Glucose-Induced Intracellular Ion Changes in Sugar-Sensitive
Hypothalamic Neurons
IAN A. SILVER 1 AND MARIA ERECIŃSKA2
1
Department of Anatomy, School of Veterinary Science, University of Bristol, Bristol BS2 8EJ, UK; and 2 Department of
Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
INTRODUCTION
It generally is accepted that the two main centers that
control feeding behavior in animals are located in the basal
hypothalamus, with the lateral hypothalamic area (LHA)
and the ventromedial hypothalamic nucleus (VMH) exhibiting a reciprocal relationship. Although the LHA, among
its other vegetative functions, plays a key role in stimulation
of appetite and food intake, the VMH exerts the opposite
effect (Bray and York 1979; Nagai and Nakagawa 1992).
One of the major endogenous regulatory factors that modulates the activity of these centers is the concentration of
blood glucose. More than 25 years ago, Oomura and cowork-
ers (1969) found that Ç30% of LHA neurons responded to
a rise in extracellular glucose level with a decrease in firing
rate (i.e., inhibition of activity), whereas Ç40% of VMH
neurons showed an increase in discharge frequency (i.e.,
stimulation of activity), under the same conditions. It also
was demonstrated that local application of glucose hyperpolarized the sensitive LHA cells, an effect that was prevented
by ouabain and azide (Oomura et al. 1974). In view of this
sensitivity to a blocker of the Na/K ATPase (ouabain) and
an inhibitor of energy production (azide), combined with
an apparent lack of change in membrane permeability, it
was postulated that glucose exerted its inhibitory influence
via stimulation of the Na/K pump activity.
In slices of nucleus tractus solitarius, which also contains
neurons sensitive to changes in extracellular glucose concentration, those that were stimulated by a rise in sugar level
(VMH-type) became depolarized by 3–10 mV and their
membrane conductance increased by 50–82% (Mizuno and
Oomura 1984). Conversely, a decrease in glucose to 3 mM
hyperpolarized these same cells and reduced their membrane
conductance. The authors postulated that such behavior most
likely was due to alterations in the membrane permeability
to potassium. Similar changes in membrane voltage consequent on either a rise or a fall in glucose level were demonstrated more recently in VMH cells in a hypothalamic slice
preparation (Ashford et al. 1990). Moreover, cell-attached
recordings from these neurons revealed the presence of potassium channels which were closed by a rise in either [ATP]
or [glucose]. It was suggested that closure of the ATPsensitive K channels is responsible for the increased firing
seen in hyperglycemia.
Our earlier investigations on neuronal responsiveness
to changes in their external environment ( Silver and Erecińska 1990, 1992, 1994 ) showed that most central nervous system (CNS) neurons are remarkably insensitive to
alterations in the extracellular concentration of glucose
and that the latter has to fall õ0.2 mM before ion gradients
begin to collapse ( Silver and Erecińska 1994 ) . The sensitivity of the hypothalamic cells to small perturbations is
thus in sharp contrast to the general pattern of neuronal
behavior. Yet except for the very few reports cited above,
there is no information on the mechanisms responsible for
this unique ‘‘reactivity.’’ The current study was undertaken to characterize the ionic changes occurring in LHA
and VMH neurons in intact rat brain, during physiological
alterations of blood glucose level, to gain some insight
into the molecular basis of glucose sensing by the CNS.
0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society
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Silver, Ian A. and Maria Erecińska. Glucose-induced intracellular ion changes in sugar-sensitive hypothalamic neurons. J.
Neurophysiol. 79: 1733 – 1745, 1998. In the lateral hypothalamic
area ( LHA ) of rat brain, Ç30% of cells showed sensitivity to
small changes in local concentrations of glucose. These ‘‘glucose-sensitive’’ neurons demonstrated four types of behavior,
three of which probably represent segments of a continuous
spectrum of recruitment in response to ever more severe changes
in blood sugar. Type I cells showed maximum activity °5.6 mM
blood glucose but became completely silent at hyperglycemia of
10 – 12 mM ( normoglycemia 7.6 { 0.3 mM; mean { SD ) . Type
II and III neurons exhibited a wider range of response. Type
IV cells ( 5 – 7% of glucose-sensitive neurons ) paralleled the
behavior of sugar-sensitive cells in ventromedial hypothalamic
nucleus ( VMH ) . In VMH, Ç40% of cells responded to changes
in blood glucose over a range of concentrations from 3.6 to 17
mM, by increasing their firing rate as sugar level rose and vice
versa. Ionic shifts during increases in blood ( brain ) glucose
levels were similar in LHA types I – III but fastest in I and
slowest in III. [ Na / ]i fell by 5 – 9 mM, [ K / ]i rose by 6 – 8 mM,
and plasma membrane hyperpolarized by 5 mV. [ Ca 2/ ]i declined
by 15 – 20 nM in line with membrane hyperpolarization. In VMH
and type IV LHA cells, [ K / ]i fell 3 – 8 mM and plasma membrane depolarized 03 to 05 mV as blood / brain glucose concentration increased from 7.6 / 2.4 to 17.6 / 4.2 mM, whereas [ Ca 2/ ]i
increased from 125 to 180 nM as a consequence of falling membrane potential. During falls in blood / brain sugar concentration
the effects in both VMH and LHA cells were reversed. The
findings are consistent with the ionic shifts in types I – III LHA
cells being dependent on alterations in Na / K-ATPase activity,
whereas those in VMH and type IV LHA cells could be caused
by modulation of ATP-dependent K / channels. A possible
mechanism for linking the effects of small changes in glucose
to ATP generation, which could bring about the above phenomena, is the interposition of a ‘‘glucokinase-type’’ enzyme in a
role similar to that which it has in glucose-sensing pancreatic
b-cells.
1734
I. A. SILVER AND M. ERECIŃSKA
Hypoglycemia was induced by subcutaneous injection of insulin
(25 IU/kg) while reversal of hypoglycemia, or induction of hyperglycemia, were achieved by slow intravenous injection of 0.5 g
glucose in 3 ml water.
METHODS
Preparation of the rats
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Electrodes
Ion-specific probes were either double-barreled microelectrodes
(tip diameter 0.08–0.15 mm, mean 0.10 mm) fabricated on a standard electrode puller from theta configuration borosilicate glass
capillaries (1.5 mm OD, 1.0 mm ID) or multibarreled electrodes
made from three to five glass capillaries (containing solid fibers)
annealed together. The barrels to be filled with ion sensors were
rendered hydrophobic by siliconization with the vapor of dimethyldichloro- or tri-n-butylchlorosilane and subsequently baked at
1507C for 1 h. The tips of electrodes used for intracellular recording
were ground to a short chisel point on a dry, rapidly rotating,
optical flat glass disk incorporating very fine diamond dust in such
a manner that the ‘‘glucose’’ barrel (see further) opened on the
most proximal part of the bevel. The reference barrels for recording
membrane potentials/cell discharges were back-filled with filtered
3 M KCl. In multibarreled electrodes, a glucose barrel was filled
with 1% (55 mM) glucose in ACSF. This barrel was used as a
searching tool for the preliminary identification of glucose-responsive cells (see Experimental design). Ion-sensitive barrels were
filled at the tip only by back-injection with a short (150–350
mm) column of liquid ion sensor and then with the appropriate
electrolyte. In cases where there was difficulty in filling the electrodes, these were placed in a teflon jig that fitted the sockets on
the rotor of a Beckman Microfuge and centrifuged for 10–15 s at
10,000 g, which usually removed any air bubbles and forced ion
sensor or electrolyte into the tips of the electrodes unless they were
blocked by debris or silicone deposit. The sensors used were: for
potassium, valinomycin as Fluka ‘‘cocktail A’’ (Wuhrmann et al.
1979) (Fluka No. 60031); for calcium, the ligand ETH 129
(N,N,N *,N *-tetracyclohexyl-3-oxapentanediamide) in the form of
the ‘‘cocktail’’ described by Schefer et al. (1986) (Fluka 21193);
and for sodium, Fluka 71176 Sodium ionophore I-Cocktail A containing the neutral lipophilic ionophore ETH 227 [N,N *,N9triheptyl-N, N *,N9-trimethyl-4,4 *,49-propylidynetris(3-oxabutyramide)] (Steiner et al. 1979). The ion-sensitive barrels were backfilled with the appropriate electrolyte for potassium, sodium, or
calcium, i.e., 0.5 M KCl, 0.5 M NaCl, or a mixture of 0.1 M Mg
acetate, 100 mM CaCl2 and 80 mM KCl, respectively. The last
was found to be more reliable for use with the ETH 129 ligand
for intracellular, calcium-sensitive probes than a simple solution
of CaCl2 .
Ion-sensitive electrodes were calibrated at 37.57C, in terms of
ionic concentration, in electrolyte mixtures that closely simulated
their expected intracellular counterparts. Details of calibration procedures have been given by Silver and Erecińska (1990). All
electrodes were calibrated individually, and only those showing a
near-Nernstian response to changes in appropriate ion concentration and a lack of sensitivity to other ions were used for experimental measurements. For calcium-sensitive electrodes, a log/linear
response of between 25 and 29 mV/decade and for Na / and K /
electrodes 51–59 mV/decade were considered acceptable. Probes
giving voltage changes outside these ranges, or that were nonlinear,
were discarded. The impedance of all electrodes was measured
before use: reference barrels were routinely 20–40r10 6 V and ionsensitive barrels always ¢10 9 V. The typical 87% response time
of ion-selective electrodes in the recording system ranged from
500 ms (Ca 2/ ) to 3 s (Na / ) and 5 s (K / ) for an increase and 700
ms (Ca 2/ ) to 5 s (Na / ) and 8 s (K / ) for a decrease, to an
order of magnitude step change of concentration. All results were
calculated from standard calibration curves constructed for the various cations as described earlier and are given as (free) ion concen-
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Measurements were made on 296 white Sprague-Dawley-derived rats of both sexes; body weight 250–300 g anesthetized by
intraperitoneal injection of pentobarbital sodium (60 mg/kg as a
6% aqueous solution; Nembutal, Abbott Laboratories) together
with atropine sulfate (0.3 mg/kg; Roche). Supplementary pentobarbitone was administered as required. Local analgesic (lignocaine 2%; Xylocaine, Astra Pharmaceuticals) was infiltrated subcutaneously over the scalp and at the prospective pressure points of
the head holder. The level of anesthesia was assessed constantly
by reference to the electrocorticogram (ECoG), electrocardiogram
(EKG), and blood pressure traces, and supplementary doses of
anesthetic given when required.
A tracheostomy was performed. The caudal artery was cannulated and connected to a Gould-Statham P50 miniature blood pressure transducer. Catheters were inserted into the left femoral vein
and artery. The venous line was used for administration of fluids,
collection of venous samples and return of blood from the glucose
measuring device (see further text).
EKG was recorded from left and right forelimbs via subcutaneous needle electrodes. The animal was placed in a modified Baltimore stereotaxic head holder and positioned for use of stereotaxic
coordinates as described by Swanson (1992). A median longitudinal skin incision was made over the skull, the periosteum reflected
on each side of the central suture and two stainless steel miniature
bone screws inserted into the frontal bones to serve as ECoG
electrodes. A hole 2.8 mm wide and 4.0 mm long was drilled with
the medial edge of the long axis parallel to and 0.5 mm lateral to
the midline and the rostral edge 0.8 mm caudal to the bregma
point. This opening was enclosed in an oval polycarbonate ring
(4.0 1 2.5 mm ID, 2.5 mm depth, 1-mm-wall thickness), which
was fixed to the skull with cyanoacrylate to form a well that was
flushed with warm (387C) artificial cerebrospinal fluid (ACSF)
during recording. The stereotaxic coordinates used were 0.8–4.3
mm caudal and 1.0–2.5 mm lateral to the bregma point for LHA
and 1.8–3.3 mm caudal and 0.6–1.2 mm lateral to bregma for
VMH. The depth of the recording points varied according to the
cranio-caudal and lateral position of the electrodes but were within
the range 7.0–9.5 mm for LHA and 8.3–9.8 mm for VMH. The
underlying dura was removed, and the hole sealed with warm 4%
agar in ACSF. Before recordings were made from cells, the agar
was removed and replaced by a small C-shaped, teflon-coated pressure foot (2.8 mm OD; 2 mm ID) derived from the design of
Phillips (1956) to minimize brain movement due to cardiovascular
and respiratory pulsation. Before intracellular recordings began
the rat was given a muscle relaxant (pancuronium bromide, 70
mg/kg; Pavulon, Organon-Teknika, Cambridge, UK) to minimize
respiratory movements. An artificial pneumothorax was created
and ventilation with 30% O2 in N2 and minimum tidal volume,
using a Harvard small animal respiratory pump, was given to provide an end-tidal PCO2 of 4.6–5.26 kPa measured by an infrared
CO2 analyzer. Periodic blood samples (20 ml) were taken from the
caudal artery, and blood gas analyses performed to check the endtidal measurements. The pia-arachnoid was perforated and electrodes inserted with a micromanipulator through the open center
of the pressure foot. After insertion of ion-specific electrodes the
hole in the skull was resealed with warm agar solution. In some
animals (25%), a second hole, 1.5 mm diam, was drilled in the
skull over the cingulate cortex and the dura removed. A glucosespecific, extracellular microelectrode (tip ° 1.0 mM) was inserted
into the brain by means of a micromanipulator and sealed in position with dental acrylic, after which the micromanipulator was
removed. A ground-reference electrode of sintered Ag/AgCl was
placed subcutaneously on the rat’s head.
GLUCOSE SENSITIVITY OF HYPOTHALAMIC NEURONS
1735
trations. This assumes that ionic strength and hence activity coefficients in calibration fluids and intracellular compartments are of
comparable magnitudes.
Glucose measurements
TABLE
1.
Responsiveness of LHA neurons to various stimuli
Glucose-Sensitive
Neurons
Stimulus
Pinch to tail
Cold on tail
Aromatic oil
Breathing 10% CO2
Firing
Response
Type I
Type II / III
GlucoseInsensitive
Neurons
F
f
0
F
f
0
F
f
0
F
f
0
12
0
157
15
0
154
3
0
166
24
0
145
73
23
21
51
18
48
27
3
87
35
0
82
403
51
137
378
34
179
298
44
249
518
0
73
Responsiveness of cells was measured as a change in firing rate as described in Experimental procedures. LHA, lateral hypothalamic area; F,
increase; f, decrease; 0, no change in firing rate. The definition of types I–
III of neurons is given in the text. Of 877 cells evaluated, 591 were glucose
insensitive, 169 were type I, and 117 were types II and III.
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FIG . 1. Extracellular records of action potentials from 2 neurons in the
lateral hypothalamic area (LHA), 1 sensitive to local, electroosmotic application of glucose (A, h ) but not to a peripheral sensory input (ice on the tail;
B, h ) the other insensitive to local glucose application (C) but responsive to
cold stimulus on the tail (D). Measurements of activity were made as
described in METHODS . Where indicted by h, 1% glucose solution was expelled from the glucose-filled barrel by electroosmosis (10 s at 10 r 10 09
A) according to the method of Oomura et al. (1969, 1974).
Microfuge capillary tubes. An aliquot of 20 ml was centrifuged
immediately, and the plasma kept on ice at 07C until analyzed for
glucose content in a standard clinical hexokinase-based colorimetric system (Technico Instruments, Tarrytown, NY) to check that
the in-line electrode was functioning reliably. Total blood sugar
was determined periodically from the other 20 ml by the method
of Trinder (1969) using an Instrumentation Laboratories (Warrington, Cheshire, UK) pediatric blood-glucose analyzer.
Recording
The ion-sensitive electrodes and their reference barrels were
connected via chlorided silver wires to a capacitance-compensated,
high-impedance ( ú10 14 V ) DC amplifier, the output of which
drove a storage oscilloscope, impulse rate meter, audioamplifier,
chart recorder and, via an A-to-D converter, an Apple Quadra
microcomputer running LabView 2.2 (National Instrumentation)
virtual instrumentation. Intracellular recordings always included
the membrane potential (the voltage from the reference barrel measured against ground potential), which was subtracted electronically from the output of the ion-sensitive probe. The output from
the reference barrel also was used to record action potentials because the response of the ion-selective barrels was too slow for this
purpose. Membrane resistance was measured routinely by current
injection through the reference barrel.
A minimum recording period of 30 s was required before intracellular ion measurements were regarded as a reliable source of
data, but recordings were made from most cells for ¢2–4 min.
Recordings of extracellular action potentials from spontaneously
active neurons were made via the reference barrels of the ionsensitive electrodes before cells were penetrated (see Experimental
design).
ECoG and EKG were recorded via appropriate standard amplifiers (Gould Electronics BV, Bilthoven, Netherlands).
Identification of anatomic location of recording sites
Reference barrels of electrodes were filled with electrolyte containing a 10% solution of the anionic fluorescent marker dye, Procion yellow, (M-4RS, I.C.I. Organics). At the end of a successful
intracellular recording, dye was injected iontophoretically into the
site with 100-ms current pulses of 2 r 10 08 A at 5 s 01 for 30–60 s,
solely to assist identification of its anatomic position. The location
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Extracellular hypothalamic glucose concentration was measured
continuously by means of glucose-sensitive, ferrocene-coated,
amperometric needle microelectrodes inserted into the diencephalon and fixed to the skull with dental acrylic (see preceding sections). These glucose sensors were as described by Silver and
Erecińska (1994) and are based on oxidation of glucose to gluconic
acid and hydrogen peroxide by glucose oxidase bound to a platinum
black surface with immediate electrolysis of the H2O2 to O2 and
H2O at the positively charged metal surface. The 1-mm tips of the
electrodes were coated in four thin layers of cellulose diacetate,
dried, and dipped into a 1% aqueous solution of ascorbate oxidase
(Boehringer-Mannheim, Indianapolis, IN). This latter is necessary
to destroy any ascorbate in the vicinity of the electrode that might
affect the measurement of glucose. Covering of an electrode tip
by a water-permeable membrane, within which the linear diffusion
path of the substrate is confined to ¢95%, virtually eliminates not
only ‘‘stirring artifacts’’ but also the effects of edema and local
hematomas. These probes were polarized at /0.6 V against a silver/silver chloride reference electrode and calibrated in standard
solutions of 0, 1, 2, 4, 8, 16, and 32 mM glucose in 5% bovine
serum albumin at 377C as described previously (Silver and Erecińska 1994). The performance of this electrode is linear within the
range of glucose concentrations 0.1–40 mM and is insensitive
to changes in oxygen concentration (Frew and Hill 1987). The
calibrations of ferrocene-based electrodes remained stable over
many days in sterile, protein-containing test solutions at 377C. The
microglucose electrodes also were used to test the effectiveness of
electroosmotic ejection of glucose from the glucose barrels of the
multibarreled electrodes.
Blood glucose was measured continuously in a small arteriovenous bypass system located between the femoral artery and the
femoral vein. The blood flowed from the arterial catheter through
a 1-mm channel in a siliconized, thermostatted polycarbonate block
that incorporated a flat-ended, glass-insulated, glucose oxidaseferrocene electrode. Blood samples (40 ml) were collected routinely at 15-min intervals, and 20 ml every 4 min (marked f in
Figs. 2 and 7) during blood-glucose perturbations, into Beckman
1736
I. A. SILVER AND M. ERECIŃSKA
TABLE 2. Changes in firing rates, intracellular ion concentrations and membrane potentials in LHA neurons type II and IV during
stepwise increases in glucose level
D Glucose, mM
Blood
Type II
Baseline
/2
/5
/10
Type IV
Baseline
/2
/5
/10
Brain
Firing Rate, spikes/s
[Na/]i, mM
[Ca2/]i, nM
[K/]i, mM
E, mV
/0.3
/1.0
/1.8
20
14
5
0
{
{
{
{
5
4
2
0
23.8
22.0
20.2
17.8
{
{
{
{
1.2
1.4*
1.2
1.2
115
110
103
98
{
{
{
{
5.7
6.1*
5.1
6.2
70.4
73.4
76.4
80.2
{
{
{
{
2.3
2.2
2.2
1.7
068.6
070.6
072.4
075.8
{
{
{
{
1.4
1.7
1.2
0.8
/0.3
/1.0
/1.8
14
19
25
31
{
{
{
{
6
6
4
6
22.8
24.2
25.6
27.0
{
{
{
{
1.2
1.2*
1.4
1.1
140
151
161
174
{
{
{
{
4.8
3.7
5.7
2.2
69.6
68.0
66.0
63.2
{
{
{
{
1.5
1.7*
1.3
0.7
067.2
065.2
064.4
061.8
{
{
{
{
1.4
1.2
1.2
0.7
of the fluorescent spots also provided a means of estimating any
brain shrinkage or distortion during subsequent processing and
reconstruction. Although Procion yellow has limitations as a
marker, other dyes that are more intensely fluorescent or localize
more precisely have a greater tendency to block the tips of intracellular microelectrodes.
At the end of each experiment, an electrode was left at the final
recording site, the animal killed with an anesthetic overdose, and
the brain perfused through the carotid arteries with ice-cold 4%
glutaraldehyde. The animal, in the headholder was placed in a cold
room at 47C. After 12 h, the electrode was removed, and the brain
extracted from the skull and placed in cold 4% glutaraldehyde for
a further 48 h. It then was cut serially in a cryostat to give 40-mm
sections, which were stained with cresyl violet or toluidine blue.
Recording points were identified from stereotaxic coordinates,
electrode tracks, and microscopic examination of fluorescent markers under UV excitation. The anatomic positions of cells from
which recordings were made were identified by reference to the
appropriate sections in the rat brain maps of Swanson (1992).
Experimental design
Spontaneously active cells in the LHA and VMH were located
by recording their (positive-going) extracellular action potentials
through the reference barrel of the ion-sensitive probes. Responsiveness of these cells to various nonspecific peripheral stimuli was
tested by application of cold (ice) and light pinch to the tail of the
rat, followed by 10-s exposure of the nose at a distance of 1 cm
to a neurosurgical swab (1 cm2 ) soaked in clove oil, and finally
10 s 10% CO2 in the respired air. Approximately 65% of all cells
FIG . 2. Continuous recordings (except for break as indicated at /2 min) of blood and brain glucose levels and
single-unit discharge frequency recorded extracellularly,
) and III ( – – – )
from glucose-sensitive types I (
cells in the LHA of rat brain. On the x axis, insulin F, time
at which the rat was given a subcutaneous injection of
insulin, 25 IU/kg. First glucose F (at 25.5 min), intravenous injections of 0.5 g glucose in 3 ml water over 1.5
min; 2nd glucose F (at 29.2 min), same amount glucose
administered but over 3 min. f, time points at which blood
samples were collected to check the accuracy of the inline blood-glucose monitor. Concentrations measured were
within experimental error the same as those obtained with
the indwelling glucose electrode and shown in the figure.
One-second segments of records of extracellular action
potentials from the type 1 cell are shown for times 0, 20
min, and 23 min at blood glucose concentrations of 7.6,
3.5, and 2.2 mM, respectively.
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Intracellular ion concentrations were measured with double- or multibarreled microelectrodes as described in Experimental procedures. Membrane
potential (E) and firing rate were measured through the reference barrels of the ion-selective probes. Values are means { SD for five cells in each
category. All values are statistically significant compared with baseline, P õ 0.05, except those marked *. Baseline glucose concentration in blood was
7.6 { 0.3 (n Å 175) and that in brain 2.4 { 0.13 (n Å 133).
GLUCOSE SENSITIVITY OF HYPOTHALAMIC NEURONS
1737
TABLE 3. Changes in firing rates, intracellular ion concentrations and membrane potentials in LHA neurons type II and IV during
stepwise decreases in glucose level
D Glucose, mM
Blood
Type II
Baseline
02
04
06
Type IV
Baseline
02
04
06
Brain
Firing Rate, spikes/s
[Na/]i, mM
[Ca2/]i, nM
[K/]i, mM
E, mV
00.3
01.1
02.2
21
23
33
0
{
{
{
{
6
3
10
0
22.4
24.4
28.0
37.0
{
{
{
{
1.9
1.9*
2.0
2.3
111
138
169
321
{
{
{
{
5.9
17
12
88
70.0
67.6
63.2
54.0
{
{
{
{
1.8
2.0*
2.1
2.0
067.8
066.2
064.0
047.3
{
{
{
{
1.2
0.7
1.1
1.5
00.3
01.1
02.2
19
5
1
0
{
{
{
{
8
4
1
0
23.0
21.6
20.4
19.4
{
{
{
{
1.4
1.0*
0.8
0.8
140
129
113
106
{
{
{
{
2.8
2.6
3.0
3.9
69.6
71.4
73.0
74.6
{
{
{
{
1.7
1.9*
1.4
1.2
068.2
069.6
071.0
072.4
{
{
{
{
1.7
1.7*
1.8
1.4
then were tested for glucose sensitivity by expelling glucose solution from the glucose-filled barrel by electroosmosis (10 s at 10 r
10 09 A) (Oomura et al. 1969, 1974). Cells that responded to local
glucose application almost invariably proved subsequently to be
sensitive to systemic changes in blood glucose; this indicated that
they were responsive to sugar concentration and not to an artifact
produced by the electrodes. If a cell responded to local glucose
application by increased or decreased firing rate, the electrode was
advanced in an attempt to penetrate it. This was successful with
30–40% of cells from which intracellular ion concentrations were
recorded. Some cells that were not impaled continued to fire actively as the electrode tip moved past them. Those that remained
FIG . 3. Records from a triple-barreled microelectrode
showing action potentials and cell membrane potential (top),
together with intra- and extracellular concentrations (in mM)
of potassium (middle) and calcium (bottom) in response to
increased glucose concentration. These records are from a
LHA type II cell. ⇓, time of penetration of the cell. Left of
⇓, extracellular recordings; right of ⇓, intracellular recordings.
Top: from reference barrel of the electrode and recorded on
a time scale (seconds) that shows individual action potentials.
Middle and bottom: displayed on a longer time scale (minutes) because of the relatively slow response times of the ionsensitive electrodes. One minute after the cell was penetrated,
intravenous glucose infusion was started. Three segments of
each trace were taken when blood glucose concentrations
were those shown on the x axis, namely baseline (7.7 mM),
/2 (9.6 mM), and /5 (12.5 mM). Insets: changes in [K / ]
and [Ca 2/ ] at altered glucose concentrations shown in expanded scale on the x axis.
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Intracellular ion concentrations were measured with double- or multi-barreled microelectrodes as described in Experimental procedures. Membrane
potential (E) and firing rate were measured through the reference barrels of the ion-selective probes. Values are means { SD for five cells in each
category. All values are statistically significant compared with baseline, P õ 0.05, except those marked *. Baseline glucose concentration in blood was
7.6 { 0.3 (n Å 175) and that in brain 2.4 { 0.13 (n Å 133).
1738
I. A. SILVER AND M. ERECIŃSKA
Statistical evaluation
Statistical evaluation of the data were performed using either
Student’s t-test or t statistics with correction for multiple comparisons. Post hoc analysis was done with Dunnett’s test. The justification for using this method was that in those experiments where
systemic glucose changes were imposed, one type of neuron was
recorded from a single rat and hence the samples were independent
of each other.
RESULTS
Responses of LHA neurons to changes in blood glucose
concentration
The specificity of the response to an increase in blood
glucose level, measured as a change in the rate of firing,
was investigated in 877 LHA neurons (Table 1); 33% (286
cells) were sensitive to glucose, whereas 60% (591 cells)
were not. Most of the glucose-insensitive cells responded to
a number of other stimuli, such as cold applied to the tail
(Fig. 1), scent of an aromatic oil, or 10% carbon dioxide
in the inspired air, usually with an increased discharge frequency. The glucose-sensitive neurons, which apparently
were scattered randomly throughout the LHA, exhibited a
nonuniform behavior: cells termed type I (see further for
the basis of classification) were highly selective for glucose
because the majority did not respond to other stimuli (Fig.
1). Types II and III cells were less ‘‘specific’’ in that a
number of them also responded to cold, pinch or increased
CO2 . Type IV cells were not sensitive to stimuli other than
glucose.
In normoglycemia, LHA neurons fired spontaneously 6–
33/s, and there was no obvious correlation between this
(basal) rate of firing and the magnitude of response to a
change in external glucose concentration. However, based
on the type or sensitivity of this response, cells could be
classified into four categories; types I–III were inhibited by
a rise in [glucose] above baseline (Figs. 1–6; Table 2),
whereas type IV cells were stimulated (Table 2). Type I
FIG . 4. Records from a triple-barreled microelectrode
showing action potentials and cell membrane potential
(top), together with intra- and extracellular concentrations
(in mM) of sodium (middle) and calcium (bottom) in response to hypoglycemia. These records are from a LHA
type II cell. ⇓, time of penetration of the cell, intra- and
extracellular recordings same as in Fig. 3. M designates the
withdrawal of the electrode from the cell. Top: from the
reference barrel of the electrode and recorded on a time
scale (seconds) that shows individual action potentials. Middle and bottom: [K / ] and [Na / ], displayed on a longer
time scale (minutes) because of the relatively slow response
times of the ion sensitive electrodes. 30 s after the cell
was penetrated intravenous insulin was administered (see
METHODS ). Four segments of each trace were taken when
blood glucose concentrations were those shown on the x
axis, i.e., baseline (7.6 mM), 01 (6.6 mM), 02 (5.6 mM),
and 04 (3.6 mM).
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stable were used for extracellular recording of relatively long term
firing rate changes, in response to systemic alterations in blood
sugar (Figs. 2 and 7). Changes in blood and brain glucose concentration were produced by systemic injection of glucose solution or
insulin as described above. Ideal intracellular recordings were those
in which changes in ionic concentrations and membrane potentials
could be followed in a series of individual cells during a control
period and through administration of, and recovery from, an imposed alteration in blood sugar. While it is possible to produce
relatively rapid increases in blood and brain glucose levels by
intravenous injection of glucose solution, lowering of blood (and
brain) sugar concentration after insulin administration inevitably
takes many minutes during which there is frequently damage to
cells impaled in the control period. For this reason, it was possible
to gather complete sets of data during development of hypoglycemia from only relatively few cells, and it was necessary to supplement these observations with measurements on cells from which
recordings had not been made in the control period but which
provided data only during development of hypoglycemia and/or
its reversal.
All procedures on animals were carried out as specified in licenses issued under the (British) Animals (Scientific Procedures)
Act 1986.
GLUCOSE SENSITIVITY OF HYPOTHALAMIC NEURONS
1739
neurons, which were predominant ( Ç60% of all glucosesensitive cells), became completely inhibited (i.e., stopped
firing) at a blood glucose of 10–12 mM, (brain glucose
3.2–3.4 mM; Fig. 5) i.e., only slightly above the physiological level in rats (7.6 { 0.3 mM, n Å 133, which corresponds
to brain glucose concentrations of 2.4 { 0.13 mM, n Å
116). Type II neurons stopped firing when blood [glucose]
reached Ç17 mM (brain glucose of 4.2 { 0.2 mM; Table
2), whereas type III still generated some discharges when
blood glucose rose ú17 mM (Fig. 6). Type IV cells (Table
2), which behaved ‘‘anomalously’’ and increased their firing
rate in hyperglycemia, constituted only 5–7% of glucosesensitive LHA neurons.
Early alterations in firing frequency also occurred when
[glucose] was lowered (Fig. 2, 4–6; Table 3). Type I cells
/
2/
/
FIG . 5. Cell firing rates (top), intracellular [Na ], [Ca ], and [K ]
(middle three), and cell membrane potential (E, bottom) from glucosesensitive type I LHA cells at blood sugar levels higher (right) and lower
(left) than the baseline control value. Intracellular ion concentrations were
measured with double or multibarreled ionselective electrodes as described
in METHODS . E and cell action potential activity were measured through
the reference barrels of the ionselective probes. Values given for each point
represent means { SD for 5 cells at each of the blood sugar levels below
the symbols. Baseline blood sugar level (0) is 7.6 { 0.3 (n Å 175).
Corresponding brain glucose concentrations can be found in Table 2. Values
are means { SD for 5 cells in each category. All values are statistically
significant compared with baseline, P õ 0.05, except those marked ns.
/ 9k27$$ap04 J392-5
were again the most sensitive: they increased their firing rate
almost to a maximum when blood glucose level fell from 7.6
to 5.6 mM (which corresponds to a decrease in brain glucose
from 2.4 to 2.1 mM). Type II and III neurons showed more
gradual changes, with type III exhibiting persistent activity at
a brain [glucose] õ0.6 mM. Examples of cell firing dynamics
recorded extracellularly from type I and type III neurons, in
response to continuous changes in blood (and brain) glucose
concentrations, are displayed in Fig. 2. The different behavior
patterns of the two cell types in relation to changes in glucose
concentration is clear. However, both increase their firing rates
as blood/brain sugar falls (brain glucose level between 1.5
and 0.5 mM) until a ‘‘disaster’’ level is reached (brain glucose
õ0.5 mM—the exact value depending on the individual animal) where cell activity ceases.
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/
2/
/
FIG . 6. Cell firing rates (top), intracellular [Na ], [Ca ], and [K ]
(middle three), and cell membrane potential (E, bottom) from glucosesensitive type III LHA cells at blood sugar levels higher (right) and lower
(left) than the baseline control value. Intracellular ion concentrations were
measured with double or multibarreled ionselective electrodes as described
in METHODS . E and cell action potential activity were measured through
the reference barrels of the ionselective probes. Values given for each point
represent means { SD for 5 cells at each of the blood sugar levels below
the symbols. Baseline blood sugar level (0) is 7.6 { 0.3 (n Å 175).
Corresponding brain glucose concentrations can be found in Table 2. Values
are means { SD for 5 cells in each category. All values are statistically
significant compared with baseline, P õ 0.05, except those marked ns.
1740
I. A. SILVER AND M. ERECIŃSKA
TABLE 4. Changes in firing rates, intracellular ion concentrations and membrane potentials in VMH neurons during stepwise
increases and decreases in glucose level
D Glucose, mM
Blood
Baseline
Hyperglycemia
/2
/5
/10
Hypoglycemia
02
04
06
Firing Rate, spikes/s
[Na/]i, mM
[Ca2/]i, nM
11 { 2
24.8 { 1.6
125 { 4.3
72.4 { 2.7
067.8 { 1.7
/0.3
/1.0
/1.8
16 { 3
27 { 1
33 { 2
24.3 { 0.7*
23.5 { 1.3
22.3 { 1.2
137 { 4.6*
154 { 6.3
180 { 5.6
71.0 { 2.7*
70.6 { 2.6*
69.2 { 2.7
066.4 { 1.5*
065.6 { 1.4
064.6 { 0.8
00.3
01.1
02.2
6{2
1{1
0{0
24.0 { 1.3*
23.1 { 1.4
22.5 { 1.5
124 { 4.8
118 { 5.5
108 { 5.0
73.5 { 2.4*
75.0 { 2.3
76.4 { 2.3
069.3 { 1.9*
070.9 { 2.1
072.5 { 2.3
Brain
[K/]i, mM
E, mV
Simultaneous measurements of firing rates and the concentration of one or more intracellular ions, together with membrane electrical potentials, were made in ú100 glucose-sensitive LHA neurons. Because it was not possible to record [K / ]i ,
[Na / ]i , and [Ca 2/ ]i , simultaneously in every cell, a few ion
measurements from different cells of the same type have been
combined. Examples of records from type II cells are shown
in Figs. 3 and 4. In Fig. 3, the top trace is from the reference
barrel and shows extracellular action potentials followed by
cell penetration (⇓) and recording of membrane potential and
intracellular action potentials. The middle and bottom traces
are from the potassium and calcium-sensitive barrels, respectively, of the same electrode. Because of the relatively slow
responses of the ion-sensitive probes, recordings from these
are displayed on a longer time scale. One minute after the cell
was penetrated, the rat was given glucose intravenously, and
as the blood glucose level rose above the baseline value of 7.7
mM, changes in cell firing rate and ion concentrations occurred
that are shown as excerpts that correspond to the increase in
blood sugar indicated on the x axis. (The same traces but at
higher amplification are shown in the insets). Figure 4 shows
continuous changes in [K / ]i and [Na / ]i together with excerpts
of action potentials during decreases in blood glucose after
injection of insulin. Similar protocols were followed for other
cells and the results are summarized as means { SD in Tables
2 and 3 and Figs. 5 and 6.
FIG . 7. Continuous simultaneous traces of blood and
brain glucose concentrations together with extracellular
record of firing frequency of a glucose-sensitive cell
in the ventromedial hypothalamic nucleus (VMH). F,
injections of insulin and glucose; f, blood sampling
points as in Fig. 2. Concentrations measured were within
experimental error the same as those obtained with the
indwelling glucose electrode and shown in the figure.
One-second segments of records of extracellular action
potentials from the cell are shown for times 0, 18 min,
and 33 min at blood sugar levels of 7.5, 5.1, and 17.2
mM, respectively.
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Intracellular ion concentrations were measured with double- or multi-barreled microelectrodes as described in Experimental procedures. Membrane
potential (E) and firing rate were measured through the reference barrels of the ion-selective probes. Values are means { SD for either 16 (baseline) or
8 (all other) measurements. All values are statistically significant compared with baseline, P õ 0.05, except those marked *. Baseline glucose concentration
in blood was 7.6 { 0.3 (n Å 175) and that in brain 2.4 { 0.13 (n Å 133). VMH, ventromedial hypothalmic nucleus.
GLUCOSE SENSITIVITY OF HYPOTHALAMIC NEURONS
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FIG . 8. Records from a triple-barreled microelectrode showing action
potentials and cell membrane potential (top), together with intra- and extracellular concentrations (in mM) of sodium (middle) and potassium (bottom) in response to hyperglycemia. These records are from a VMH cell.
⇓, time of penetration of the cell, extracellular recordings to left, intracellular
recordings to right. ⇑, withdrawal of the electrode from the cell. Top: from
the reference barrel of the electrode and recorded on a time scale (seconds)
that shows individual action potentials. Middle and bottom: [K / ] and
[Na / ], displayed on a longer time scale (minutes) because of the relatively
slow response times of the ion-sensitive electrodes. One minute after the cell
was penetrated, intravenous glucose infusion was started. Three segments of
each trace were taken when blood glucose concentrations were those shown
on the x axis, namely baseline (7.6 mM), /2 (9.6 mM), and /4 (11.6
mM).
Responses of VMH neurons to changes in blood glucose
concentration
Of the 87 VMH neurons tested, 38 (43%) responded to
a rise in external glucose concentration by an increase in
firing rate. These cells were not sensitive to other stimuli
employed. Only five of these glucose-sensitive neurons were
of type I, i.e., those in which the change in the discharge
rate reached a maximum after a relatively small rise in the
sugar level (from 7.4 to 8–10 mM in the blood). Most
neurons, 85%, were of type III and generated progressively
higher discharge rates when blood glucose was gradually
raised to ¢15 mM. No cells were encountered whose activity
was inhibited by hyperglycemia.
Lowering external [glucose] had the opposite effect; even
a very small decrease in blood sugar level, from 7.6 to 7.0
mM, reduced the rate of firing. At 5.6–6.0 mM blood glucose concentration, the discharge frequency fell by 40–60%,
whereas at 3–4 mM, ú90% of cells were silent (Table 4).
An example of typical changes in neuronal activity, recorded
extracellularly, during a continuous cycle from normo-,
through hypo- to hyperglycemia, is shown in Fig. 7.
Concentrations of intracellular ions were measured with
microelectrodes in 38 glucose-sensitive VMH neurons dur-
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Intracellular ion concentrations measured in hypothalamic
neurons of normoglycemic animals were very similar, if not
identical, and were close to the values determined by us
previously in cells in other regions of the brain (Erecińska
and Silver 1992, 1994; Silver and Erecińska 1990, 1992).
Neurons that were inhibited by a rise in [glucose] (97 cells
of types I–III) showed very similar behavior patterns: the
concentration of potassium rose whereas that of calcium and
sodium fell (Figs. 5 and 6; Table 2). These alterations were
relatively small: 6–10 mM for sodium and potassium and
15–20 nM for calcium. Membrane potentials became more
negative by 4–7 mV, cells hyperpolarized and action potentials became fewer and larger.
In the very few neurons that were stimulated by a rise in
glucose (type IV), changes in ion concentrations were in
the opposite directions: sodium and calcium increased and
potassium decreased while the cell depolarized by a few
millivolts. An interesting difference between the type IV
neurons and all other glucose-sensitive LHA cells was the
resting concentration of calcium, which was Ç30 nM higher,
140 versus 110 nM (Table 2).
Gradual changes in intracellular ion concentrations also
were seen when blood glucose level was lowered progressively (Figs. 4–6; Table 3). The internal concentrations of
sodium and calcium rose, whereas that of potassium fell and
cells depolarized; all responses of type I cells were significant at a smaller decrement in [glucose] (decrease in blood
glucose concentration by 2 mM; Fig. 5) than those of types
II and III (Fig. 6; Table 3). The early increases and/or
decreases in intracellular [ions] ( D brain glucose 00.3 and
01.1 mM) were again relatively small. However, in severe
hypoglycemia, when glucose concentration in brain fell to
Ç0.2 mM and lower ( D brain glucose 02.2 mM), ‘‘catastrophic’’ changes began to occur: there were rapid rises in
internal concentrations of sodium and calcium and a simultaneous loss of potassium with a fall in membrane potential
as described previously (Silver and Erecińska 1992, 1994)
During decreases in blood (brain) glucose, type IV LHA
neurons exhibited changes in ion concentrations that were
opposite to those in types I–III; [Na / ]i and [Ca 2/ ]i declined, [K / ]i rose, and the plasma membrane hyperpolarized
by 3–4 mV (Table 3). When the recordings carried out at
the lowest brain [glucose]e were continued for 3–5 min after
the 0.2 mM level was reached, in three of five cells listed
in Table 3, there were rapid falls in [K / ]i (to 52.3 { 4.5
mM) and membrane potential (to 054.3 { 4.7 mV) and
increases in [Na / ]i (to 33.3 { 4.0 mM) and [Ca 2/ ]i (to
289 { 42 nM). In the remaining two cells, similar but slower
changes were observed (results not shown).
Responses of intracellular ions to physiological changes
in [glucose] were determined in 26 neurons classified as
sugar-insensitive. The basal (normoglycemic) concentrations of [Na / ]i (24–27 mM), [K / ]i (67–71 mM), [Ca 2/ ]i
(105–120 nM), and cell membrane electrical potentials
( 065 to 069 mV) were not altered when blood glucose was
either raised to 15–17 mM (4–5 mM in brain) or reduced
to 1.5–2 mM (0.3–0.4 mM in brain). However, in very
severe hypoglycemia (brain sugar level õ0.3 mM), catastrophic ion movements were seen, similar to those that
occurred under comparable conditions in glucose-sensitive
cells.
1741
1742
I. A. SILVER AND M. ERECIŃSKA
ing increases and decreases in blood sugar level. With hyperglycemia, [K / ]i and [Na / ]i fell, whereas [Ca 2/ ]i rose and
cells depolarized by a few millivolts (Fig. 8 and Table 4).
During hypoglycemia, intracellular potassium concentration
increased, whereas that of sodium and calcium decreased.
The plasma membrane hyperpolarized (Fig. 9 and Table 4).
DISCUSSION
The present study is, to the best of our knowledge, the
first work that examines, in a systematic manner and in
considerable detail, the changes in intracellular concentrations of [Na / ]i , [K / ]i , and [Ca 2/ ]i and membrane potential
as well as in neuronal firing rate that occur in LHA and
VMH cells in intact rat brain in response to alterations in
blood (and brain) glucose level. It shows that 30–45% of
the neurons in hypothalamic areas LHA and VMH are endowed with exquisite sensitivity to increases/decreases in
glucose concentration. In both these regions, alterations in
cell firing rate and internal ion concentrations were observed
at increments/decrements in the extracellular sugar level as
small as 0.2–0.3 mM (or blood glucose decreases/increases
of 2 mM). This behavior indicates that these ‘‘sensors’’ can
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FIG . 9. Records from a triple-barreled microelectrode showing action
potentials and cell membrane potential (top), together with intra- and extracellular concentrations (in mM) of potassium (middle) and calcium (bottom) in response to hypoglycemia. These records are from a VMH cell. ⇓
and ⇑ and traces as in Fig. 8. Thirty seconds after the cell was penetrated,
intravenous insulin was administered (see METHODS ). Four segments of
each trace were taken when blood glucose concentrations were those shown
on the x axis, i.e., baseline (7.6 mM), 02 (5.6 mM), 04 (3.6 mM), and
05 (2.6 mM). Last (5th) segment shows the recovery of the measured
parameters after glucose infusion followed by withdrawal of the electrode.
respond to physiological shifts in sugar concentration and
therefore have the capacity to be an integral part of the
body homeostatic mechanisms. However, not all cells, in
particular those in the LHA, exhibit the same degree of
sensitivity. Although for the sake of simplicity in presentation of the data we categorized the LHA glucose-sensing
neurons into four classes, our findings suggest that there is
a continuous spectrum of responsiveness and that the
stronger the stimulus (change in glucose concentration) the
larger is the number of cells that are recruited to react. Some
of the glucose-sensitive cells of types II and III also responded to peripheral sensory stimuli, and it is possible that
such inputs may modulate their response to glucose.
Consistent with the data in the literature (Oomura et al.
1969), the majority of glucose-sensitive LHA cells hyperpolarized and decreased their firing rate when blood glucose
level was raised and depolarized and increased discharge
frequency when glucose concentration fell. Concomitantly,
hyperglycemia was accompanied by a reduction in [Na / ]i
and a rise in [K / ]i , whereas the opposite events took place
in hypoglycemia. This pattern of responses is consistent with
stimulation and inhibition, respectively, of activity of the
plasma membrane Na/K ATPase, as postulated by earlier
authors (Oomura et al. 1969). The question that was not
addressed previously by others but that can be answered
based on our results, is how a change in external glucose
level is ‘‘translated’’ into altered pump function.
The major determinants of the Na/K pump activity are
the concentrations of intracellular sodium and extracellular
potassium as well as [ATP] and the products of its hydrolysis, [ADP] and [Pi]. The first three are stimulatory, whereas
the latter two are inhibitory (De Weer 1970; Garay and
Garrahan 1975; Glynn and Karlish 1975; Robinson and
Flashner 1979). Transport of glucose from extracellular
space into neurons is considered to occur via facilitated diffusion, which does not require sodium (Lund-Andersen
1979). Thus enhanced uptake of this sugar in hyperglycemia
should not elevate [Na / ]i as indeed is indicated by our data.
(Although an apparent lack of even transient increases in
the intracellular level of sodium could argue against the
existence in LHA sensor cells of a separate, Na-linked glucose carrier small changes occurring in õ3 s might have
been missed due to the relatively slow response of the Na
electrode.) Similarly, an elevation in [K / ]i under the same
conditions (Table 2; Figs. 3, 5, and 6) suggests that [K / ]e
is unlikely to rise, whereas a possible decrease could reduce,
but not augment, the pump activity. By contrast, a rise in
ATP (and consequent decreases in [ADP] and [Pi]), if it
occurs, should be stimulatory, whereas a decline should be
inhibitory. The suggestion that such a mechanism for
Na/K ATPase regulation exists is based on the findings
(Ikehara et al. 1984; Soltoff and Mandel 1984; Tessitore et
al. 1986) that the activity of this enzyme in several types of
intact cells (in contrast to membrane preparations isolated
therefrom) does not attain a maximum at the physiological,
intra-cellular ATP level (2.5–6 mM for various cell types;
2.5–3 mmol/g wet weight, or 3.0–3.5 mM in brain) (Erecińska and Silver 1989, 1994; Siesjö 1978), even though both
nucleoside triphosphate binding sites should be saturated
with the substrate [Km for ATP of Ç10 mM at the catalytic
site and Ç0.5 mM at the regulatory site (Glynn and Karlish
GLUCOSE SENSITIVITY OF HYPOTHALAMIC NEURONS
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An alternative is that fuel-sensitive LHA neurons, like
liver and pancreatic b-cells (Meglasson and Matschinsky
1984), possess glucokinase, a low-affinity (high Km ) hexokinase. This enzyme is not inhibited by glucose-6-phosphate
and exhibits a Hill coefficient of 1.2–1.5 (Meglasson et al.
1983; Storer and Cornish-Bowden 1976), which amplifies
metabolic changes in response to increases/decreases in substrate concentration. It has been suggested that the glucokinase is responsible for glucose-induced energetic alterations
in b-cells (Meglasson and Matschinsky 1986) in which
[ATP] (or [ATP]/[ADP]) controls activity of the ATPdependent K / -channel (Ashcroft 1988; Dunne and Petersen
1991). Our conjecture that glucose-sensitive LHA neurons
contain an analogous sensor mechanism is supported by the
recent finding that certain cells in the medial hypothalamus
express a glucokinase gene, glucokinase mRNA, and glucokinase activity (Jetton et al. 1994). By modulating the concentration of cytosolic [ATP]/[ADP] LHA glucokinase
could alter the activity of the plasma membrane Na pump,
which in many systems (Lynch and Balaban 1987; Takai
and Tomita 1986; Weiss and Lamp 1989), including brain
(Lipton and Robacker 1983; Raffin et al. 1988, 1992; Roberts 1993), is particularly sensitive to glycolytically generated ATP.
The glucose-sensitive VMH cells depolarized and increased their discharge frequency when glucose level was
raised and hyperpolarized and decreased their firing rate
when [sugar] was reduced. Concurrently, [K / ]i fell in hyperglycemia and rose in hypoglycemia. These changes are
consistent with the closure and opening in the two conditions
respectively, of the ATP-sensitive K / -channels, as indeed
has been demonstrated directly by electrophysiological studies (Ashford et al. 1990). The VMH ATP-sensitive potassium channels have a single channel conductance of Ç150
pS, and their Ki for ATP ( Ç3 mM) is considerably higher
than that in skeletal (0.5 mM) and cardiac muscles (0.1–
0.5 mM) and pancreatic b-cells (15–20 mM) (Ashcroft
1988). This high Ki for ATP, very close to the concentration
of the latter in ‘‘resting’’ brain (Erecińska and Silver 1989,
1994; Siesjö 1978), means that small changes in energy
level could alter potassium permeability and thus membrane
potential in these neurons, very sensitively. Although no
information currently exists on how glucose-responsive
VMH cells control their energy metabolism, the existence
of a glucokinase-like enzyme, similar to that outlined above
for LHA cells, would allow for a continuous spectrum of
changes in [ATP] ([ATP]/[ADP]) and account for our
experimental findings. It is worth pointing out that this mechanism is very similar to that which has been postulated to
control potassium channel activity, and hence physiological
function, of fuel-sensing pancreatic b-cells (Ashcroft 1988;
Dunne and Petersen 1991).
Our results also show that alterations in [Na / ]i and [K / ]i ,
which occur in the sensitive LHA and VMH neurons in
hyper- and hypoglycemia, are accompanied by changes in
intracellular calcium concentration. The pattern of behavior,
a rise during depolarization and a decline on hyperpolarization, is consistent with an increase in the number of opened
Ca 2/ -channels when the membrane potential falls. These
changes may be important because it has been reported recently that [Ca 2/ ]i controls the fraction of sodium channels
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1975; Robinson 1976)]. If such dependence on energy also
occurs in LHA neurons and if changes in [glucose] continuously modify the intracellular concentration of ATP, then
activity of the pump will increase above the basal level in
hyperglycemia and decrease in hypoglycemia in agreement
with our results. Although this scenario could account for
the findings, it explains neither how a small change in glucose level brings about an alteration in intracellular energy
content nor why there is a difference between glucose-sensitive LHA cells and the majority of CNS neurons that do not
respond metabolically to small (physiological) alterations in
sugar concentration.
It generally is accepted that cerebral metabolism of glucose, which is one of the determinants of tissue ATP level,
is phosphorylation- and not transport-limited (Furler et al.
1991; Lund-Andersen 1979; Pardridge 1983; Robinson and
Rapoport 1986). Two conclusions follow: that under physiological, normoglycemic conditions extra- and intracellular
concentrations of glucose are similar and that activity of
the phosphorylating enzymes, and not the neuronal plasma
membrane transporters (Bell et al. 1993; Vannucci et al.
1997), is the rate-controlling step. The first is supported by
the findings that total cerebral glucose content (which is
mainly intracellular) (e.g., Duffy et al. 1972; Gjedde and
Diemer 1983; Lewis et al. 1974; Mason et al. 1992) is very
close to that in the extracellular space (Silver and Erecińska
1994). With respect to the second, glucose enters neurons
predominantly on GLUT3, which has a relatively high affinity for its substrate (Km õ 2 mM) (Colville et al. 1993);
thus the transporter is likely to operate at a velocity not
much below the Vmax . Sugar phosphorylation is catalyzed
by hexokinases and brain contains almost exclusively the
type I enzyme, which, with a Km for glucose of 10–50
mM (Wilson 1985), should be saturated under physiological
conditions.
It is clear that the above considerations, which are based
on measurements in bulk tissue, apply to the majority of
CNS neurons (which are not glucose sensitive) but not to
LHA cells. In the latter, it is either the transport or phosphorylation of glucose that must be distinct. Moreover the velocity of either or both of these processes would have to vary
over a broad range of sugar concentrations to modulate cellular metabolic rate and ATP content. Recent computer modeling studies (Sweet and Matschinsky 1995) of glucose metabolism in pancreatic b-cells, which also respond sensitively
to alterations in the ambient glucose level (Meglasson and
Matschinsky 1986), show that for transport not to be limiting
its velocity has to exceed phosphorylation by a factor of
5. When the maximal rate of transport approaches that of
phosphorylation, the former becomes a very sensitive effector of fuel usage (Sweet and Matschinsky 1995). There
is no information on what relations exist between the two
processes in glucose-sensing hypothalamic neurons, but
there is no indication in our results that transport of glucose
involves a process other than facilitated diffusion, which
varies linearly with changes in substrate concentration. Consequently, alteration of metabolism by Dglucose of 0.2–0.3
mM would be expected to be very small. Hence in our
opinion, although glucose transport is a potential site of
control, it seems unlikely to play a major role in affecting
energy metabolism of glucose-sensitive hypothalamic cells.
1743
1744
I. A. SILVER AND M. ERECIŃSKA
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-28329.
Present address of M. Erecińska: Dept. of Anatomy, School of Veterinary
Science, University of Bristol, Bristol BS2 8EJ, UK.
Address reprint requests to I. A. Silver.
Received 19 June 1997; accepted in final form 12 December 1997.
/ 9k27$$ap04 J392-5
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