Organization of the basal forebrain in the cat: localization of -enkephalin, substance P, and choline acetyltransferase immunoreactivity

The present study uses immunocytochemical techniques to determine whether cholinergic basal forebrain neurons in the cat are in a position to receive a homogeneous pattern of inputs, or if specific immunocytochemically defined afferent systems are localized to only selected regions of the basal forebrain. Monoclonal antibodies against choline acetyltransferase (ChAT) were used to identify the location of putative cholinergic neurons which are known to project to the cerebral cortex. In addition, polyclonal antibodies against substance P (SP) or enkephalin (Enk) were used on either adjacent or on the same histological sections reacted for ChAT to identify the neuropeptide plexuses that provide input to the basal forebrain. ChAT-immunoreactive (ChAT-IR) perikarya were located throughout the vertical limb, genu and horizontal limb of the diagonal band of Broca. ChAT-IR neurons also were located within the substantia innominata (SI), within the peripallidal zone around the globus pallidus, and were intercalated within the internal capsule. Enk-IR and SP-IR were used to determine the distribution of putative peptidergic terminals within the basal forebrain. Extensive Enk-IR and SP-IR terminal label was localized within the globus pallidus and the surrounding peripallidal zones, as well as within the SI, whereas the components of the diagonal band of Broca demonstrated negligible Enk-IR and SP-IR label. These data predict that the subdivisions of the cholinergic basal forebrain in the cat do not share a uniform afferent system, and only selective portions of this cholinergic system are in an anatomical position to receive a major direct input from the identified subcortical peptidergic afferents. The segregation of afferents has important consequences in the selective control of cortical function by the cholinergic basalocortical pathway.     

Distribution of parvalbumin-containing neurons and lectin-binding perineuronal nets in the rat basal forebrain

In sections of rat brain treated forWisteria floribunda agglutinin (WFA) labelling the occurrence of parvalbumin (PARV)-, calbindin (CALB)- or choline acetyltransferase (ChAT) immunoreactivity was analyzed in the basal forebrain using dual-peroxidase and double-fluorescence methods. Only PARV-immunoreactive (-ir) neurons were surrounded by WFA-labelled, i.e.N-acetylgalactosamine-containing, perineuronal lattice-like structures known as perineuronal nets. The distribution of these nets and PARV-ir cells in the rat basal forebrain was documented to obtain detailed data on their co-existence. A remarkable diversity distribution of both markers was observed, as PARV-ir neurons are only associated with nets in the medial septal nucleus, the nuclei of the diagonal band and the magnocellular preoptic nucleus, but not in the ventral pallidum or the substantia innominata/nucleus basalis complex. These differences in the neuronal microenvironment may reflect system-related specializations of neurons within the basal forebrain nuclei.     

Cholinergic and non-cholinergic neurons of cat basal forebrain project to reticular and mediodorsal thalamic nuclei

Choline acetyltransferase immunohistochemistry combined with the retrograde transport of horseradish peroxidase showed that the reticular and mediodorsal thalamic nuclei of the cat receive an important input from cholinergic and non-cholinergic neurons of substantia innominata and adjacent structures in the basal forebrain.     

Effect of basal forebrain somatostatin and parvalbumin neurons in propofol and isoflurane anesthesia

AimsThe basal forebrain (BF) plays an essential role in wakefulness and cognition. Two subtypes of BF gamma‐aminobutyric acid (GABA) neurons, including somatostatin‐expressing (GABA^SOM) and parvalbumin‐positive (GABA^Parv) neurons, function differently in mediating the natural sleep–wake cycle. Since the loss of consciousness induced by general anesthesia and the natural sleep–wake cycle probably share similar mechanisms, it is important to clarify the accurate roles of these neurons in general anesthesia procedure.MethodsBased on two transgenic mouse lines expressing SOM‐IRES‐Cre and PV‐IRES‐Cre, we used a combination of genetic activation, inactivation, and chronic ablation approaches to further explore the behavioral and electroencephalography (EEG) roles of BF^SOM and BF^Parv neurons in general anesthesia. After a single intravenous injection of propofol and the induction and recovery times of isoflurane anesthesia, the anesthesia time was compared. The changes in cortical EEG under different conditions were also compared.ResultsActivation of BF GABA^SOM neurons facilitates both the propofol and isoflurane anesthesia, manifesting as a longer anesthesia duration time with propofol anesthesia and a fast induction time and longer recovery time with isoflurane anesthesia. Moreover, BF GABA^SOM‐activated mice displayed a greater suppression of cortical electrical activity during anesthesia, showing an increase in δ power bands or a simultaneous decrease in high‐frequency power bands. However, only a limited and nuanced effect on propofol and isoflurane anesthesia was observed with the manipulated BF GABA^Parv neurons.ConclusionsOur results suggested that BF GABA^SOM neurons play a critical role in propofol and isoflurane general anesthesia, while BF GABA^Parv neurons appeared to have little effect.     

Cholinergic systems in the rat brain: III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia, and basal forebrain

The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.     

Effect of high-dose methyl-prednisolone on brainstem encephalopathy and basal ganglia impairment complicating cat scratch disease

Cat scratch disease (CSD) is a zoonotic illness caused by the Gram negative bacillus Bartonella henselae characterized by a small skin lesion at the site of a bite, lick or scratch by a cat, commonly followed by regional lymphadenopathy 1 or 2 weeks later. We report herein on severe neurological complications of CSD combining brainstem encephalopathy and basal ganglia impairment. This 12-year-old female acutely presented to a local hospital with profound coma and a prolonged tonic posturing of extremities. On the neurological examination she was deeply comatose with pin-point pupils and lack of vestibulo-ocular responses, suggestive of brainstem encephalopathy, along with marked rigid hypertonicity suggestive also of basal ganglia impairment. Initially suspecting Herpes simplex encephalitis or acute disseminated encephalomyelitis she was promptly started with high-dose methyl-prednisolone and acyclovir. Her parents apparently reported that she was scratched by a kitten some 4 weeks prior to her present admission and as such, suspecting CSD, she was begun with doxycycline and rifampicin. Her serology had proven positive for IgM antibodies to Bartonella henselae establishing the diagnosis. She regained consciousness after 4 days and the signs of brainstem and extra-pyramidal impairment also gradually abated and disappeared after 10 days. A follow-up exam after a month disclosed mild extra-pyramidal abnormalities which disappeared after 3 months. Although extremely rare, CSD should be also considered in a patient presenting with a severe encephalopathy and associated basal ganglia impairment. The prompt administration of high-dose methyl-prednisolone upon admission may have contributed to the favorable outcome in our patient and therefore should be advocated in any patient presenting with profound encephalopathy regardless the underlying etiology recovered later.     

Basal forebrain neurons have axon collaterals that project to widely divergent cortical areas in the cat

Basal forebrain neurons with axon collaterals that project to widely divergent cortical areas were identified using retrograde transport of two labels. A proportion of neurons in the basal forebrain have axon collaterals that project to both anterior (precruciate gyrus) and posterior (marginal and suprasylvian gyri) cortical areas or to medial (precruciate gyrus) and lateral (ectosylvian and anterior suprasylvian gyri) cortical areas. These branched fibers originate from cells located predominantly in the basal nucleus of Meynert. The existence of such neurons suggests that individual basal forebrain cells are capable of influencing widespread neocortical zones in the cat.     

Intense immunoreactivity for Mn-superoxide dismutase (Mn-SOD) in cholinergic and non-cholinergic neurons in the rat basal forebrain

The immunohistochemical localization of manganese (Mn)-superoxide dismutase (Mn-SOD) was studied in the rat basal forebrain using polyclonal antibodies to Mn-SOD. Neurons of the basal forebrain exhibit a high density of Mn-SOD immunoreactivity. Double immunostaining with a monoclonal antibody to choline acetyltransferase demonstrated that both cholinergic and non-cholinergic neurons in the basal forebrain are intensely immunoreactive for Mn-SOD.     

Basal forebrain and anterior thalamic contributions to acetylcholinesterase activity in granular retrosplenial cortex of rats

Histochemical studies demonstrate that granular retrosplenial cortex (cortical areas 29b and c) of the adult rat displays a characteristics laminar pattern of acetylcholinesterase (AChE) activity. While some AChE-positive axons are found in all cortical layers, most intense staining occurs in two bands that correspond to layers I and III. The present studies were directed toward identifying the neural systems underlying this AChE activity. Unilateral electrolytic or excitatory amino acid induced lesions of the basal forebrain, including the nucleus of the diagonal band, result in reductions of AChE staining throughout ipsilateral granular retrosplenial cortex; particularly noteworthy are the reductions in layer I and the deeper cortical layers. AChE staining remains in superficial layer I and in layer II. Placement of lesions in the anterior thalamus, including all of the anterior dorsal nucleus, results in reduction of AChE histochemical staining in the outer part of layer I and especially in layer III. Staining remains in much of layer I and in the deepest band of layer III. Placement of electrolytic lesions in the hypothalamus or the midbrain tegmentum produce no detectable change in the pattern of AChE in retrosplenial cortex. These results indicate that AChE activity in granular retrosplenial cortex is found primarily within afferent axons from the basal forebrain system and from anterior dorsal thalamus, and these two systems of afferents display distinct laminar patterns of termination.     

Basal forebrain efferents reach the whole cerebral cortex of the cat

Efferent projections from the basal forebrain to the cat's cerebral cortex were traced with the retrograde horseradish peroxidase technique. Different areas of the cerebral cortex of 51 cats were injected with small amounts of horseradish peroxidase. The entire basal forebrain was screened for labeled neurons. Following all injections, retrogradely labeled neurons could be detected in either the medial septum, or the vertical and horizontal limb of the diagonal band of Broca, or the substantia innominata, or in several of these structures. All three basal forebrain structures project heavily to allocortical regions, but only weakly to neocortical regions. An exception is the medial prefrontal cortex which is densily innervated by the substantia innominata (i.e., comparably dense as allocortical regions are innervated by the substantia innominata). Large injections into he basal temporal cortex (including the perirhinal cortex) and into the insular cortex also led to a considerable number of labeled cells in the substantia innominata. The results indicate a widespread innervation of the cat's cerebral cortex by the basal forebrain. This diffuse projection to the cortex has recently been found also in monkeys and rats. Anatomical and functional implications of these projections in the cat are discussed and related to findings in other species.     

Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons

Nerve growth factor (NGF) has recently been implicated as a trophic agent in the survival and maintenance of basal forebrain cholinergic neurons. To test the hypothesis that NGF may play a role in the age-related degeneration of basal forebrain neurons and decline of cerebral cholinergic function, we have used a monoclonal antibody to the NGF receptor, 192 IgG, to immunocytochemically visualize and compare rat basal forebrain neurons responsive to NGF in aged (30 months) and young adult (10 months) rats. In a subpopulation of aged rats, NGF receptor-immunoreactive cells in the basal forebrain appear vacoulated and shrunken, and the neuropil staining is markedly reduced. While no substantial decline in cell density is apparent in Nissl-stained sections, the number of NGF receptor-positive cell profiles within the vertical limb of diagonal band nuclei is reduced by an average of 32% in aged rats. Marked reduction in the expression of NGF receptors in aged rats may signify loss of capacity of the basal forebrain neurons to bind and transport NGF from their terminals in the hippocampus and cortex, subsequent decrease in NGF delivered to the cell bodies, and eventual cellular dysfunction and death of neurons in aging.     

Chemical anatomy of the human ventral striatum and adjacent basal forebrain structures

Calbindin D-28k (CB), calretinin (CR), substance P (SP), limbic system-associated membrane protein (LAMP), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) were used as chemical markers to investigate the organization of the ventral striatum (VST) and adjacent structures in healthy human individuals. No clear boundary could be established between the dorsal striatum and the VST, and the core/shell subdivisions of nucleus accumbens (Acb) could be distinguished only at the midrostrocaudal level of the VST. The CB-poor shell displayed intense immunostaining for SP and CR but only weak staining for LAMP. By contrast, the core was weakly stained for SP and CR and moderately stained for LAMP and CB. There was no difference between shell and core with regard to the cholinergic markers. The Acb harbored numerous ChAT- and CR-immunoreactive cell bodies, the latter being distributed according to a marked, mediolaterally increasing gradient. The size of the ChAT- and CR-immunoreactive perikarya in the Acb varied according to their location in the core and shell. The VST was surrounded by a chemically heterogeneous group of cell clusters referred to as interface islands. The CR-rich caudal portion of the VST merged with the bed nucleus of the stria terminalis dorsally and the diagonal band of Broca ventromedially, the latter two structures displaying complex immunostaining patterns. The claustrum was markedly enriched in LAMP and harbored different types of CR- and CB-immunopositive neurons. These results demonstrate that the neurochemical organization of the human VST is strikingly complex and exhibits a greater heterogeneity than the dorsal striatum.     

Behavioural, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system

Use of the selective immunotoxin; 192 IgG-saporin, is helping to elucidate the role of the cholinergic system in cognition by overcoming the problems of interpretation associated with the use of non-specific lesioning agents. In separate studies, we have compared the long- and short-term effects of single site and combined saporin lesions of the nucleus basalis magnocellularis and medial septal area, on spatial learning and memory in radial arm and water maze tasks. At 11 months, only rats with combined lesions showed deficits in both radial and water maze tasks, although terminal cholinergic deafferentation was substantial and extensive tissue loss was seen at the injection sites in both single and combined lesions. However, the extensive tissue loss with long-term lesions suggested that behavioural deficits were not solely attributable to cholinergic deafferentation. In contrast, when rats with combined lesions were tested 5 months after lesioning, no deficits were apparent, although there was almost complete loss of choline acetyltransferase- and nerve growth factor receptor-immunoreactivity in the basal forebrain with no tissue damage at the injection sites. This study supports existing literature that selective loss of cholinergic neurons in the basal forebrain does not produce behavioural impairments in standard tasks of learning and memory, but deficits are apparent when damage is non-selective as occurs late after lesioning, confounding interpretation of behavioural data. It further highlights potential problems with this immunotoxin in long-term studies.     

Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebrain neurons

Recent data indicate that the neurons of the cholinergic basal forebrain (CBF) respond to nerve growth factor (NGF) with increased survival under experimental conditions and have NGF receptors which mediate the binding and retrograde transport of NGF from axon terminals to somata. Focal intraparenchymal injections of retrograde tracing agents into neuropil demonstrate that the distribution of axons from cholinergic nuclei to cortex and hippocampus is topographically restricted and largely ipsilateral. Monoclonal antibody 192, a well-characterized antibody which recognizes only the rat NGF receptor, was labelled with 125I and injected into a lateral ventricle of adult rats. Highly specific bilateral transport to numerous neurons of the CBF system was demonstrated by autoradiography. This result directly demonstrates that suitably targeted antibodies can be taken up by specific neuronal populations following intraventricular injection and implies that CBF neurons may be influenced by relatively high molecular weight substances injected into cerebrospinal fluid.     

The effect of prefrontal stimulation on the firing of basal forebrain neurons in urethane anesthetized rat

The basal forebrain (BF) contains a heterogeneous population of cholinergic and non-cholinergic corticopetal neurons and interneurons. Neurons firing at a higher rate during fast cortical EEG activity (f>16Hz) were called F cells, while neurons that increase their firing rate during high-amplitude slow-cortical waves (f<4Hz) were categorized as S-cells. The prefrontal cortex (PFC) projects heavily to the BF, although little is known how it affects the firing of BF units. In this study, we investigated the effect of stimulation of the medial PFC on the firing rate of BF neurons (n=57) that were subsequently labeled by biocytin using juxtacellular filling (n=22). BF units were categorized in relation to tail-pinch induced EEG changes. Electrical stimulation of the medial PFC led to responses in 28 out of 41 F cells and in 8 out of 9 S cells. Within the sample of responsive F cells, 57% showed excitation (n=8) or excitation followed by inhibitory period (n=8). The remaining F cells expressed a short (n=6) or long inhibitory (n=6) response. In contrast, 6 out of the 8 responsive S cells reduced their firing after prefrontal stimulation. Among the F cells, we recovered one cholinergic neuron and one parvalbumin-containing (PV) neuron using juxtacellular filling and subsequent immunocytochemistry. While the PV cell displayed short latency facilitation, the cholinergic cell showed significant inhibition with much longer latency in response to the prefrontal stimulus. This is in agreement with previous anatomical data showing that prefrontal projections directly target mostly non-cholinergic cells, including GABAergic neurons.     

The basal ganglia in extrapyramidal dysfunction

The rapid advances in knowledge of basal ganglia circuitry and function in recent years have allowed the construction of a functional scheme to explain many facets of known pathologic states. The dichotomy of Parkinson's disease; akinesia with increased tone, and the mirror effects in Huntington's disease; hemiballismus and tardive dyskinesia, hyperkinesia with decreased tone are explained as due to two outputs of the system with an intervening inhibitory neuron which reverses the sign. The two outputs control different motor functions; pallidothalamic involved primarily with movements and nigrobrainstem involved primarily with muscle tone.     

Regional analysis of age-related changes in the cholinergic system of the hippocampal formation and basal forebrain of the rat

In an attempt to clarify conflicting reports of age-related changes in cholinergic systems of the rat hippocampal formation and basal forebrain, we compared aged (40 months) and adult (12 months) male rats using quantitative, regional receptor autoradiography in addition to radiolabelled assays of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). The activities of ChAT and AChE in Ammon's horn/subiculum are 24% and 38% lower, respectively, in the aged brains. There is also a drop in both ChAT (38%) and AChE (28%) activities in the septum, and a 46% drop in ChAT activity in the nucleus basalis of aged rats. In the septal pole of the hippocampal formation there is no significant change with age in binding of the muscarinic antagonist, tritiated quinuclidinyl benzylate (3H-QNB) in any hippocampal subregion. However, specific binding in the temporal pole is higher in the subiculum (40%), CA (27%), and dentate gyrus (25%) of the aged animals. Because some of the neurons of the diagonal band of Broca project to the temporal areas of the hippocampal formation by way of a ventral pathway, it is possible that with age this septohippocampal pathway is selectively affected. Particularly in Ammon's horn and the subicular regions of the aged rat hippocampus, postsynaptic muscarinic receptors may upregulate to compensate for decreases in presynaptic cholinergic activity.     

额叶皮质损伤对大鼠基底前脑胆碱能神经元的影响

本实验探讨了前额叶皮质局限性损伤对大鼠学习、记忆功能及基底前脑胆碱能神经元的影响。用外科手术造成大鼠一侧前额叶皮质局限性损伤后不同时间、用Ｙ型迷宫检测学习、记忆功能、用组织化学技术检测基底前脑含乙酰胆碱酸酶（ＡChE)活性神经元。实验观察到前额叶皮质损伤后１周,动物学习,记忆功能有所障碍,损伤同侧的基底前脑胆碱能神经元有所减少,但均无统计学意义,损伤后２,３,４周,动物学习、记忆障碍明显,损伤同侧基底前脑胆碱能神经元明显减少（Ｐ＜0.05),且两者变化相平行。结果表明单侧前额叶皮质局限性损伤不仅可引起动物学习、记忆功能障碍,且可引起同侧基底前脑胆碱能神经元丢失,且两者发展相平行,提示基底前脑胆碱能神经元逆行性变性在动物额叶皮质损伤引起的学习、记忆障碍中起作用。     

Hypertrophy of basal forebrain neurons and enhanced visuospatial memory in perinatally choline-supplemented rats

The effects of choline supplementation during two time-frames of early development on radial-arm maze performance and the morphology of basal forebrain neurons immunoreactive for the P75 neurotrophin receptor (NTR) in male and female Sprague–Dawley rats were examined. In the first experiment, rats were supplemented with choline chloride from conception until weaning. At 80 days of age, subjects were trained once a day on a 12-arm radial maze for 30 days. Compared to control littermates, supplemented rats made fewer working and reference memory errors; however, the memory enhancing effects of choline supplementation were greater in males than females. A morphometric analysis of NTR-immunoreactive cell bodies at three levels through the medial septum/diagonal band (MS/DBv) of these rats revealed that perinatal choline supplementation caused the somata of cells in the MS/DBv to be larger by 8–15%. In a second experiment, choline supplementation was restricted to embryonic days 12–17. A developmental profile of NTR immunoreactive cell bodies in the MS/DBv of 0-, 8-, 16-, 30- and 90-day old male and female rats again revealed that cell bodies were larger in choline-supplemented rats than controls. As in the behavioral studies, the effect of choline supplementation was greater in male than female rats. These data are consistent with the hypothesis that supplementation with choline chloride during early development leads to an increase in the size of cell bodies of NTR-immunoreactive cells in the basal forebrain and that this change may contribute to long-term improvement in spatial memory.