Research reportGABAA receptor modulation of temperature sensitive neurons in the diagonal band of Broca in vitro
Introduction
Neurons in the basal forebrain, including those in the horizontal limb of the diagonal band of Broca (HDB), have been hypothesized to be involved in both behavioral state regulation and thermoregulation. Electrical stimulation in the HDB of cats evokes sleep [21], and lesions of the HDB suppress sleep [24]. In freely moving adult cats and rats, a group of HDB neurons exhibits increased discharge rates during non-REM sleep as compared to waking 2, 14, 19, 23. Activation of dopaminergic neurons of the ventral tegmental area with neurotensin decreases core body temperature in rats and this effect is blocked by dopaminergic antagonist application into the diagonal band [12]. In addition, the medial septal region, which is closely associated with the HDB and sends projections through the HDB, is involved in thermoregulatory responses such as shivering and vasoconstriction 4, 10. Therefore, specific neuronal populations within the HDB appear to be involved in both thermoregulatory and behavioral state regulation.
HDB neuronal populations involved in the regulation and integration of behavioral state and thermoregulation include those that respond to temperature and those containing the neurotransmitter γ-aminobutyric acid (GABA) [11]. Temperature sensitive neurons have been categorized as: (1) warm sensitive neurons (WSNs), (2) cold sensitive neurons (CSNs) and (3) temperature insensitive neurons (TINs) 6, 11, 13. We have previously shown in the slice preparation that the HDB, like the preoptic area/anterior hypothalamus (POAH), contains a high proportion of WSNs [11]. Warm sensitivity was preserved during synaptic blockade in 32% of that sample. A recent in vivo study in the rat identified HDB WSNs that displayed increased discharge rates during non-REM sleep as compared to waking, and CSNs having decreased firing rates during spontaneous non-REM sleep compared to waking [2]. It has also been shown in vivo that local warming induced activation of HDB and POAH WSNs promotes sleep and causes increased slow wave activity in the cortical EEG during sleep 17, 20. Thus, it has been hypothesized that activation of POAH and diagonal band WSNs induces non-REM sleep.
HDB and POAH GABAergic neurons have been postulated to play a role in the promotion of sleep 9, 22. A GABAergic projection, as labeled by glutamic acid decarboxylase (GAD), has been demonstrated from the HDB to the posterior lateral hypothalamus [10]. It has also been shown that GABA release in the posterior lateral hypothalamus increases during non-REM sleep as compared to waking and REM sleep [18]. Activation of HDB and POAH WSNs, by local warming, decreases the activity of waking-related posterior lateral hypothalamic neurons [15]. Based on these findings, WSNs in these regions are hypothesized to be GABAergic projection neurons. These neurons may work antagonistically with HDB arousal-related neurons, presumably CSNs, to control behavioral state [2].
The mRNAs of several GABAA receptor subunits have been found in HDB neurons, potentially GABAergic projection neurons, that contain parvalbumin, a GABAergic neuronal marker [7]. Also, GAD65, the synthesizing enzyme for GABA which has been associated with GABA terminals, is found in the HDB [25]. It has been shown that a bilateral injection of the GABAA agonist muscimol into the HDB decreases non-REM sleep and increases wakefulness [16]. Since WSNs are hypothesized to be the sleep promoting neurons of the HDB, we predicted that the GABAA agonist, muscimol, would decrease the warm sensitivity and firing rate in warm sensitive HDB neurons in vitro.
Section snippets
Methods
HDB horizontal slices were prepared from Sprague–Dawley rats (40–120 g). Animals were entrained to a 12 h/12 h light/dark cycle (lights on 0600–1800) and maintained at an ambient temperature of 24±2.0°C with food and water provided ad libitum. Rats were lightly anesthetized via halothane inhalation and then decapitated. The brains were quickly excised and placed in 0–4°C oxygenated (95% O2, 5% CO2) artificial cerebral spinal fluid (aCSF). The aCSF composition [13]was (in mM): KCl 5.0, NaCl
Results
Complete experimental recordings, including baseline, drug application and washout, lasted an average of 81 min. Fig. 1, Fig. 2 show three complete representative recordings using the 15 μM microwire method. The microwire technique, similar to the technique widely used in the chronic rat and cat and used in a previous in vitro study from our laboratory, consistently yielded well-isolated, stable unit recordings with signal to noise ratios of ≥3:1 (Fig. 1, Fig. 2) [11]. In control experiments
Discussion
Several regions of the brain contain neurons that are responsive to changes in local temperature. The importance of thermosensitive neurons in the HDB is related to the proposed interplay between behavioral state and the thermoregulatory function of these neurons (see Section 1). Activation of HDB WSNs by local warming facilitates non-REM sleep, and a bilateral injection of the GABAA agonist, muscimol, into the HDB decreases non-REM sleep 16, 20. Both GABAergic neurons and GABAergic terminals
Acknowledgements
This research was supported by PHS grant MH47480, HL60296 and the Medical Research Service in the Department of Veterans Affairs.
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