Supplemental Materials and Methods
SI test (continued)
The apparatus has an open roof and is approximately 0.9 m long x 0.9 m wide with 0.3 m walls. All tests
were videotaped. The “experimental” rat and an unfamiliar “partner” rat were individually habituated to the box
for a 5 min period 24 h prior to each test. During the SI test, the two rats were placed together in the center of the
box for 5 min. A baseline SI test was performed 72+ h after i.v. catheterization, but prior to osmotic minipump
implantation. Another SI test was performed 5 days following minipump infusions and immediately following
saline or sodium lactate infusions. The total duration (sec) of non-aggressive physical contact (grooming, sniffing,
crawling over and under, etc.) initiated by the “experimental” rat was quantified over the 5 min duration by SDF,
whom was blind to treatments.
Immunohistochemistry (continued)
Briefly, sections were washed in PBS and then incubated in 1% H2O2 in PBS for 20 min. Sections were
washed in PBS then PBST then incubated 12-16 h in PBST with primary antibody. The following day, sections
were incubated 2 h in the appropriate secondary antibody: biotinylated swine anti-rabbit IgG (c-Fos, TH; Cat no.
E0353; DAKO, diluted 1:200), biotinylated rabbit anti-sheep IgG (ChAT, Cat. no. BA6000, Vector Laboratories,
diluted 1:500) or biotinylated horse anti-mouse IgG (GAD67 and NeuN, Cat. no. BA-2000, Vector Laboratories,
diluted 1:500). Sections were washed again for 30 min in PBST then incubated 1.5 h in an avidin-biotin-peroxidase
complex (Cat no. PK-6100, Vector Laboratories, diluted 1:200). Substrates for chromogen reactions were SG (cFos; Cat. no. SK-4700, Vector Laboratories) or 0.01% 3,3’-diaminobenzidine tetrahydrochloride (TH, ChAT,
GAD67 and NeuN; DAB, Cat. no. D-5637, Sigma) in PBS containing 0.003% H2O2, pH 7.4. Substrate reactions
were run for 20 min for c-Fos, TH, ChAT, GAD67 and NeuN.
Data generation for immunohistochemistry (continued)
Quantification of the number of c-Fos-ir cells in the PAG (at -7.04, -7.60 and -8.30 mm bregma) was
performed at 400 x magnification (i.e., 40x objective + 10x eyepiece) by centering the field of view over a
subregion of the PAG and counting only the c-Fos-ir cells in this field of view (regional counts did not overlap and
represented a 0.25 mm2 area/side).
For analysis of c-Fos-ir cell counts in some brain regions with a small size or an irregular outline, high
resolution photographs were taken at 200 x magnification (i.e., 20x objective + 10x eyepiece) and placed in a
graphic illustration program (CorelDraw 11.633; Viglen Ltd.). An outline of each brain region, and outlines
depicting subdivisions of some regions including the BNST/LSV, amygdala, DMM, PVN, PBN and ventrolateral
medulla was exported from a standard rat brain stereotaxic atlas (Paxinos and Watson, 1997) and overlaid to scale
onto the respective region (see Fig. S2b, S2d, S3c, S3d for illustrated examples). A dot was placed over each cFos-ir nucleus to prevent over or under counting. Out-of-focus c-Fos-ir nuclei were verified at 400 x magnification
using a binocular brightfield microscope. The area of each region or subregion was calculated with an image
analysis program (SPOT 3.5.5 for Windows digital imaging software; Silicon Graphics, Mountain View, CA,
USA). For bilaterally distributed brain regions (e.g., amygdala) the areas listed below are the added areas for each
side counted. The regions analyzed this way and the calculated area for each region are as follows: analyses of the
OVLT and SFO were conducted at 0.00 and -0.92 mm bregma representing 0.042 and 0.035 mm2, respectively;
analysis of the parabrachial nucleus (PBN) was conducted at -9.30 mm bregma representing 0.40 mm2; analysis of
subregions of the BNST [i.e., BSTM and BSTL] was conducted at +0.20 mm bregma representing an area of 0.179
and 0.134 mm2, respectively and at -0.26 mm bregma representing 0.110 and 0.178 mm2, respectively; analysis of
subregions of the LSV was conducted at +0.20 and -0.26 mm bregma representing 0.366 and 0.090 mm2,
respectively; analysis of subregions of the rostral amygdala [i.e., CeC, CeL, CeM and BLA] was conducted at -2.12
mm bregma representing 0.146, 0.067, 0.147 and 0.177 mm2, respectively; analysis of subregions of the caudal
amygdala [i.e., CeC, CeL, CeM, BLA, BLP and LA] was conducted at -2.56 mm bregma representing 0.060, 0.039,
0.117, 0.131, 0.091 and 0.030 mm2, respectively; analysis of the raphe pallidus [(RPa) representing 0.006
mm2/level] was conducted at -11.60, -11.80, -11.90 and -12.30 mm bregma; analysis of subdivisions of the
paraventricular hypothalamus (PVN) was conducted at -1.80 mm bregma [PVN, dorsal part (PaDC, representing
0.008 mm2), PVN, lateral magnocellular part (PaLM, representing 0.023 mm2), PVN, medial parvocellular part
(PaMP, representing 0.027 mm2) and PVN, ventral part (PaV, representing 0.018 mm2)]; analysis of the
ventrolateral medulla was conducted at -11.80, -13.30, -13.68 and -14.20 mm bregma [nucleus ambiguus (Amb,
representing 0.006 mm2), pre-Bötzinger complex (PreBo, representing 0.009 mm2), lateral paragigantocellular
nucleus (LPGi, representing 0.082 mm2), caudal ventrolateral medulla (CVLM, representing 0.010 mm2) and lateral
reticular nucleus (LRt, representing 0.103 mm2)]; analysis of the dorsomedial medulla was conducted at -13.30, 13.68 and -14.20 mm bregma [rostral nTS (at -13.30 mm bregma representing 0.046 mm2), mid-rostrocaudal nTS
(at -13.68 mm bregma representing 0.022 mm2), caudal nTS (at -14.20 mm bregma representing 0.006 mm2),
including the area postrema (AP, representing 0.124 mm2), dorsal motor nucleus of the vagus (DMV, representing
0.009 mm2), and hypoglossal nucleus (12, representing 0.055 mm2)].
Analysis of the number of c-Fos/TH-ir neurons was conducted at 15 anatomical levels from -8.80 to -14.20
mm bregma (see Fig. S1). Analysis of the numbers of c-Fos/ChAT-ir neurons was conducted in the DMV at -13.68
mm bregma and the Amb at -12.80 mm bregma to verify whether or not altered numbers of c-Fos-ir cells in these
regions were acetylcholinergic parasympathetic premotor neurons. For nuclei containing catecholaminergic or
acetylcholinergic neurons, one coronal section was selected from each animal for each anatomical level and the
number of c-Fos/TH or c-Fos/ChAT double-positive neurons and the total number of TH or ChAT-positive neurons
sampled were counted throughout relevant nuclei or subnuclei at each level studied. For nuclei with
catecholaminergic or acetylcholinergic neurons, all double immunostaining (c-Fos/TH and c-Fos/ChAT) and single
immunostaining (c-Fos, TH and ChAT) cell counts were done at 400 x magnification using a Leica DME binocular
microscope by an observer (PLJ), who was blind to the experimental treatment of each rat.
An estimation of c-Fos induction in neuronal populations was conducted in a brain region showing a robust lAG+lactate effect (i.e., the PBN). Neurons were identified with neuronal marker n, which is expressed in most
neurons of adult mammals (e.g., rodents) in the central and peripheral nervous system (Mullen et al., 1992). In
order to identify neurons, quantification of the number of c-Fos-ir cells in the PBN (-9.30 mm bregma) was
performed at 400 x magnification (i.e., 40x objective + 10x eyepiece) by centering the field of view over the
ventrolateral subregion of the PBN (see Fig. S3d) and counting the total number of NeuN-ir neurons, c-Fos/NeuNir neurons and single c-Fos-ir cells in this field of view (represented a 0.25 mm2 area).
Details of missing values for overall ANOVAs with repeated measures
For single immunostaining c-Fos cell counts there were a total of 37 missing values out of 1584 total values
(approximately 2.3%). Missing values for c-Fos/TH-ir cell counts were identical to those for TH-ir cell counts and
included 24 missing values out of 695 total values (approximately 3.5%). Missing values for c-Fos/ChAT-ir cell
counts were identical to those for ChAT-ir cell counts and included 1 missing value out of 24 total values
(approximately 4%).
Intra- and inter-rater analyses
Intra- and inter-rater analyses of the number of c-Fos-ir cells were conducted on the CeA (at -2.56 mm
bregma) and in the OVLT. The intra-rater analyses were conducted by PLJ. The inter-rater analyses were
conducted by PLJ and PEK. Both PLJ and PEK were blind to the experimental treatments.
Photography
Photomicrographs were obtained using a Leica brightfield microscope using N plan 5X, 10X, 40X and 63X
objective lenses (model DMLB, Leica, Bannockburn IL), an Insight digital camera (Diagnostics Instruments Inc.,
Sterling Heights MI) and SPOT 3.5.5 for Windows digital imaging software (Silicon Graphics, Sunnyville CA).
Photographic plates were prepared in CorelDraw 11.633 for Windows (COREL Inc, Eden Prairie MN).
Supplemental Figure 1 - Diagrammatic representation of a mid-sagittal rat brain section illustrating the
topographical distribution of brain nuclei and cell groups studied in experiment 1. Medullary and pontine
catecholaminergic cell groups [tyrosine hydroxylase- (TH-) immunoreactive (ir)] are indicated by shading (dark
gray, noradrenergic cell groups; light gray, adrenergic cell groups). The CVOs (i.e., AP, OVLT and SFO) are
outlined by black dotted lines, and other brain regions are outlined by dashed black lines. Cell counts of c-Fos-ir
cells, TH-ir neurons, and c-Fos/TH-ir neurons were conducted at 8 forebrain, 5 pontine and 11 medullary levels that
are represented on the figure by vertical lines with numbers at the end of these lines indicating mm from bregma.
Cell counts of choline acetyltransferase (ChAT)-ir and c-Fos/ChAT-ir neurons in the Amb and DMV were
conducted at -12.80 and -13.68 mm bregma, respectively. Abbreviations: ac, anterior commissure; cc, corpus
callosum; opt, optic tract; HC, hippocampus; scp, superior cerebellar peduncle. See methods for further
abbreviations.
Table S1 – Secondary 2-way ANOVA results for numbers of c-Fos-ir cells in each brain region using isomer and
lactate as main factors from experiment 1. a) results for c-Fos single immunostaining with Columns l representing
brain regions and rostrocaudal levels (mm from bregma) and columns 2-4 representing ANOVA results for main
effects of lactate and l-AG, and the interaction between lactate and l-AG . b) c-Fos/TH-ir double immunostained
cells, and c) c-Fos/ChAT double immunostained cells. See methods for abbreviations.
Table S2 - Mean numbers of c-Fos-ir cells in the rostral amygdala -2.12mm from bregma. following either i.v.
infusions of saline (Sal) or sodium lactate (Lac) in rats with prior infusions of d- or l-allylglycine (d- or l-AG) into
the DMH/PeF region from experiment 1. Column 1 indicates brain regions; numbers in parentheses indicate
anatomical levels relative to bregma. Numbers in columns 2-5 represent the number of c-Fos-ir cells. The titles
above columns 2-5 indicate treatment groups, d-AG/Sal, d-AG/Lac, l-AG/Sal or l-AG/Lac, respectively. Values
below columns 2-5 are the mean ± S.E.M. Abbreviations: see methods section for full list of abbreviations.
Table S3 - Mean numbers of c-Fos-ir cells in anatomically defined subdivisions of the central nervous system that
did not respond following either i.v. infusions of saline (Sal) or sodium lactate (Lac) in rats with prior infusions of
d- or l-allylglycine (d- or l-AG) into the DMH/PeF region from experiment 1. Column 1 indicates brain regions;
numbers in parentheses indicate anatomical levels relative to bregma. Numbers in columns 2-5 represent the
number of c-Fos-ir cells. The titles above columns 2-5 indicate treatment groups, d-AG/Sal, d-AG/Lac, l-AG/Sal or
l-AG/Lac, respectively. Values below columns 2-5 are the mean ± S.E.M. Abbreviations: see methods section for
full list of abbreviations.
Table S4 - Mean numbers of catecholaminergic neurons that did not respond following either i.v. infusions of saline
(Sal) or sodium lactate (Lac) in rats with prior infusions of d- or l-allylglycine (d- or l-AG) into the DMH/PH
region in experiment 1. Column 1 represents abbreviated brain regions and numbers in parentheses are mm caudal
to bregma. Below these are the no. of tyrosine hydroxylase (TH) neurons also immunoreactive for c-Fos. No
significant differences in the total number of TH-ir neurons were observed between groups in any subdivision
analyzed (see results section for details). The titles above columns 2-5 represent rats treated with d-AG/Sal, dAG/Lac, l-AG/Sal or l-AG/Lac, respectively. Values below columns 2-5 are the mean ± S.E.M. Abbreviations: see
methods section for full list of abbreviations.
Figure S2 - Neural circuits implicated in DMH/PeF - mediated suppression of parasympathetically-mediated
baroreflex from c-Fos responses in experiment 1. Graph illustrating numbers of c-Fos-ir cells in subdivisions of the
a) amygdala (see methods section for amygdala subregion abbreviations) and c) dorsomedial medulla (DMM)
following i.v. infusions of saline or lactate in rats previously treated with d- or l-AG infusions into the DMH/PeF
region. Values of bars represent the mean, whereas error bars represent S.E.M. (* P < 0.05). Drawings illustrate cFos-ir cells in b) subdivisions of the amygdala [dashed black lines indicate subregions of the amygdala and solid
black lines indicate the external capsule (ec)] and d) subdivisions of the dorsomedial medulla [dashed black lines
indicate the nucleus of the solitary tract (nTS), solid black lines indicate the area postrema (AP), dorsal motor
nucleus of the vagus (DMV) and hypoglossal nucleus (12) according to a standard stereotaxic atlas (Paxinos and
Watson 1997) and gray solid lines indicate the outline of the brain section, blood vessels and central canal (cc)] of a
single rat from each treatment group. Scale bars: b, 200µm; d, 400 µm.
Figure S3 – Evidence of sympathetically mediated responses following sodium lactate infusion in l-AG-treated rats
and neural circuits implicated in DMH/PeF - mediated sympathoexcitation from experiments 1. Graph illustrating
numbers of c-Fos-ir cells in subdivisions of the a) paraventricular hypothalamic nucleus (PVN, see methods for
abbreviation of subregions) and in the b) parabrachial nucleus (PBN) following i.v. infusions of saline or lactate in
rats previously treated with d- or l-AG infusions into the DMH/PeF region. Values of bars represent the mean,
whereas error bars represent S.E.M. (* P < 0.05). Drawings illustrating c-Fos-ir cells (each black dot = 1 c-Fos-ir
cell) in subregions of the c) paraventricular hypothalamic nucleus (PVN) [dashed black lines indicating subregions
and solid gray line indicating the 3rd ventricle] and d) parabrachial nucleus (PBN) [dashed lines indicating the
lateral and medial PBN (LPBN and MPBN), solid black line indicating the superior cerebellar peduncle (scp) and
solid gray lines indicating the outline of the brain section] from a single rat in each treatment group from
experiment 1. Scale bars: c, 150 µm; d, 400 µm.
Figure S4 - Evidence of peripheral sympathetic involvement in l-AG+lactate mediated cardiovascular responses, ab) The effect of intravenous infusions of atenolol on lactate-induced cardiovascular responses in l-AG
treated rats in experiment 4. Changes in a) mean arterial blood pressure (MAP in mm Hg) and b) heart rate
[HR in beats/min (BPM)] from pre-i.v. infusions (baseline) to post-i.v. infusions of Sal or Lac in l-AG
treated rats. Mean values of baseline MAP and HR + S.E.M. are listed in a legend for each group. Symbols
represent the means, whereas error bars represent S.E.M. [time effects, black * (versus t = -5 min); Group
effects, for a) open † (Lac+l-AG+Atenolol versus Sal+l-AG+Atenolol), black † (Lac+l-AG versus Sal+lAG+Atenolol); for b) black † (l-AG+Lac versus Sal+l-AG+Atenolol ), open † (Lac+l-AG versus Lac+lAG+Atenolol), P < 0.05]. Gray shading on graphs indicates the onset and duration of Sal or Lac infusions.
c) Illustration of cannula placements for osmotic minipump infusions of l-AG in the DMH/PeF region from
-2.80 to -3.30 mm bregma for experiment 4. Black circles represent histological placements in the
DMH/PeF region. No differences were noted between baseline levels of MAP, RR and HR for each group
(data not shown).
Figure S5 - Small box in a) indicates the area of the PBN (i.e., ventrolateral PBN, vlPBN) where immunoreactive
cells were counted; b) graph depicts the no. of c-Fos immunopositive (+)/neuronal marker n [NeuN (+), neuron-like
responses] cells (see left hand axis and bars with black lines) and total numbers of NeuN(+) cells (see right hand yaxis and bars with gray lines) from all 4 treatment groups (x-axis). Numbers above bars indicate percentage of
colocalization; c) graph depicts the no. of single c-Fos (+)/NeuN immunonegative (-) cells (glia-like) from all
treatment groups (x-axis). Photomicrographs of the blue/black nuclear c-Fos staining and brown cytoplasmic NeuN
stainining of the vlPBN at low (photo to left) and high (photo to right) magnification are listed for d) d-AG+lactate
treated rats and e) l-AG+lactate treated rats. High magnification photographs were taken from low magnification
photographs illustrated in d) and e) as black box. Scalebar: 50 μm for low magnification photographs and 17 μm for
high magnification photographs.
References
Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development
116:201-211.
Paxinos G, Watson C (1997) The Rat Brain Stereotaxic Coordinates. San Diego: Academic Press