripheral amino acid mobilization, hepatic gluconeogenesis, reduced insulin sensitivity, increased utilization of fatty acids and reduced glycolysis (5-9). These effects of Cort secretion are associated with a rapid shift in metabolic substrate utilization that increases and preserves glucose availability to the brain (44). However, it is important to note that these actions are not responsible for the rapid elevation of glucose that occurs in response to acute glucose deficit. The latter hyperglycemic response is mediated by adrenal medullary epinephrine secretion and is eliminated by adrenomedullary denervation (32,33). In addition, hypothalamic DSAP injections that reduce the Cort response to glucoprivation do not impair the adrenomedullary hyperglycemic response (13).Previous neurochemical data strongly support a role for CA neurons in stimulating Cort secretion in response to some stressors (29-30, 45-47). Hypothalamic CRH neurons are major innervation targets for C1 neuron axons (48). CA subpopulations within the C1 cell group co-express NPY and/or cocaine-and- amphetamine-regulated transcript (CART) (31). Both NPY and CART are involved in control of Cort secretion. Of all NPY neurons in the brain, C1 neurons provide the largest percentage of NPY innervation to the PVH (mainly the parvocellular region) (30). Similarly, CRH neurons in the parvocellular PVH are densely innervated by terminal varicosities that contain the epinephrine biosynthetic enzyme, phenethanolamine-N-methyl transferase, and co-express CART (49, 50). Currently, the relative importance of co-release of CA with NPY and/or CART is not known.Previously we demonstrated that nanoliter injections of 5TG into A1/C1, increase Cort secretion (15). We also demonstrated that Cort secretion in response to glucoprivation is impaired in rats in which hypothalamically-projecting CA neurons are retrogradely ablated following hypothalamic DSAP treatment (13). In addition, a recent report indicates that optogenetic stimulation of C1 CA neurons increases plasma Cort in DBH-Cre+ mice (51). Our current results, showing that activation of DREADD-transfected C1 neurons evokes Cortsecretion in rats are consistent with these prior reports and collectively confirm the presence of a VLM to PVH CA pathway that increases Cort secretion in response to glucose deficit. CNO injections most effectively triggered Cort secretion in rats with DREADD transfection of A1/C1 and C1m neurons, suggesting a relatively restricted distribution of CA neurons mediating this response. Retrograde lesions of hindbrain CA neurons by hypothalamic DSAP injections impair the Cort response to glucoprivation, but not the circadian rhythm of Cort secretion or the response to swim stress (13). Therefore, it is possible that the Cort response elicited in this study by CNO activation of VLM CA neurons is specifically related to glucoregulation. However, it is also possible that at least some CA neurons may elicit Cort secretion in response to inputs from neurons responsive to some nonglucoprivic stressors (37).Pharmacogenetic activation of C1 neurons triggered downstream activation of neurons that receive CA neuron projections. For example, activation of C1 neurons resulted in increased c-Fos expression in the PVH. In addition, some non-CA neurons, but no A2 neurons, in the dorsal medulla were activated following CNO injection of A1/C1 DREADD transfected rats. In addition to demonstrating activation of downstream targets, our results also indicate that A2 neurons are not excited by projections from VLM CA neurons. Indeed if A2 neurons do receive projections from A1 or C1 neurons within our transfection sites, these projections must be inhibitory.None of the CRRs measured in this experiment were activated by CNO in rats in which A1 neurons were transfected caudal to the A1/C1 group. Although this result encourages a focus on the role of C1 neurons in activation of glucoprivic feeding, Cort and adrenal medullary responses, we cannot conclude that the A1 neurons in the A1/C1 cluster do not contribute to these responses. Neurons throughout the extent of the A1 group respond to both glucoprivation and, after A1 DREADD transfection, to CNO. A1 neurons contribute to the noradrenergic innervation of hypothalamic magnocellular neurons in the supraoptic nucleus of the hypothalamus (4, 52-54) and are involved in a number of neuroendocrine responses that may not be directly related to glucoregulation. For example, we found previously that the same hypothalamic DSAP lesions that abolished insulin-induced food intake and Cort secretion also impaired the vasopressin response to hypovolemic hypotension. However, hypoglycemia did not stimulate vasopressin secretion in controls, indicating that at least some of these A1 are controlled by nonglucoprivic signals (55), as is also true for C1 neurons (56).Although responses to profound glucoprivation require hindbrain CA neurons, it is still not known whether glucoprivic signals activate these neurons directly or whether CA neurons are downstream of other glucose-sensitive cells. However, glucoprivic feeding and elevation of blood glucose can be elicited even in rats in which all connections between forebrain and hindbrain have been severed by midcollicular decerebration (57-59). In addition, these responses can be elicited by fourth, but not lateral ventricular administration of the antiglycolytic agent, 5TG, after acute blockade of the cerebral aqueduct (60). In addition, localized nanoliter injections of 5TG into specific hindbrain CA sites robustly stimulate these and other counter-regulatory responses (12, 15). Therefore, glucose-sensing cells of sufficient potency to drive these reflex responses must be located in the hindbrain. However, it is also clear that critical interaction occurs between CA neurons and forebrain sites, which in some cases may be bidirectional. For example, it is well known that orexin stimulates feeding. However, we have shown that CA neurons activate orexin neurons during glucoprivation and in response to CNO in A1/C1 DREADD transfected rats (26), but that retrograde lesion of CA neurons by PVH or lateral hypothalamic DSAP injection abolishes feeding induced by either lateral or fourthventricular injection of orexin (26,27). Similarly, activation of orexin neurons stimulates a hyperglycemic response via an orexinergic lateral hypothalamic to VLM pathway (61) and optogenetic stimulation of the PVH elicits a hyperglycemic response by a PVH to VLM pathway (37). Possibly such interactions occur during glucoprivic conditions, as well as under circumstances other than urgent glucoprivation, to engage and co-ordinate appetitive responses such as food seeking, to facilitate co-ordination of ongoing behaviors with anticipated or ongoing increases in glucose utilization, and possibly also to expand the participation of hindbrain glucoregulatory mechanisms in multiple aspects of metabolic homeostasis. In short, the signaling elements that activate CA neurons required for CRRs, the circuitry controlled by these neurons, and the mechanisms that maintain activation of the required circuitry during glucoprivation are important questions that will require additional work. In summary, the data reported here show that selective activation of CA neurons in the C1 cell group is sufficient to activate three CRRs that are crucial for maintenance and/or restoration of blood glucose in the face of glucose deficit. Taken together with previous work showing that selective immunotoxin DSAP lesion of these neurons permanently CNO agonist abolishes all three of these responses to glucose deficit (13,16,19), our findings focus attention on VLM CA neurons as crucial mediators of CRRs. Finally, our results also are consistent with the hypothesis that CA neuron dysfunction may play a role in the pathogenesis of HAAF, a complication of insulin therapy (1,10) in which protective and restorative responses to glucose deficit are impaired (62, 63).