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 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.

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 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 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.

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.