1) The study examined orexin neurons, which are involved in arousal, food intake, and metabolism. It found that these neurons rely on lactate produced by astrocytes as their primary energy source. 2) Experiments showed that lactate was able to restore the firing activity of orexin neurons when glucose was removed. Blocking lactate transport or depleting astrocyte-produced lactate inhibited neuronal firing. 3) Orexin neurons were found to act as "lactate sensors," with their firing rates dependent on extracellular lactate concentrations. This suggests lactate provides an important signaling role in the brain beyond merely serving as an energy substrate.

1. Sachin Mehta
Analysis of article: “ATP-Sensitive Potassium Channel-Mediated Lactate
Effect on Orexin Neurons: Implications for Brain Energetics during
Arousal” Matthew P. Parsons and Michiru Hirasawa. 2010. The Journal of
Neuroscience. 30(24): 8061-8070.
http://www.jneurosci.org/content/30/24/8061.full
Introduction
The brain utilizes a significantly large proportion of glucose in the body.
While it is still not entirely confirmed, the widespread belief is that glucose
uptake is performed by certain “glucosensing neurons” in the hypothalamus and
brainstem (Levin et al., 2004). They maintain this homeostasis by excitatory or
inhibitory effects. However, these cells are not the only glucose-metabolizing
cells in the brain. Astrocytes have been identified as the primary cell type to
utilize glucose, and they produce lactate as an additional substrate.
In the last decade or so, new evidence has emerged suggesting that lactate
is one of the primary sources of energy and regulates certain metabolic factors.
The extent of its role in certain areas of the brain is not completely understood.
Parsons and Hirasawa conducted their research on a group of cells known as
orexin neurons, which are known to play an important part in food intake,
autonomic function and wakefulness. These qualities make them great
candidates to consider, as they obviously require energy input and metabolism
for function. Previous research has shown that orexins are stimulated by glucose
and lactate, and perhaps require this lactate as their fundamental source of

2. energy. This study showed that orexin neurons sense certain levels of astrocyte-
produced lactate, and in turn, modulate their neuronal activity and responses to
these levels.
Experimental System
This research used a combination of electrophysiology on rat and mice
brains, immunohistochemistry, data and drug analyses to learn more about
orexin neurons. The rats and mice were anesthetized and decapitated to obtain
coronal hypothalamic slices. Glucose concentrations were measured using
patch-clamp recordings on the hypothalamic slices. Additional patch-clamp
tests were used to measure the firing (action potential) characteristics of the
orexin neurons. These values were used to deduce orexin neuron concentration
in comparison to the high presence of melanin-concentrating hormone (MCH) in
the same area. Phenotypic characterizations of the orexin neurons were
performed by voltage ramps and current injections.
Immunohistochemical techniques were performed to detect antibody
signaling at different parts of the sectioned brains. Rabbit anti-MCH and goat
anti-orexin A antibodies were mixed together to determine the localizations of
certain orexins and MCH. Additionally, KATP (ATP-sensitive potassium)
channel subunits were identified using rabbit anti-Kir6 antibodies.
Immunofluorescence was then visualized using confocal microscopy.

3. Data analysis was an important part of this study. Researchers used
Synaptosoft software to analyze certain action potential properties, such as
current, frequency and membrane potential. T-tests were also performed in
order to deem the significance of the data.
Finally, a variety of drugs were used in conjunction with the
electrophysiology techniques in order to understand structures and functions of
the key players in orexin neuron metabolism.
Experiments and Results
In order to determine the role of lactate and whether or not it was
preferred by orexins, cell-firing experiments showed the necessity for glucose
and lactose. 4-CIN, an inhibitor of MCT’s (monocarboxylate transporters--
needed for lactate transport across membrane) was used, and showed an
inhibition of firing activity. Next, the behavior of orexins in a glucose-free
environment was shown to completely shut off firing activity, revealing the true
importance of glucose as an energy substrate. Lactate supply was then used to
bring back complete firing activity. The mechanism by which this occurs is
unclear, as it is possible that the lactate produced by nearby astrocytes was
enough for this restoration. The researchers then tested the necessity of
endogenous astrocyte-produced lactate on the orexin neurons. Hypothalamic
slices were drained of all remaining glucose and other energy substrates by glial
toxin fluoroacetate (FAC), and the firing rate was examined in conjunction with a

4. lactate and glucose supply. The results showed that astrocytes convert the
glucose to lactate. This lactate is then used by orexins and is responsible for
maintaining any spontaneous firing activity.
KATP channels were examined because they are known to be crucial in
lactate transport. Their structures were investigated using immunofluorescence
labeling. These techniques show that KATP channels of the orexin neurons
consist of Kir6.1 and SUR1 subunits, which are modulated depending on the
specific metabolic activities of the cells. Glibenclamide blocked the
hyperpolarization of the KATP channels, indicating that these channels are
necessary for firing activity and that KATP channels control lactate levels.
Lastly, the researchers determined that these orexin neurons are capable
of acting as “lactate sensors.” They tested this idea by depleting all lactate from
the hypothalamic slices, and testing the firing rate. The orexins based their firing
rates on the level of lactate available, implying that their activity is concentration-
dependent.
Conclusion
The research performed by Parsons and Hirasawa showed that lactate
produced by astrocytes is needed and preferred as an energy source by orexin
neurons. Because lactate seemed to induce an excitatory effect on the firing
frequency, it was concluded that orexin neurons act as concentration-dependent
lactate sensors. That is, these neurons can perceive a change in the concentration

5. of extracellular lactate levels, which can alter their cellular effects and expression.
Lactate also plays a crucial role in sustaining a normal resting membrane
potential when combined with glucose and KATP current. Lastly, the
researchers concluded that orexin neurons contain a relatively generous amount
of intracellular lactate, which serves as an endogenous energy supply in the
absence of glucose.
Significance
This study sheds light on the importance of lactate as an energy substrate
and a paracrine factor. It is capable of providing signaling effects to orexin
neurons, indicating brain activity and energy supply. High lactate levels result
in stimulated orexin and KATP channel activity. Furthermore, orexin neurons
are essential in the astrocyte-coupling process and can provide additional
neuroprotection. Because orexin neurons play a significant role in the
physiology of certain processes, such as wakefulness and food intake,
understanding even more thoroughly how and what purpose lactate serves the
brain will provide us with a key to treating GI disorders and sleep pattern phases
(Shram et al., 2002). Finally, since this area of study is relatively new, much more
research is needed to truly understand the extent and importance of lactate
processes in the brain.

6. Literature Cited
Levin BE, Routh VH, Kang L, Sanders NM, Dunn-Meynell AA (2004).
Neuronal glucosensing: what do we know after 50 years? Diabetes
53:2521–2528.
Shram N, Netchiporouk L, Cespuglio R (2002). Lactate in the brain of the
freely moving rat: voltammetric monitoring of the changes related to the
sleep–wake states. Eur J Neurosci 16:461– 466.