c-Fos protein expression is increased in cholinergic neurons of the rodent basal forebrain during spontaneous and induced wakefulness

It has been proposed that cholinergic neurons of the basal forebrain (BF) may play a role in vigilance state control. Since not all vigilance states have been studied, we evaluated cholinergic neuronal activation levels across spontaneously occurring states of vigilance, as well as during sleep deprivation and recovery sleep following sleep deprivation. Sleep deprivation was performed for 2 h at the beginning of the light (inactive) period, by means of gentle sensory stimulation. In the rodent BF, we used immunohistochemical detection of the c-Fos protein as a marker for activation, combined with labeling for choline acetyl-transferase (ChAT) as a marker for cholinergic neurons. We found c-Fos activation in BF cholinergic neurons was highest in the group undergoing sleep deprivation (12.9% of cholinergic neurons), while the spontaneous wakefulness group showed a significant increase (9.2%), compared to labeling in the spontaneous sleep group (1.8%) and a sleep deprivation recovery group (0.8%). A subpopulation of cholinergic neurons expressed c-Fos during spontaneous wakefulness, when possible confounds of the sleep deprivation procedure were minimized (e.g., stress and sensory stimulation). Double-labeling in the sleep deprivation treatment group was significantly elevated in select subnuclei of the BF (medial septum/vertical limb of the diagonal band, horizontal limb of the diagonal band, and the magnocellular preoptic nucleus), when compared to spontaneous wakefulness. These findings support and provide additional confirming data of previous reports that cholinergic neurons of BF play a role in vigilance state regulation by promoting wakefulness.     

c-Fos protein expression is increased in cholinergic neurons of the rodent basal forebrain during spontaneous and induced wakefulness

It has been proposed that cholinergic neurons of the basal forebrain (BF) may play a role in vigilance state control. Since not all vigilance states have been studied, we evaluated cholinergic neuronal activation levels across spontaneously occurring states of vigilance, as well as during sleep deprivation and recovery sleep following sleep deprivation. Sleep deprivation was performed for 2 h at the beginning of the light (inactive) period, by means of gentle sensory stimulation. In the rodent BF, we used immunohistochemical detection of the c-Fos protein as a marker for activation, combined with labeling for choline acetyl-transferase (ChAT) as a marker for cholinergic neurons. We found c-Fos activation in BF cholinergic neurons was highest in the group undergoing sleep deprivation (12.9% of cholinergic neurons), while the spontaneous wakefulness group showed a significant increase (9.2%), compared to labeling in the spontaneous sleep group (1.8%) and a sleep deprivation recovery group (0.8%). A subpopulation of cholinergic neurons expressed c-Fos during spontaneous wakefulness, when possible confounds of the sleep deprivation procedure were minimized (e.g., stress and sensory stimulation). Double-labeling in the sleep deprivation treatment group was significantly elevated in select subnuclei of the BF (medial septum/vertical limb of the diagonal band, horizontal limb of the diagonal band, and the magnocellular preoptic nucleus), when compared to spontaneous wakefulness. These findings support and provide additional confirming data of previous reports that cholinergic neurons of BF play a role in vigilance state regulation by promoting wakefulness.     

Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats

Administration of estradiol to gonadectomized female, but not male rats, is associated with increased activity of choline acetyltransferase in the medial aspect of the horizontal diagonal band nucleus, the frontal cortex, and CA1 of the dorsal hippocampus. Four other basal forebrain cholinergic nuclei did not show changes in choline acetyltransferase activity after estradiol. These data have implications for possible benefits of estradiol administration to patients with senile dementia of the Alzheimer's type.     

Distribution of anxiogenic-induced c-Fos in the forebrain regions of developing rats

Summary. An anxiogenic or a pharmacological stressor, N-methyl--carboline-3-carboxamide (FG-7142), (20mg/kg, intraperitoneally injected) induced a dense nuclear c-Fos-like immunoreactivity in the pyriform cortex, cingulate and retrosplenial cortex, layers II–VI of the neocortex, lateral habenula, lateral septum, paraventricular nucleus of the thalamus, striatum, central and medial nucleus of the amygdala, but a sparse c-Fos immunostaining in the hippocampus and layer I of the neocortex in the forebrain of 56-day-old rats. Among these regions, the 8-day-old rats expressed much fewer c-Fos-positive cells in the neocortex, lateral habenula, lateral septum and medial nucleus of the amygdala than the young adult rats following the FG-7142 injection. These differences in the regional distribution of a neuronal activity marker, c-Fos, could reflect the postnatal development of neuronal populations or neuron circuits involved in stress and/or emotional response in the forebrain.Received January 27, 2003; accepted May 13, 2003 Published online July 30, 2003     

Cholinergic limbic projections and behavioral role of basal forebrain nuclei in the rat

The purposes of the present study were to identify cholinergic non-neocortical projections of the basal forebrain and to determine the role of this region in the regulation of estrogen-dependent reproductive behaviors in the rat. Bilateral electrolytic lesions were placed in an area encompassing the horizontal limb of the diagonal band, as well as portions of the substantia innominata and magnocellular preoptic nucleus, and choline acetyltransferase (CAT) activity was assayed in microdissected brain areas seven days after lesion. Compared to sham surgery, lesions of this region significantly reduced CAT activity in the basal amygdala (34%), dorsal hippocampus (14%), cingulate cortex (25%), piriform cortex (36%), and entorhinal cortex (34%). Other limbic and midbrain structures do not appear to receive significant cholinergic innervation from this locus since no reductions in CAT were detected after bilateral lesions. These included the anterior hypothalamus, ventromedial hypothalamus, mammillary nucleus, habenula, subiculum, ventral hippocampus, insular cortex, central gray, and interpeduncular nucleus. Behaviorally, female rats with bilateral lesions of the basal forebrain displayed an unusually high incidence of rejection behavior in response to attempted mounts by stimulus male rats in sexual behavior tests. There was no effect of basal forebrain lesions on the incidence of lordosis exhibited by these females. The dissociation of rejection and lordosis suggests that distinct neural pathways mediate the occurrence of these reproductive behaviors and that rejection behavior may be regulated by basal forebrain pathways.     

GABAergic control of basal forebrain cholinergic neurons and memory

The involvement of the GABAergic innervation of basal forebrain neurons in the rats' conditional visual discrimination performance was examined. Performance in such a task is based on the subjects's ability to retrieve information about response rules, and previous experiments have demonstrated that basal forebrain lesions interfere with this ability. Following the acquisition of the task, chronic guide cannulae were stereotaxically implanted into the substantia innominata of both hemispheres, and the animals were retrained. Administration of the GABAA-agonist muscimol into the substantia innominata (0, 25, 50 ng/0.5 microliters/hemisphere) dose-dependently decreased the number of correct responses, increased the number of errors of omission, increased response latency, but did not affect side bias. Systemic co-administration of the cholinesterase inhibitor physostigmine (0, 0.1, 0.2 mg/kg; i.p.) exclusively interacted with the effects of muscimol on correct responding. Specifically, physostigmine dose-dependently intensified and attenuated the muscimol-induced reduction in correct responding. Although it cannot be excluded that alternative neuronal mechanisms were involved in the mediation of the effects of muscimol and their interaction with physostigmine, these findings support previous evidence indicating that the activity of basal forebrain cholinergic neurons is controlled by a GABAergic input, and that this neuronal link is involved in mnemonic processing.     

c-Fos antisense oligodeoxynucleotide offsets behavioral nociceptive responses and both up-regulations of c-Fos protein and dynorphin a (1-8) in dorsal horn: a study using the formalin test in rats

The formalin test was used to elicit acute and chronic pain in rats, and antisense oligodeoxynucleotide (AS-ODN) was used as a tool to modulate the expression of nociceptive behavioral and neurochemical responses. AS-ODN complementary to c-Fos mRNA was administered intrathecally (i.t.) 4 h before formalin injection in the experimental group. Normal saline or reverse AS-ODN was pre-administered i.t. at the same time in two control groups (saline and reverse AS-ODN). The results showed that the acute phase of nociceptive behavior showed no change by AS-ODN administration, whereas the tonic phase of nociceptive licking and biting behavior was significantly suppressed by AS-ODN as compared with the saline or the reverse AS-ODN group, respectively (p < .05 and p < .01). At the same time, both Fos-like immunoreactive (FLI) neurons and density of dynorphin-like immunoreactivities (DLI) were decreased significantly (p < .05 and p < .01) in the AS-ODN group as compared with that in two control groups. The results indicate that the long-lasting nociceptive responses elicited by sustained noxious inputs are based on the up-regulation of c-Fos gene expression, which in turn induces the upregulation of Dyn A production. It is proposed that intensified Dyn A production in the dorsal horn may be pivotal for the appearance of chronic pain.     

Subchronic haloperidol increases CB(1) receptor binding and G protein coupling in discrete regions of the basal ganglia

The present study was designed to test whether chronic neuroleptic treatment, which is known to alter both expression and density of dopamine D(2) receptors in striatal regions, has effects upon function and binding level of the cannabinoid CB(1) receptor in the basal ganglia by using receptor autoradiography. As predicted, subchronic haloperidol treatment resulted in increased binding of (3)H-raclopride and quinpirole-induced guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) in the striatum when compared to that measured in control animals. This increased D(2) receptor binding and function after 3 days washout was normalized after a 2-week washout period. Effect of haloperidol treatment was studied for CB(1) receptor binding and CP55,940-stimulated [(35)S]GTPgammaS in the striatum, globus pallidus, and substantia nigra. (3)[H]CP55,940 binding levels were found in rank order from highest to lowest in substantia nigra > globus pallidus > striatum. Furthermore, subchronic haloperidol treatment resulted in elevated binding levels of (3)[H]CP55,940 in the striatum and the substantia nigra and CB(1) receptor-stimulated [(35)S]GTPgammaS bindings in the substantia nigra after 3 days washout. These increased binding levels were normalized at 1-4 weeks after termination of haloperidol treatment. Haloperidol treatment had no significant effect on CB(1) receptor or [(35)S]GTPgammaS binding levels in globus pallidus. The results help to elucidate the underlying biochemical mechanism of CB(1) receptor supersensitivity after haloperidol treatment.     

Ontogeny of L-DOPA-induced locomotion: Expression of c-Fos in the brainstem and basal ganglia of rats

Neonatal rats suspended in harnesses, limbs hanging freely, and injected with 100 mg/kg l-3,4-dihydroxyphenylalanine (L-DOPA), engage in a behavior (air stepping) that closely resembles spontaneous locomotion. Rats no longer demonstrate this response after postnatal day 20 (P20). In the present experiment, an immunohistochemical analysis of the immediate early protein c-Fos was performed as a marker for cellular activity in the brains of suspended rat pups treated with l-DOPA at P15 and P25. Control rats were injected with saline at each age and subjected to the same behavioral protocol. Only P15 rat pups injected with L-DOPA engaged in air stepping and expressed the highest levels of c-Fos reactivity in output nuclei of the basal ganglia, as well as the pedunculopontine (PPN) and cuneiform (Cnf) nuclei. Twenty-five-day-old rats, which did not air step, exhibited reduced c-Fos labeling in these areas as well as in the locus coeruleus (LC). Our findings suggest that excitation of the basal ganglia resulted via afferents from the PPN and/or Cnf, which may develop before reciprocal inhibitory connections are fully mature. We propose that a circumscribed portion of the midbrain, which overlaps with the physiologically defined mesencephalic locomotor region (MLR), is necessary for the production of L-DOPA-induced locomotion. We propose further that this action is induced against a background of heightened arousal during the first three postnatal weeks but comes under inhibitory control in rat pups older than 20 days of age.     

Differential control of cortical activity by the basal forebrain in rats: a role for both cholinergic and inhibitory influences

Using microdialysis and high-performance liquid chromatography, we measured acetylcholine (ACh) release simultaneously from two cortical sites in anesthetized rats. One site was always in the somatosensory cortex, and the other was in either the visual or the motor cortex. After baseline measurements were obtained, selected sites in the basal forebrain (BF) were stimulated to increase ACh release. Some BF sites provoked more release in one microdialysis probe than in the other, suggesting some degree of corticotropic organization of the cholinergic projections from the BF. BF sites optimal for release from the visual cortex were separated from optimal sites for release from the somatosensory cortex by greater distances than were the best sites for release from the somatosensory and the motor cortex. Stimulation of a single BF site often provoked similar release from the latter two cortical areas. Electrical stimulation of the BF also modified cortical neuronal activity. Activation of some BF sites provoked an intense discharge of many neurons in the vicinity of the cortical recording electrode, and the same stimulus site in the BF provoked release of large amounts of ACh in the cortex. Stimulation of other BF sites produced strong inhibition of ongoing cortical activity and no increase in cortical ACh release. When other sites were stimulated, they had no effect or they generated stereotyped bursting patterns in the cortex without any observable effect on ACh release. BF sites that generated inhibition of cortical neural activity were generally located near the sites that activated the cortex and provoked release of ACh. These data suggest an elaborate control of the sensory cortex by a mechanism involving both gamma-aminobutyric acid-containing and cholinergic neurons of the BF. J. Comp. Neurol. 381:53-67, 1997. © 1997 Wiley-Liss, Inc.     

Learning-dependent dynamics of beta-frequency oscillations in the basal forebrain of rats

Cholinergic, GABAergic and glutamatergic projection neurons of the basal forebrain (BF) innervate widespread regions of the neocortex and are thought to modulate learning and attentional processes. Although it is known that neuronal cell types in the BF exhibit oscillatory firing patterns, whether the BF as a whole shows oscillatory field potential activity, and whether such neuronal patterns relate to components of cognitive tasks, has yet to be determined. To this end, local field potentials (LFPs) were recorded from the BF of rats performing an associative learning task wherein neutral objects were paired with differently valued reinforcers (pellets). Over time, rats developed preferences for the different objects based on pellet‐value, indicating that the pairings had been well learned. LFPs from all rats revealed robust, short‐lived bursts of beta‐frequency oscillations (～25 Hz) around the time of object encounter. Beta‐frequency LFP events were found to be learning‐dependent, with beta‐frequency peak amplitudes significantly greater on the first day of the task when object–reinforcement pairings were novel than on the last day when pairings were well learned. The findings indicate that oscillatory bursting field potential activity occurs in the BF in freely behaving animals. Furthermore, the temporal distribution of these bursts suggests that they are probably relevant to associative learning.     

Pontine and basal forebrain transections disinhibit brown fat thermogenesis in neonatal rats

Bignall, Heggeness and Palmer (1975) were the first to demonstrate increases in metabolic heat production following midpontine transection in neonatal rats. Subsequent work in adult rats has shown that this procedure disinhibits thermogenesis by brown adipose tissue (BAT). Bignall and his colleagues also found that hypothalamic ablation did not result in increased thermogenesis in 5-day-olds, leading them to conclude that thermoregulation depends on more caudal structures at that age. We have also found that midpontine transection disinhibits BAT thermogenesis and, furthermore, have extended that finding to newborn pups. When transections were made in the basal forebrain, however, we also found profound and rapid increases in brown fat thermogenesis. These results suggest the presence of at least two sources of inhibition of BAT thermogenesis in newborn rats: one located in the rostral pons-caudal midbrain and one located in the basal forebrain.     

Antisense oligodeoxynucleotide-induced suppression of basal forebrain NMDA-NR1 subunits selectively impairs visual attentional performance in rats

It is generally agreed that basal forebrain neuronal circuits contribute to the mediation of the ability to detect, select and discriminate signals, to suppress the processing of irrelevant information, and to allocate processing resources to competing tasks. Rats were trained in a task designed to assess sustained attention, or in a cued discrimination task that did not tax attentional processes. Animals were equipped with guide cannula to infuse bilaterally antisense oligodeoxynucleotides (ODNs) against the N-methyl-D-aspartate (NMDA) NR1 subunits, or missense ODNs, into the substantia innominata of the basal forebrain. Infusions of antisense or missense ODNs did not affect cued visual discrimination performance. Infusions of antisense ODNs dose-dependently impaired sustained attention performance by selectively decreasing the animals' ability to detect signals while their ability to reject nonsignal trials remained unchanged. The detrimental attentional effects of antisense infusions were maximal 24 h after the third and final infusion, and performance returned to baseline 24 h later. Missense infusions did not affect attentional performance. Separate experiments demonstrated extensive suppression of NR1 subunit immunoreactivity in the substantia innominata. Furthermore, infusions of antisense did not produce neurotoxic effects in that region as demonstrated by the Fluoro-Jade METHOD: The present data support the hypothesis that NMDA receptor (NMDAR) stimulation in the basal forebrain, largely via glutamatergic inputs originating in the prefrontal cortex, represents a necessary mechanism to activate the basal forebrain corticopetal system for mediation of attentional performance.     

Decreased forebrain [S]TBPS binding and increased [H]muscimol binding in rats that do not develop stress-induced behavioral depression

Recent evidence suggests that anxiety and its biological concomitants may be involved in the pathophysiology of depression. In the present study, the in vitro radioligand binding of [³H]flunitrazepam, [³H]muscimol and[³⁵S]t-butylbicyclophosphorothionate (TBPS) sites on the benzodiazepine /GABA chloride ionophore receptor complex (BGRC) was examined using the learned helplessness paradigm. Only rats which did not develop the syndrome showed a significant increase in [³H]muscimol binding in cerebral cortex and a decrease in [³⁵S]TBPS binding in cerebral cortex and hippocampus in comparison to naive controls. For both ligands, this represented a change inB_max rather than a change in affinity. Adrenalectomy had no impact on these alterations indicating that critical endogenous factors are not manufactured by the adrenal glands. These findings suggest that the BGRC in the forebrain may be a site mediating the ‘coping’ ability of rats that do not develop the learned helplessness syndrome. The possible involvement of neurosteroids in the effect is discussed.     

Wakefulness-inducing effects of histamine in the basal forebrain of freely moving rats

Histamine-containing neurons of the tuberomammillary nucleus (TMN) are implicated in facilitating wakefulness. They project to many brain areas, including the cholinergic basal forebrain (BF). The cholinergic magnocellular regions of the BF are important in the regulation of cortical arousal and wakefulness, and a role for histamine in this activity is suggested by in vitro data indicating histamine excites BF cholinergic and non-cholinergic neurons. To test the hypothesis that histamine induces wakefulness via actions in the BF, we performed microdialysis perfusion of different concentrations of histamine (100, 500 and 1000 microM) in the BF of Sprague-Dawley rats. A MANOVA analysis showed that histamine produced a highly statistically significant and dose-dependent increase in wakefulness and decrease in non-rapid eye movement (NREM) sleep compared with artificial cerebrospinal fluid perfusion. From a wakefulness baseline percentage time of about 12% with artificial cerebrospinal fluid, histamine perfusion increased this value to 26% (100 microM), 36% (500 microM), or 47% (1000 microM). There was no statistically significant change in rapid eye movement (REM) sleep. Histamine perfusion (500 microM) in a control site, the centromedian thalamic nucleus, did not produce any change in behavioral state. The results indicate a prominent role of histamine in wakefulness regulation via the BF and further support the hypothesis that the BF has an important role in EEG activation and wakefulness.