Invited Symposium: Role of the Basal Forebrain Neurons in Cortical Activation and Behavioural State Regulation
Several lines of evidence have implicated the cholinergic magnocellular regions of the basal forebrain (BF) in the regulation of cortical excitability and wakefulness (W). This BF region contains cholinergic and non-cholinergic cortically projecting neurons, of which the majority exhibit their highest discharge activity during wakefulness (wake-active neurons). We recently reported in vivo evidence that this BF region is a potent site for the sleep-inducing action of endogenous adenosine (AD) (Porkka-Heiskanen et al. 1997). Adenosine, a byproduct of cellular metabolism, is an inhibitory neuromodulator and putative sleep factor. In vitro studies from our lab have shown that AD exerts a tonic inhibitory control over the cholinergic neurons of the BF and mesopontine tegmentum (Rainnie et al. 1994). Furthermore, the stimulants caffeine and theophylline are powerful antagonists of AD receptors. Data from recent and ongoing experiments is presented below that addresses the following hypothetical model: During prolonged wakefulness, when neural metabolism is highest, extracellular AD accumulates selectively in the BF and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting the wake-active neurons in the BF.
Materials and Methods
Experimental Animals & Surgery.
Target Sites, Stereotaxic Coordinates.
Microdialysis Sampling Procedures.
Neurochemical Analysis of Adenosine.
Combined Unit Recording & Microdialysis Methods.
Surgical preparation of animals and implantation of electrodes for electrographic recordings has been described in the previous section. The unit recording proposed in this application is to be done in conjunction with MD perfusion of pharmacological agents. For this purpose we have used concentric microdrives implanted bilaterally in the BF. Each concentric microdrive consisted of a 19 ga MD probe cannula which was inserted in the advanceable 17 ga stainless steel cannula of a screw-driven microdrive with a 14 ga fixed outer cannula. The microwire unit recording bundle of thirteen 32 Ám and one 64 Ám insulated wires was glued to the outside of the 19 ga MD probe cannula. Within the MD probe cannula was an obturator, which was removed at a later time to allow microdialysis probe insertion. Dimensions of the steel guide cannula were as follows: The 19 ga MD cannula was O.D.= 1.1 mm and I.D. = 0.8 mm. . The advanceable stainless steel tube (17 ga) had dimensions of O.D.= 1.5 mm and I.D. = 1.2 mm. The fixed microdrive cannula had the dimensions (14 ga) of O.D.= 2.1 mm and I.D. = 1.7 mm. This concentric microdrive was constructed so that the electrode tips ended up in the middle (dorso-ventrally) of the 2 mm length of dialysate membrane, and furthermore, we estimate that the electrode tip was approximately 200 microns from the lateral surface of the MD probe.
The guide tube of both the devices was stereotaxically targeted bilaterally above the BF Substantia Innominata. At the time of surgery the drive was fixed to the skull with dental acrylic so that the tip of the guide cannula was 5 mm above the target. A bundle of fourteen insulated flexible Formvar-insulated stainless steel microwires was attached with SuperGlue to the guide cannula so as to protrude 4 mm beyond the cannula and thus reach about 1 mm above the target. Unit recording began after post-operative recovery and adaptation to the testing cage. Microwires were advanced in steps of 40-80 Ám until resolvable single units were encountered, and then the MD probe was placed in the dialysis cannula. At least 12 hours after probe insertion, perfusion with ACSF was begun, and baseline spontaneous unit activity during active waking (AW), quiet waking (QW), and slow wave sleep (SWS) of resolvable units was recorded. Then the pharmacological agent to be tested was perfused through the MD cannula, and unit activity and electrographic data were recorded.
Signal Processing of Unit Activity.
Analysis of single unit activity across behavioral states.
Measurement of extracellular AD in cat.
AD levels during Spontaneous Sleep Cycles.
AD levels during Prolonged Wakefulness. As we reported in 1997 (Porkka-Heiskanen et al. 1997), AD levels rise in the feline BF during 6 hours of sleep deprivation (Figure 2a). During the recovery period, after deprivation ended, BF AD levels gradually decreased over the next 2-3 hours, a time when the behavioral state was mainly SWS. Next, we were somewhat surprised to not find a similar rise in the VA/VL during 6 h sleep deprivation (Figure 2b). This led us to add several more brain regions to be tested. Preliminary data exist measuring AD concentrations in 6 different brain regions during 6 hr total sleep deprivation produced by gentle handling/playing (EEG verified), and during the post-deprivation recovery sleep period (3 h) (AD levels are expressed as a % of the AD level measured in a baseline W sample). The clearest pattern emerged in the BF, where AD levels rose throughout the period of prolonged W and then declined in recovery sleep (see details in Porkka-Heiskanen et al. 1997). Although less steep, the rise in AD levels in the cortex site appeared similar to the BF (currently, N=4). However, in the thalamus (VA/VL), DRN, PPT, and POA, AD levels did not rise during 6 h sleep deprivation.
In summary, during spontaneous sleep-wake cycles in cat, AD levels fluctuated similarly in the brain areas tested. During prolonged W, AD levels steadily increased in the BF; a similar pattern, although less pronounced, was seen in cortex. However, a rise with prolonged W was not seen in other areas, suggesting that during prolonged waking AD levels may not be similarly elevated in all brain areas, as would be expected if AD were a sleep-inducing factor linked to metabolism that was acting globally. Many factors determine the level of extracellular AD, and it is beyond the scope of the present paper to fully explain the variations observed. Nonetheless, these data, taken together with our behavioral data on local perfusion with AD transport inhibitors (described below), suggest that AD is a physiological sleep factor and support the hypothesis that high brain levels of AD promote drowsiness and the transition from W to sleep by acting on the cholinergic zone of BF.
BF drug perfusions and sleep behavior in cat.
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Unit Recording Combined with MD in the Basal Forebrain. Data support the hypothesis that W-selective cells in the BF (possibly cholinergic EEG activating neurons) are inhibited by AD agonists. Cyclohexyladenosine (CHA, an A1 agonist; 0.1, 1.0, 10.0 ÁM) has produced a dose-dependent decrease in the neuronal discharge in the 4 neurons recorded to date. Figure 6 shows the A1 agonist-induced inhibition of discharge in 4 neurons, relative to their discharge rate during the preceding ACSF perfusion period.
Discussion and Conclusion
The powerful behavioral state-altering effects of AD could hypothetically occur primarily via the cholinergic neurons' widespread and strategic efferent targets in the cortical and thalamic systems that are known to be important for the control of cortical activation. Our recent preliminary data in cat strengthen the hypothesis that the BF region mediates the sleep-promoting actions of endogenous AD following prolonged wakefulness.
Thus, we find that during 6 h of prolonged wakefulness AD levels rise sharply in the cholinergic zone of BF, but not in any other subcortical structures tested to date. This site-specific accumulation of AD during prolonged wakefulness suggests that the sleep-promoting effects of AD may be mediated by an AD inhibition of the BF cholinergic arousal system. The marked regional variations in AD levels observed indicate further the existence of precise physiological regulation of AD levels, by, as yet, unidentified mechanisms. Finally, using the novel method that combines single unit recording and MD, we now show at the level of the single neuron that AD agonists inhibit the discharge activity of wake-selective BF neurons. Taken together these data support the hypothesis that AD and antagonists at AD receptors alter behavioral state via their actions in the cholinergic zone of the BF. It remains important to determine if AD's ability to reduce the discharge of BF cholinergic neurons is by itself necessary and sufficient for AD to induce sleep, or whether AD also acts on non-cholinergic arousal-related neurons in the BF.
Berman AL (1968) The brainstem of the Cat. Madison, WI: The University of Wisconsin Press.
Berman AL, Jones EG (1982) The thalamus and basal telencephalon of the Cat. Madison, WI: The University of Wisconsin Press.
Porkka-Heiskanen, T, Strecker, RE, Thakkar, M, Bjorkum, AA, Greene, RW and McCarley, RW. Adenosine: A mediator of the sleep-inducing effects of prolonged wakefulness, Science 1997; 276:1265-1268.
Rainnie DG, Grunze HCR, McCarley RW, Greene RW. Adenosine Inhibits Mesopontine Cholinergic Neurons: Implications for EEG Arousal. Science 1994; 263:689-692.
Thakkar, M, Strecker, RE and McCarley, RW. Behavioral state control through differential serotonergic inhibition in the mesopontine cholinergic nuclei: A simultaneous unit recording and microdialysis study, J. Neurosci. 1998; 18:5490-5497.
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|McCarley, RW; (1998). Adenosinergic Modulation Of Basal Forebrain (BF) Neurons In The Control Of Behavioral State. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/semba/mccarley0388/index.html|
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