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From the *Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy; and Istituto di Cura San Camillo, Ospedale/IRCCS, Venezia – Lido, Italy.
Address correspondence and reprint requests to Ulderico Freo, MD, Institute of Anesthesiology and Intensive Care, Department of Pharmacology and Anesthesiology, University of Padova, Via C. Battisti 267, 35121 Padova, Italy. Address e-mail to ulderico.freo{at}unipd.it.
Abstract
BACKGROUND: Midazolam has hypnotic and sedative activities, which may be mediated by different neuronal structures. We investigated the time course effect of a hypnotic dose of midazolam on conscious motor behavior and on patterns of brain metabolism.
METHODS: Loss of nociceptive reflexes and impairment of spontaneous locomotor activity were used as indices for the hypnotic and sedative effects of midazolam, and the regional cerebral metabolic rates for glucose (rCMRglc) were used as indices of neuronal effects of midazolam. Locomotor activity was measured with a monitor and rCMRglc were measured with the quantitative autoradiographic [14C]2-deoxyglucose procedure in 62 brain regions of Fischer-344 rats at 2, 30, 60, 120, and 180 min after IV administration of saline or midazolam 5 mg/kg.
RESULTS: After midazolam administration, rats were anesthetized at 2 min, awake but severely impaired at 30 min and slowly recovering motor activity thereafter. Anesthesia was associated with widespread rCMRglc decreases (59 areas affected, 38% mean decrease). Recovery of consciousness was associated with normalizing rCMRglc in visual, auditory, and somatosensory cortices and in the locus coeruleus (47 regions affected, 31% decrease). Recovery of motor activity was paralleled by slow rCMRglc normalization in the frontal motor, limbic, and thalamic regions (at 60, 120, and 180 min 31, 17, 4 areas affected, 26, 20, and 15% decreases from control values).
CONCLUSIONS: Whereas the hypnotic effects of midazolam may result from inhibition of brain structures involved in arousal and sensory processing, its sedative effects may result from inhibition of subcortical motor and limbic regions.
Midazolam is a benzodiazepine 
-aminobutyric acid type A (GABAA) agonist that is featured in anxiolytic, anticonvulsivant, muscle relaxant, and sedative-anesthetic activities.1 Because it rapidly enters the brain and is rapidly metabolized, midazolam is regarded as a rapid, short-acting drug. The pharmacokinetic–pharmacodynamic relationships of midazolam's actions have been extensively investigated with behavioral and electrophysiological techniques, and convincing correlations have been shown between plasma concentrations, binding affinity to GABAA receptors, and some behavioral effects of midazolam.2
Midazolam, however, alters cognitive, emotional, and motor behavior for some time after anesthesia and at doses that are not sedative.3 The hypnotic effects of midazolam are reduced in mice lacking the 
subunits of GABA receptors4 but also in mice with reduced N-methyl-d-aspartate receptor 
1 subunits.5 The anticonvulsivant effects of midazolam have no relationship with its affinity for GABAA receptors.6 Also, midazolam reduces release of acetylcholine in the hippocampus,7 of dopamine in the striatum,8 and of epinephrine in medial prefrontal cortex.9 Altogether, the data suggest that midazolam may act through different mechanisms, which makes it difficult to ascribe behavioral effects of midazolam to a single neuronal function. Electrophysiological studies provide sensitive and continuous information on the global effect of midazolam on the central nervous system but do not provide information about the neuroanatomical substrata of its behavioral effects.
The 2-deoxy-d-glucose ([14C]DG) autoradiographic method allows the simultaneous measurement of regional cerebral metabolic rates for glucose (rCMRglc). rCMRglc are putative indices of integrated neuronal function.10,11 Brain regions in which metabolism is altered often correspond to known neural circuits that are implicated in specific behaviors. The method showed that behavior is coupled to regional energy metabolism after administration of several drugs and has provided useful insights on psychoactive drugs in animal and human studies.10–14 We thought that midazolam could produce time-dependent, regionally specific changes in rCMRglc because, after a hypnotic dose of midazolam, a behavioral spectrum can be observed (i.e., loss of consciousness and nociceptive reflexes, motor sedation). To investigate this hypothesis, we measured rCMRglc at different times after a hypnotic dose of midazolam and related the findings to behavioral measures.
METHODS
Materials
Experiments were performed on male, 3-month-old, Fischer-344 rats (Charles River, Como, Italy). Rats were group-housed (three per cage) and were accorded at least 1 wk in a humidity and temperature-controlled vivarium (22°C) with free access to chow and water. [14C]-2-deoxy-d-glucose specific activity 50 to 55 mCi/mmol was obtained from New England Nuclear (Boston, MA). Midazolam hydrochloride was a kind gift of Hoffman La Roche (Basel, Switzerland) and was dissolved in sterile isotonic saline 0.9% (w/v) to a concentration of 5 mg/mL.
Behavior
All studies were performed in accordance with the guidelines for animal care of the United States and were approved by the IRB of University of Padova (Italy).
Loss of motor responses to tail pinch and to corneal touch and impairment of locomotor activity served as indices of, respectively, hypnotic and sedative effects of midazolam. Nociceptive reflexes and spontaneous motor activity were determined after IV tail injection of saline or midazolam 5 mg/kg to groups of six rats each, different from those used in the rCMRglc study.
Nociceptive reflexes were assessed in 10-s periods in the first minute after midazolam administration and then in 30-s periods until recovery. Locomotor activity was measured in an apparatus consisting of wire mesh plastic cages (length x width x height, 44 x 24 x 20 cm). Horizontal locomotor activity was measured in 10-min periods as the total of sequential interruptions of 2 or more infrared light beams located 1 cm from the cage floor (Digiscan Animal Activity Monitor, Omnitech Electronics Inc., Columbus, OH). Rats were habituated to the activity apparatus before testing for 60 min sessions in 3 separate days. At the end of the third habituation session, locomotor activity was measured for 20 min before and for 180 min after treatment.
rCMRglc Measurement
rCMRglc were measured in groups of six rats injected IV with saline at 2, 60, and 180 min before [14C]DG (controls) or with midazolam 5 mg at 2, 30, 60, 120, or 180 min before [14C]DG.
The [14C]DG experiments were performed as described in previous publications.12,15
The day before experiment, rats were anesthetized with isoflurane 1.5% and catheters were implanted in the right femoral artery and vein. Catheters were tunneled under the dorsal skin up to the base of the skull. Catheters protruded approximately 2 cm from a small incision and were filled with a heparinized solution. On the following day, rats were brought with their cage from the vivarium to the laboratory and catheters were extended to allow injection of [14C]DG, blood sampling, and measurement of physiological variables. [14C]DG (125 µCi/kg) was injected as an IV bolus at the times and doses reported above. During the following 45 min, 12 arterial blood samples were collected at fixed time-points and later assayed for glucose (Glucose Analyzer II, Beckman, Irvine, CA) and 14C (Model LS9000, Liquid Scintillation Spectrometer, Beckman) concentrations. Body temperature was measured intermittently by a rectal thermoprobe and was kept between 35.5°C and 37°C by a thermostatic device (Indicating Controller, model 73ATA, Yellow Springs, OH) that activated a heating element when the temperature decreased to <35.5°C. Before and at 3, 15, and 40 min after [14C]DG arterial blood gases were assessed by sampling (pH Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) and arterial blood pressure and heart rate by connecting the arterial catheter to a pressure transducer (Model PM-2A, Honeywell, Minneapolis, MN).
Rats were killed at 45 min after [14C]DG by an IV overdose of sodium pentobarbital (60 mg in 1 mL of saline solution). The brains were rapidly removed and frozen in 2-methylbutane cooled to –55°C. Later, they were sliced in coronal sections (20-µm thick) in a cryostat (Bright Model 5030, Hacker Instruments, Fairfield, NJ), maintained at –20°C, and dried immediately on a hotplate. Autoradiographs were obtained by exposing SB-5 radiograph films (Eastman Kodak, Rochester, NY) to the brain sections together with [14C]methylmethacrylate standards (Amersham, Arlington Heights, IL) for 7 days.
Densitometry of the autoradiographs was performed with a semiautomated microdensitometer in 62 brain regions. Six separate determinations of optical density were made at each region in both left and right sides of the brain, and their means were averaged. Each anatomical region evaluated was defined by comparison with rat brain atlases.16 rCMRglc was calculated from brain and plasma radioactivity and plasma glucose concentrations, using standard equations and constants.10,11
Statistical Analysis
Pairwise statistical comparisons for rCMRglc values were analyzed by Dunnett multiple comparison test by comparing the mean of each midazolam-treated group to saline-injected controls. For behavioral and physiological measures, posttreatment values were compared with pretreatment baseline values with the Student's t-test. Statistical significance was taken in all cases to be P < 0.05.
RESULTS
Behavior
Before treatment, motor activity was not different (P < 0.05) between the saline control group (i.e., mean counts ± sem, 25 ± 3) and the midazolam-treated group (27 ± 3 counts).
After saline administration, motor activity was not significantly different from baseline (at 2, 30, 60, 120, and 180 min 28 ± 5, 24 ± 4, 25 ± 5, 24 ± 6, and 23 ± 6 counts).
After midazolam administration, at 2 min, animals appeared anesthetized, with a regular breathing and with no motor activity, either spontaneously or in response to corneal touch or to tail pinch (times of loss of reflexes 4.8 ± 0.1 and 5.1 ± 0.2 min). Thirty minutes after midazolam administration, rats were awake but severely ataxic and had impaired motor activity (6 ± 2.1 counts, 76% decrease from baseline). Then, rats progressively recovered (at 60, 120, and 180 min 17 ± 5, 21 ± 4, and 23 ± 6 counts, 36, 24, and 14% decreases from baseline) (Fig. 1).
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Physiological Variables
Before treatment, mean arterial blood pressure (125 ± 8 mm Hg), heart rate (443 ± 24 bpm), body temperature (36.5°C ± 0.2°C), arterial pH (7.39 ± 0.02), Po2 (93 ± 2 mm Hg) and Pco2 (37 ± 2), and plasma glucose (115 ± 8 mg/dL) concentrations were similar to the means previously reported12,16 and were not altered significantly after administration of saline or midazolam.
rCMRglc
Values of rCMRglc in control and midazolam-treated rats are presented in Table 1. rCMRglc was determined in 62 brain regions of animals given [14C]DG at 2, 60, and 180 min after saline administration and 2, 30, 60, 120, and 180 min after midazolam 5 mg. rCMRglc were similar among control animals injected with saline at different times (rCMRglc data from saline-injected animals at 2 and 180 before [14C]DG min are not shown) and were similar to those previously reported for awake, young male rats.12,16
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Anesthetic loss of nociceptive reflexes was associated with almost universal reductions of rCMRglc. Recovery of nocidefensive reflexes and of consciousness was associated with rCMRglc normalization in visual, auditory, and somatosensory areas and in the locus coeruleus and motor recovery by gradual rCMRglc normalization in frontal cortical, limbic, and thalamic regions (Fig. 2).
DISCUSSION
Midazolam produced a quick and complete suppression of motor activity and widespread cerebral hypometabolism. Consciousness recovered rapidly along with brain metabolism in primary cortical sensory and brainstem areas. Spontaneous motor activity then recovered slowly in parallel with brain metabolism in primary cortical sensory and brainstem areas.
Measurements of rCMRglc by the [14C]DG method under-estimates the peak effects, such as the hypnotic effect, of short-acting compounds like midazolam for which a steady-state effect on metabolism is not maintained during the 45-min tracer uptake period. Under-estimation is minimized, however, by administering the tracer at the time of peak effect, because the amount of the [14C]DG available for incorporation decreases with time. Under steady-state conditions, about half of [14C]DG is incorporated in the first 10–15 min, when midazolam's effects on rCMRglc are greatest.10,11
Concurrent metabolic and behavioral alterations have been reported after administration of several other drugs acting on different neurotransmitter systems and are thought to reflect drug kinetic features.12–14 Midazolam easily enters from the plasma into the brain because it is highly lipophilic, nonionized at physiological pH,17 and largely unbound to plasma proteins.2 In experimental animals, after administration of midazolam 5–10 mg/kg, plasma and brain concentrations peak rapidly within a few minutes and then, given midazolam's rapid metabolism, decline in parallel with a half-life in 20–30 min range.18,19 In the present study, the kinetics of midazolam's hypnotic effects (i.e., short action coincident with the brain concentration and electroencephalogram profiles) were unlike those of neurotransmitter antagonists and resembled instead those of receptor agonists, which usually dissociate readily from receptors as the brain concentration decreases, and substantiates its putative action as a GABAA receptor agonist.18
Whether anesthesia is due to a global, nonspecific metabolic depression or to an effect on specific brain areas or networks is still a research question. One study has demonstrated that anesthetics can produce sedative effects by interacting with specific sleep pathways. Direct injection of propofol in the hypothalamus-induced sedation that could be counteracted in a precise fashion by systemic administration of the GABA antagonist, gabazine.20 In our study, rCMRglc were profoundly decreased during anesthesia and back to normal values at awakening in cortical and brainstem areas involved in facilitating arousal and processing21 of multiple sensory inputs.21 In rats, the GABAergic anesthetic propofol dose-dependently decreased rCMRglc in subcortical and cortical regions.22 In humans, at sedative doses that cause unresponsiveness, midazolam and propofol decreased rCMRglc and cerebral blood flow in cortical regions only.23,24 At surgical doses, propofol decreased regional cerebral blood flow (rCBF) in thalamus and midbrain.25 The locus coeruleus is enriched by GABAA receptor 2 and 3 subunits and, almost unique among brain regions, by GABAA and 
subunits.26 GABA injections within the locus coeruleus induce sleep.27 Within the locus coeruleus, midazolam may act as an agonist of GABA receptors or adrenergic receptors28 to reduce neuronal electrical activity29 and norepinephrine release.9 Hence, our data suggest that interfering with sensory signals in primary cortical areas and in subcortical areas promoting sensory modulation may be pertinent to the hypnotic effects of midazolam.
At 60–120 min after its administration plasma and brain concentrations of midazolam are expected to be rather low and probably in the range of those found after an anxiolytic dose of midazolam. At 60–120 after midazolam administration, motor activity was impaired and cerebral metabolism was still depressed in frontal, limbic, and thalamic regions. In rats, benzodiazepines and nonbenzodiazepine GABA agonists, such as diazepam, muscimol, and 4,5,6,7-tetrahydroisoxazolo (4,5-e)-pyridin-3-ol (THIP), decreased rCMRglc, maximally in thalamic and hypothalamic nuclei.30,31 In humans, lorazepam at sedative doses decreased electroencephalographic activity and rCMRglc in the thalamus32,33 and these effects were partially reversed by the benzodiazepine antagonist, flumazenil.34,35 Based on these and other studies, the thalamus has been proposed as the key structure for the hypnotic and sedative effects of GABA drugs. However, the observed effect in a brain region heavily interconnected, such as the thalamus, may result not from a direct drug-receptor interaction but, because of synaptic propagation, from primary target areas such as the limbic and frontal cortices. Molecular biology studies showed that 2 and 5 GABA subunits that play a role in the anxiolytic actions of benzodiazepines and emotional and spatial learning are present in high densities in the amygdala and the hippocampus.36 Hence, topographic distribution and kinetics of rCMRglc decreases during postanesthetic sedation differ from those during anesthesia and may reflect midazolam's interaction with specific GABA subunits.34–37
In conclusion, a hypnotic dose of midazolam determined biphasic behavioral and cerebral metabolic effects. The data suggest that decreased neuronal activity in sensory processing areas may mediate the fast resolving unconsciousness and decreased activity in subcortical motor and limbic regions mediate the subsequent prolonged sedation. Future studies with drugs selective for specific GABA receptor subtypes may further elucidate the specific role of these brain regions in the behavioral effects of midazolam.
Footnotes
Accepted for publication January 14, 2008.
REFERENCES
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