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Overview: The Study of Neuromodulators in Neural Circuits and Behavior

alphabet soup

Neuromodulators are a special category of neurotransmitters that play profound roles in cognition, emotion, and behavior. Neuromodulators differ from classical neurotransmitters in that they are typically expressed by unique clusters of neurons, they project diffusely throughout the nervous system, and they modulate postsynaptic neurons in a way that alters their responses to traditional neurotransmitters (such as GABA and glutamate). They are typically either small peptides ("neuropeptides"), such as hypocretin, melanin-concentrating hormone, neuropeptide S, and cortistatin, or derivatives of amino acids ("monoamines"), such as dopamine, histamine, norepinephrine, and serotonin. The small molecule acetylcholine functions as a neurotransmitter in the peripheral nervous system and as a neuromodulator in the central nervous system. Interestingly, all central nervous system neurons that produce neuromodulators also release traditional neurotransmitters, raising the question as to what neuromodulators contribute to neural circuits aside from classical neural signaling.

The alphabet soup of some commonly studied neuromodulators. 5HT, serotonin; ACh, acetylcholine; CST, cortistatin; DA, dopamine; Hcrt, hypocretin; His, histamine; MCH, melanin-concentrating hormone; NE, norepinephrine; NPS, neuropeptide S.

In our lab, we focus on the role that many of these neuromodulators play in various mammalian behaviors. A major focus of our lab is understanding the role of neuromodulators in arousal and sleep. Some neuromodulators, such as 5HT, Ach, DA, His, and NE are known to promote arousal, with Hcrts stabilizing wake states. Others, such as CST and MCH, are thought to promote sleep states. However, our understanding is limited, and important questions remain:

  • Is neural activity in nuclei that produce neuromodulators sufficient or permissive to promote sleep, wakefulness or state transitions?

  • What downstream neural populations are necessary or sufficient to mediate the effects of neuromodulators?

  • What are the kinetics of neurotransmission between sleep- and wake-promoting circuits in the brain in an unrestrained, behaving animal?

  • What are the consequences of neuromodulator vs neurotransmitter release from synapses on downstream neurons?

Our lab is currently attempting to answer these questions using the methods described below.

Further reading:

de Lecea (2010). A decade of hypocretins: past, present, and future of the neurobiology of arousal. Acta Physiol. 198(3):203-8. [Abstract] [PDF]

de Lecea (2008). Cortistatin--functions in the central nervous system. Mol. Cell. Endocrin. 286(1-2):88-95. [Abstract] [PDF]

Adamantidis and de Lecea (2008). Physiological arousal: a role for hypothalamic systems. Cell Mol. Life Sci. 65(10):1475-88. [Abstract] [PDF]

The role of neuromodulators in sleep/wake transitions. The balance between wakefulness and sleep is thought to depend upon a balance of activity in different neuromodulatory systems. NE, His, 5HT, and other systems tend to be active during wakefulness, with Hcrt exerting an excitatory tone on these systems. Other systems, such as the MCH system and populations of GABAergic nuclei are more active during sleep. The precise contribution of individual neuronal populations to sleep/wake states is relatively unknown.

New Approaches to Studying the Function of Neuromodulators in vivo

In order to investigate the role of neuromodulators in behavior, it is necessary to test the effects of manipulating their function.  Traditionally, neuromodulator systems have been perturbed in vivo using electrical, physical, pharmacological, and genetic methods.  Although much progress has been made using these classical techniques, considerable drawbacks prevent their use in the study of neural circuits with fine spatial and temporal precision.  Electrical and physical techniques are not spatially precise and can cause stimulation, inhibition, or inactivation of surrounding cells and processes.  Pharmacological and genetic methods have better spatial selectivity but lack temporal resolution at the scale of single action potentials. 

Strategies to manipulate neural activity in vivo. Cells in the nervous system reside in heterogeneous populations composed of different subtypes (here depicted as different colors). Electrical microstimulation techniques have high temporal precision but affect all cells and fibers surrounding the electrode. Physical probes irreversibly ablate or reversibly cool cells to reduce neural activity, but also affect all cells and fibers surrounding the electrode. Pharmacological injection of a drug might be able to target a particular cell-type based on cell-specific protein expression, but the drug could remain in the system for minutes or hours after injection. Genetic inactivation of specific cells (such as the cells depicted in blue) also lacks temporal precision and is often irreversible. Optogenetic stimulation or inhibition of cells using light allows for cell-type specific targeting of optogenetic probes (such as the cells depicted in blue) with millisecond-timescale precision of activation.

To overcome these limitations, we have added optogenetics to our repertoire of techniques for studying neuromodulatory systems.  Optogenetics consists of a set of probes that are activated by light (“opto-”) and are genetically encoded (“-genetics”), allowing for the direct control of specific populations of neurons.  For example, Channelrhodopsin-2 (ChR2) is a nonspecific cation channel that opens and depolarizes neurons when stimulated with blue light. Halorhodopsin (NpHR) is a chloride pump that hyperpolarizes neurons in response to yellow light.  These probes can manipulate neurons in vivo with high temporal precision and rapid reversibility.  Therefore, optogenetic tools can be thought of as a perfect combination of an electrode, which has high temporal precision, with a genetically encodable probe, which has high spatial res- olution. We now harness the potential of these tools to study multiple neuromodulatory systems in the brain, such as the Hcrt, LC-NE, and VTA-DA systems described below. 

Further reading:

Carter et al. (2011). Optogenetic investigation of neural circuits in vivo. Trends Mol. Med. IN PRESS

Adamantidis et al. (2010). Optogenetic deconstruction of sleep/wake circuitry in the brain. Front. Mol. Neuro. 2:31. [Abstract] [PDF]

Zhang et al. (2010). Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5(3):439-56. [Abstract] [PDF]

Optogenetic probes. Channelrhodopsin-2 (ChR2--top) is a cation channel that opens in response to blue light illumination. Halorhodopsin (NpHR--bottom) is a chloride pump that activates in response to yellow light illumination. Both probes are genetically encoded and do not require exogenous co-factors, allowing for cell-type specific targeting in vivo.

The Hypocretins

In 1998, our group discovered the hypocretins (Hcrts—also called “Orexins”), a pair of neuropeptides expressed exclusively in the brain by neurons in the lateral hypothalamus.  Hcrt neurons send diffuse projections throughout the brain and excite their postsynaptic targets by binding to one of two G-coupled protein receptors, the hypocretin receptors (HcrtRs).  Hcrts play a key role in the stability of wakefulness.  Electrophysiological recordings of Hcrt neurons show that they are relatively silent during sleep compared with wakefulness, with phasic bursts of activity preceding transitions to wakefulness.  Loss-of-function perturbation of the Hcrts or their receptors cause narcolepsy: human narcoleptics suffer from gradual loss of Hcrt neurons, narcoleptic dog populations show mutations in the hypocretin-2 receptor, and mouse models of Hcrt dysfunction (Hcrt knock-out mice or Hcrt::ataxin mice) also exhibit a narcoleptic phenotype.  When centrally administered, the Hcrts cause an increase in the time spent awake and a decrease in non-rapid eye movement (NREM) sleep and REM sleep. In addition to maintaining wakefulness, our lab has uncovered other roles for Hcrts in mediating the physiological hallmarks of stress and reinstating extinguished cocaine-seeking behavior.

Hcrt expression

The first image of the hypocretin system. This autoradiograph shows the result of an in situ hybridization experiment showing the expression of hypocretins in a coronal section from rat brain. A few thousand neurons express Hcrts in the lateral hypothalamus

Hypocretin implant

We have now applied optogenetic technology to stimulate or inhibit Hcrt neurons in vivo with high temporal resolution.  Using viral gene delivery techniques, we stably express ChR2 or NpHR in Hcrt neurons and investigate the effects of optogenetic stimulation on behavior.  Our work shows that optogenetic stimulation of Hcrt neurons at frequencies >5 Hz (but not 1 Hz) is sufficient to increase the probability of an awakening event during NREM and REM sleep.  This stimulation also increases neural activity in downstream arousal nuclei, such as the tuberomammilary nucleus and locus coeruleus.  Current projects in the lab aim at understanding the role of Hcrts in other behaviors, as well as understanding the importance of downstream targets in mediating Hcrt functions.

Further reading:

de Lecea et al. (1998). The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. PNAS 95(1):322-7. [Abstract] [PDF]

Sutcliffe & de Lecea (2002). The hypocretins: setting the arousal threshold. Nat. Rev. Neurosci. 3(5):339-49. [Abstract] [PDF]

Winsky-Sommerer et al. (2004). Interaction between the corticotropin-releasing factor system and hypocretins (orexins): a novel circuit mediating stress response. J. Neurosci. 24(50):11439-48. [Abstract] [PDF]

Boutrel et al. (2005). Role for hypocretin in mediating stress-induced reinstatement of cocaine-seeking behavior. PNAS 102(52):19168-73. [Abstract] [PDF]

Adamantidis et al. (2007). Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450(7168):420-4. [Abstract] [PDF]

Targeting the Hcrt system with optogenetics. To genetically target Hcrt neurons, we stereotaxically inject a virus carrying ChR2 driven by the Hcrt promoter into the lateral hypothalamus. To stimulate Hcrt neurons with light, we also implant a stainless steel cannula above the Hcrt field for placement of a fiber-optic cable.

Investigating Other Neuromodulatory Systems

locus coeruleus

In addition to Hcrt neurons, we have also applied optogenetic technology to the study of other neuromodulator systems. For example, the LC-NE system has been suggested to play major roles in arousal and attention, and we now study the effects of manipulating these neurons in vivo. We also target the VTA-dopamine system, the brain's major reward pathway. In the future, we will continue to use cutting-edge gene delivery strategies and genetically-encoded probes to study new neural populations and their downstream targets in behavior.

Further reading:

Carter et al. (2010). Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat. Neurosci. 13(12):1526-33. [Abstract] [PDF]

Adamantidis et al. (2010). Optogenetic deconstruction of sleep/wake circuitry in the brain. Front. Mol. Neuro. 2:31. [Abstract] [PDF]

Tsai et al. (2009). Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324(5930):1080-4. [Abstract] [PDF]



Targeting the LC-NE system with optogenetics. We used a combination of transgenics and viral delivery to target LC neurons with optogenetic transgenes. The image above shows expression of ChR2-eYFP (green) merged with immunohistochemical staining against Tyrosine Hydroxylase (red), a marker of noradrenergic neurons. We see over 99% specificity of viral targeting of LC neurons, allowing us to directly modulate the LC-NE system in vivo.

Studying the VTA-dopamine system with optogenetics. With our collaborators in the lab of Karl Deisseroth, we targeted the dopaminergic neurons of the VTA. The VTA sends strong projections to the nucleus accumbens as part of the brain's reward circuitry. The image above shows nucleus accumbens neurons (red) strongly innervated by fibers from the VTA transduced with ChR2-eYFP (green).