The Hypothalamus And Addiction

Many levels of hypothalamic function are likely to influence addiction. While most of the previous work on the role of the hypothalamus in addiction assessed effects of the HPA (hypothalamic-pituitary-adrenal) axis and various physiological states, such as stress, on addiction,25-27 the LH-restricted expression of ORX/HCRT suggests a direct role for these peptides in controlling addiction-related behaviors. Indeed, the ORX/HCRT neurons project broadly to regions throughout the brain, including mesolimbic and brainstem nuclei known to be important in the development of drug addiction.15,28-30

5.1. The LH and Drugs of Abuse

The LH, in particular, has a history of being associated with drug addiction and drug abuse behavior. The relationship between the LH and both feeding and addiction may not be surprising since behavioral studies have shown cross-sensitization between 'natural' rewards and drugs of abuse. Both drugs of abuse and food restriction profoundly modulate LH-mediated ICSS and their effects can cross-sensitize.31,32 Moreover, leptin acts to reduce heroin self-administration33 and has can modulate LH-mediated ICSS. 14

Furthermore, there are experiments that have more directly implicated the LH in the reinforcing properties of drugs. Both rats and mice will self-administer morphine and other opioid peptides directly into the lateral hypothalamus.34-36 While some studies have found that LH lesions do not block opiate self-administration,37 other groups have demonstrated that systemic self-administration of heroin can be blocked by opiate antagonist delivery directly to the LH.38 Supporting work has established that direct administration of opioids to the LH region results in a conditioned place preference to the drug.39 However, the molecular basis of this LH-mediated response to opiates has remained largely uncharacterized.

6. ORX/HCRT NEURONS RESPOND TO CHRONIC MORPHINE AND MORPHINE WITHDRAWAL

While it is clear that the LH has powerful effects on behavior, the specific neuronal cell types mediating these functions have not been identified. The LH-specific expression of both MCH and ORX/HCRT makes these neuropeptides prime candidates for mediating behaviors related to drug addiction. Toward this goal, the ORX/HCRT and MCH neurons were studied for their responses to chronic morphine and precipitated morphine withdrawal. The neurons were evaluated with two separate markers: (1) c-Fos immunoreactivity, and (2) CRE-LacZ reporter activity40 were used to assess neural response and plasticity. Both c-Fos and the CRE-LacZ reporter are highly regulated by morphine and have been used extensively to map brain regions that demonstrate activation and neuronal plasticity in response to morphine and precipitated withdrawal.41-43 Results from this work indicate that the ORX/HCRT neurons are highly responsive to morphine, with nearly 10X as many neurons showing CRE-LacZ, compared to control animals, after five days of morphine (Figure 1A) (F(3 23) = 49.93;p < 0.05).

Neuron 10x

Figure 1. LH neurons expressing ORX/HCRT respond to morphine treatment while LH neurons expressing MCH do not. Mice were treated with sham or morphine pellets (2 x 25 mg) for five days followed by injection of saline or naltrexone (100 mg/kg, subcutaneous) four hours before being perfused and analyzed by immunohistochemistry. Neuronal responses to sham surgery (Sham) chronic morphine (Morph), sham surgery plus naltrexone (Sham+Nal), or chronic morphine plus naltrexone (Morph+Nal) were evaluated by use of a CRE-LacZ reporter mouse (A) or c-Fos detection (B). Figure modified from 44. N=6 per group, *p<0.05.

Figure 1. LH neurons expressing ORX/HCRT respond to morphine treatment while LH neurons expressing MCH do not. Mice were treated with sham or morphine pellets (2 x 25 mg) for five days followed by injection of saline or naltrexone (100 mg/kg, subcutaneous) four hours before being perfused and analyzed by immunohistochemistry. Neuronal responses to sham surgery (Sham) chronic morphine (Morph), sham surgery plus naltrexone (Sham+Nal), or chronic morphine plus naltrexone (Morph+Nal) were evaluated by use of a CRE-LacZ reporter mouse (A) or c-Fos detection (B). Figure modified from 44. N=6 per group, *p<0.05.

In contrast, no c-Fos is seen in ORX/HCRT neurons (Figure 1B), consistent with previous data suggesting that the c-Fos response desensitizes over time. To further evaluate the ORX/HCRT neurons for their responsiveness, we precipitated withdrawal by administration of the ^-opioid receptor antagonist naltrexone after 5 days of morphine treatment. Under this scenario, dramatic c-Fos responses are seen (F(3,20) = 15.79; p < 0.05) while a CRE-LacZ response is seen in over 30% of the neurons (Figure 1B).

The MCH neurons of the LH were also examined and did not show molecular responses to morphine or morphine + naltrexone (Figure 1A and 1B). Independent experiments have shown that the CRE-LacZ reporter is active in the MCH neurons as demonstrated by a large percentage of MCH neurons responding to starvation (data not shown). Thus, the data suggests a very specific response of ORX/HCRT neurons to both morphine exposure and precipitated withdrawal.

It should be noted that only a subset of ORX/HCRT neurons (25-35%) responded to morphine and precipitated withdrawal (Figure 1A and 1B) and nearly 50% of the cells showing CRE-LacZ response did not express ORX/HCRT. This suggests that the ORX/HCRT neurons are heterogeneous and also that there is a set of unidentified LH neurons that appears to be responsive to morphine.

7. THE ORX/HCRT GENE IS UPREGULATED AFTER PRECIPITATED MORPHINE WITHDRAWAL

Since the ORX/HCRT neurons are responsive to morphine, the expression of the ORX/HCRT gene was tested using paradigms described above. An orexin-tau-LacZ knock-in line was used to evaluate expression of the ORX/HCRT gene, as B-galactosidase allows for better quantitative analysis of gene expression. Analysis of LH brain sections from reporter mice showed that, unlike c-Fos or the CRE-LacZ reporter gene, the Orx-tau-LacZ reporter is not upregulated after chronic morphine (Figure 2). Interestingly, however, the reporter gene is upregulated ~40% after naltrexone-induced withdrawal from morphine (F(3,19) = 5.78; p < 0.01).

Figure 2. The ORX/HCRT gene is upregulated after chronic morphine and precipitation of withdrawal, but not after chronic morphine alone. Orx-tau-LacZ mice were treated with morphine as described in Figure 1. Brain sections were tested for B-galactosidase activity and densitometry performed to assess levels of ORX/HCRT gene expression. Figure modified from 44. N=5 per group, *p<0.05.

The data suggest that regulation of the ORX/HCRT gene may be occurring in response to the neuronal state. In particular, it may be that ORX/HCRT gene in upregulated in response to the release of the peptides during precipitated morphine withdrawal. However, future studies utilizing direct measurement of peptide release or receptor activation are needed to test this prediction.

8. ORX/HCRT NEURONS EXPRESS THE ^OPIOID RECEPTOR

Previous in situ hybridization studies have indicated that the ^-opioid receptor is expressed in the LH region, but the identity of these neurons was not known. Triple immunostaining and confocal analysis has recently demonstrated that ORX/HCRT neurons express the ^-opioid receptor and may therefore be responding directly to morphine and naltrexone.44 While not all ORX/HCRT neurons express ^-opioid receptor, all neurons that respond after precipitation of withdrawal (c-Fos or LacZ positive) appear to possess ^-opioid receptor. The expression of ^-opioid receptor on

Opioid Positive Test

I I Sham iZZI Sham+Nat ^^ Morph EHMorph+Nal

Figure 2. The ORX/HCRT gene is upregulated after chronic morphine and precipitation of withdrawal, but not after chronic morphine alone. Orx-tau-LacZ mice were treated with morphine as described in Figure 1. Brain sections were tested for B-galactosidase activity and densitometry performed to assess levels of ORX/HCRT gene expression. Figure modified from 44. N=5 per group, *p<0.05.

I I Sham iZZI Sham+Nat ^^ Morph EHMorph+Nal

ORX/HCRT neurons may explain why heroin is self-administered directly to the LH region and why opioid receptor antagonists administered to the LH can block this self-administration.38

9. ORX/HCRT KNOCKOUT MICE SHOW ATTENUATED MORPHINE WITHDRAWAL

To investigate the functional role of ORX/HCRT in response to drug administration, mice lacking the ORX/HCRT peptides18 were analyzed for physical dependence to morphine. Following morphine exposure and precipitation of withdrawal, animals were evaluated for physical signs of withdrawal.44 ORX/HCRT mutant mice exhibited dramatic attenuation of specific withdrawal symptoms (Figure 3). To control for general behavior abnormalities that might confound this data, the mutant mice were tested for normal open field activity and observed to confirm an absence of cataplexy or narcolepsy during the test period.44

Hypocretin And Narcolepsy

Figure 3. ORX/HCRT knockout mice (gray bars) show attenuated physical withdrawal from chronic morphine when compared to wildtype littermates (white bars). Two days after morphine pellet (25 mg) or sham surgery, mice were injected with naloxone (1 mg/kg subcutaneous) and monitored for 25 minutes for physical signs of withdrawal listed above each graph. ORX/HCRT mutant mice showed significant attenuation in a subset of physical withdrawal signs. Figure modified from 44. N=8 per group, *p<0.01.

Figure 3. ORX/HCRT knockout mice (gray bars) show attenuated physical withdrawal from chronic morphine when compared to wildtype littermates (white bars). Two days after morphine pellet (25 mg) or sham surgery, mice were injected with naloxone (1 mg/kg subcutaneous) and monitored for 25 minutes for physical signs of withdrawal listed above each graph. ORX/HCRT mutant mice showed significant attenuation in a subset of physical withdrawal signs. Figure modified from 44. N=8 per group, *p<0.01.

10. SUMMARY OF ORX/HCRT DATA

Taken together, the above data suggest that the ORX/HCRT neurons of the LH are responsive to opiates. Expression of the ^-opioid receptor on the ORX/HCRT neurons further implies a direct mode of action on the neurons. Finally, the attenuated withdrawal seen in mutant mice indicates that the ORX/HCRT neuropeptide is required for normal development of dependence and/or expression of withdrawal (see discussion below). Together, the data support a role for the ORX/HCRT neuropeptide system in molecular adaptations to morphine exposure.

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  • Micheal
    Where to get orexin peptides tested?
    7 years ago

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