Elsevier

Brain Research

Volume 913, Issue 1, 14 September 2001, Pages 106-115
Brain Research

Interactive report
Effects of hypocretin–saporin injections into the medial septum on sleep and hippocampal theta

https://doi.org/10.1016/S0006-8993(01)02792-5Get rights and content

Abstract

Neurons containing the peptide hypocretin, also known as orexin, were recently implicated in the human sleep disorder narcolepsy. Hypocretin neurons are located only in the lateral hypothalamus from where they innervate virtually the entire brain and spinal cord. This peptide is believed to be involved in regulating feeding and wakefulness. However, to fully understand what other behaviors are regulated by this peptide it is necessary to investigate each hypocretin target site. In the present study, we focus on one hypocretin target site, the medial septum, where there is a dense collection of hypocretin-2 receptor-containing cells, and degenerating axons are present here in canines with narcolepsy [J. Neurosci. 19 (1999) 248]. We utilize a saporin toxin conjugated to the hypocretin receptor binding ligand, hypocretin-2, and find that when this toxin is injected into the medial septum, it lesions the parvalbumin and cholinergic neurons. We contrast the effects of the hypocretin–saporin with another saporin conjugated toxin, 192 IgG–saporin, that lesions only the cholinergic neurons in the basal forebrain. 192 IgG–saporin reduced theta activity, a finding consistent with previous reports [J. Neurophysiol. 79 (1998) 1633; Neurodegeneration 4 (1995) 61; Neuroscience 62 (1994) 1033]. However, hypocretin–saporin completely eliminated hippocampal theta activity by day 12, indicating that parvalbumin-containing cells in the medial septum generate theta. The daily amount of sleep and wakefulness were not different between hypocretin–saporin, 192 IgG–saporin, or saline-treated rats. The homeostatic response to 12 h prolonged wakefulness was also not affected in hypocretin–saporin lesioned rats. These findings suggest that hypocretin neurons could facilitate theta generation during episodes of purposeful behavior by activating GABAergic neurons in the MS/VDB. In this way, hypocretin, which is implicated in feeding, energy metabolism and wakefulness, serves to influence cognitive processes critical for the animal’s survival.

Introduction

Recently, the neuropeptide hypocretin, also known as orexin, was implicated in maintaining wakefulness [20], [34]. Loss of hypocretin neurons is associated with sleepiness and increased rapid eye movement (REM) sleep propensity in the sleep disorder narcolepsy [33], [40]. Consistent with such a neuronal loss, levels of hypocretin-1 are undetectable in the cerebrospinal fluid of narcoleptic patients [30]. Narcoleptic-like behavior is evident in hypocretin gene knockout mice [7] and in mice where the hypocretin neurons degenerate in adulthood [16]. In adult rats lesions of hypocretin neurons produce hypersomnolence and increased REM sleep within a few days after the lesion [13], further indicating a common function of these neurons across species. The distribution of hypocretin-containing neurons has been described in the mouse [37], rat [10], [34], cat [43], [48] and humans [4]. Neurons containing this peptide are located only in the lateral hypothalamus, from where they project to virtually the entire brain and spinal cord [4], [10], [29], [34], [37]. Hypocretin neurons were originally implicated in energy metabolism and feeding since they are located in the lateral hypothalamus. However, it is very likely that this peptide has a multifunctional role because of the widespread projection of hypocretin neurons. To understand what behaviors are regulated by hypocretin, it is necessary to investigate each of the projection sites separately.

The medial septal area [the medial septal nucleus (MS) and vertical limb of the diagonal band of Broca (VDB)] of the basal forebrain is one of the targets of the hypocretin neurons [8], [29], and the hypocretin-2 receptor is present here [41]. In canine narcolepsy this receptor is mutated [24] and a higher number of degenerating axons are found in the medial septum, and in other limbic regions such as the amygdala [38]. Septal neurons are believed to generate hippocampal theta rhythm, a nearly sinusoidal rhythm of 4–12 Hz which is present in the hippocampus during exploratory movements in waking and during REM sleep. Electrical or chemical stimulation of the medial septal area drives theta rhythm [6], [46]. The theta rhythm is present in all species in which REM sleep occurs and this rhythm is thought to be involved in learning and memory. The theta rhythm facilitates the induction of long-term potentiation in hippocampal circuits and also facilitates and controls the flow of information to the hippocampus or through the hippocampus to the targets [6], [23]. Spatial memory is impaired following disruption of the theta rhythm by chemical inactivation of the MS/VDB or following excitotoxic septal lesions [23], [28].

To determine whether the hypocretin innervation of the medial septum influences sleep–wake regulation including theta activity, in the present study we targeted the hypocretin-receptor bearing neurons in the MS/VDB for destruction using the ligand hypocretin-2 conjugated to the ribosomal inactivating enzyme, saporin. The effects of the hypocretin2–saporin were compared with another saporin toxin, 192 immunoglobulin (Ig) G–saporin, that binds to the p75 nerve growth factor receptor. Since this receptor is found exclusively on cholinergic neurons in the MS/VDB, the 192 IgG–saporin selectively lesions the cholinergic medial septal neurons [2], [3], [22]. We reasoned that by employing two different saporin conjugates, one that targets the cholinergic neurons (192 IgG–saporin) and the other (hypocretin2–saporin) that destroys hypocretin-receptor bearing neurons, the relative contribution of hypocretin on septal cholinergic versus non-cholinergic neurons in sleep–wake homeostasis and theta activity could be better understood.

Section snippets

Subjects and surgery

Fourteen male Sprague-Dawley rats (400–450 g) were anesthetized [IM injection of cocktail of acepromazine (0.75 mg/kg), xylazine (2.5 mg/kg) and ketamine (22 mg/kg)] and using a stereotaxic apparatus injected with the test substances (see next paragraph) and then implanted with electrodes to record the electroencephalogram (EEG) and electromyogram (EMG). Four stainless steel screw electrodes were positioned in the skull to sit on the surface of the cortex and were used to record the EEG. Two

Loss of ChAT- and PARV-immunoreactive cells in the MS/VDB following treatment with saporin conjugates

The extent of the lesion induced by intraseptal injection of hypocretin–saporin was verified on tissue sections stained with Neutral Red. Fig. 1 shows a drawing of a typical lesion in a single representative rat (rat #300). The lesion included the whole medial septum, vertical limb of the diagonal band of Broca (VDB), and part of the horizontal limb of the diagonal band of Broca (HDB) (Fig. 1). All the rats treated with hypocretin–saporin had a significant loss of both ChAT-positive (−87%) and

Discussion

Our findings indicate that (1) the hypocretin–saporin toxin kills both the PARV-containing neurons and ChAT-containing neurons in the MS/VDB; (2) neuronal loss in the MS/VDB is accompanied by loss of theta activity; and (3) loss of PARV- and ChAT-containing neurons in the MS/VDB does not produce a change in overall levels of sleep–wakefulness or diminish the drive to sleep following a 12-h period of prolonged wakefulness.

In the present study, we examined hypocretin function in the medial septal

Acknowledgements

We thank Dr. Mary Ann Greco and Carlos Blanco-Centurion for discussions, and Elizabeth Winston for data analysis. We also acknowledge the technical assistance provided by Munazza Malik and Jill Winston. Supported by NIH grants NS30140, AG09975, AG15853, and the Medical Research Service of the Department of Veterans Affairs.

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