Magnocellular Basal Forebrain Neurons Research Articles

Selective age-related loss of CALBINDIN-D 28k from basal forebrain cholinergic neurons in the common marmoset ( callithrix jacchus)

A significant number of the cholinergic neurons in the basal forebrain of the primate, but not the rodent brain contain the calcium binding protein calbindin-D 28k (CB). Previous experiments in our laboratory have demonstrated a substantial age-related loss of CB from the human basal forebrain cholinergic neurons (BFCN). The present study investigated the possible age-related loss of CB from the BFCN in a non-human primate species, the common marmoset ( Callithrix jacchus). Quantitative analysis of matching sections as well as unbiased stereological determination of neuronal number were used in 16 adult marmosets ranging in age between 2 and 15 years. No significant changes were observed in the number of choline acetyltransferase-positive BFCN when a group of young animals (≤4 years) was compared with a 6–8-year-old group and a 9–15-year-old group. Similarly, no age-related changes were observed in Nissl-stained magnocellular basal forebrain (putatively cholinergic) neurons. In contrast, the BFCN of the two older groups of animals displayed a significant loss of CB. The age-related loss of CB occurred in all sectors of the BFCN, but was greatest in the anterior sector of this cell group. The CB loss was neurochemically specific since the BFCN in the older groups of animals continued to express other markers such as high and low affinity neurotrophin receptors. The age-related loss of CB from the marmoset BFCN was also regionally selective as CB positive neurons in other structures, such as the cerebral cortex and the striatum displayed no apparent age-related changes. These results indicate that the marmoset BFCN display a significant and selective age-related loss of CB reminiscent of that observed in the human. Therefore, the common marmoset represents an appropriate animal model in which the consequences of BFCN CB loss can be investigated in depth. Loss of CB from the aged BFCN is likely to reduce the capacity of these neurons to buffer intracellular calcium and to leave them vulnerable to insults which can result in increased calcium levels. The vulnerability of the CB-negative BFCN in the aged marmoset to various insults which disturb calcium homeostasis remains to be investigated.

Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture.

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.

Ca2+-permeable non-NMDA glutamate receptors in rat magnocellular basal forebrain neurones.

1. Ionotropic glutamate receptor-mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch-clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks. 2. Non-NMDA and NMDA receptor-mediated events could be distinguished pharmacologically using the selective agonists (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA), and antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (AP5). 3. Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady-state EC50 values for AMPA and kainate were 2.7 +/- 0.4 microM (n = 5) and 138 +/- 25 microM (n = 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 +/- 0.08 microM (n = 5) at -70 mV. These data strongly suggest that AMPA receptors are the predominant non-NMDA receptors expressed by basal forebrain neurones. 4. At -70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor. 5. Current-voltage relationships for non-NMDA receptor-mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 microM spermine in the recording pipette, currents frequently displayed double-rectification characteristic of non-NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26-3.6; median, 1.27 (n = 15). 6. Current flow through non-NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of -70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 microM. In six out of seven cells tested, block by Cd2+ was voltage sensitive. 7. Ca2+ permeability of many of the non-NMDA receptors expressed by magnocellular basal forebrain neurones may underlie the unusual sensitivity of cholinergic basal forebrain neurones to non-NMDA receptor-mediated excitotoxicity.

Light and Electron Microscopic Localization of Presenilin-1 in Primate Brain

Several genes have been implicated in the pathogenesis of early-onset familial Alzheimer's disease. A majority of the autosomal dominant cases are linked to recently identified mutations in the presenilin-1 gene on chromosome 14. The native presenilin-1 protein in primates has not been well characterized, and its precise localization is unknown. We have studied the native presenilin-1 protein in monkey brain and peripheral tissues by using a monoclonal antibody specific for the N-terminal domain of human presenilin-1. Western blots detect polypeptide species of approximately 49 and approximately 32 kDa from COS-7 and PC12 cells transfected with full-length human presenilin-1 cDNA and from in vitro translations of the normal human presenilin-1 mRNA. A 32 kDa polypeptide is detected in monkey peripheral tissues, with the highest expression in testis and lung. In all brain regions the 32 kDa band is the predominant form of presenilin-1, and it is found in particulate subfractions. Light microscopic immunocytochemistry reveals presenilin-1 staining in all brain regions, with the strongest labeling in neurons and neuropil. In addition, weaker immunoreactivity is also present in glia and blood vessels. Neuronal staining shows significant variability, with particularly intense labeling of certain cell types, including large neocortical and hippocampal pyramidal neurons, magnocellular basal forebrain neurons, brainstem motoneurons, and some populations of interneurons. By electron microscopic immunocytochemistry, highly selective presenilin-1 staining is seen on the cytoplasmic surfaces of membranous organelles, which suggest localization to the endoplasmic reticulum-Golgi intermediate compartment, a subdomain of the endoplasmic reticulum, and some coated transport vesicles.

Modulation of Inhibitory Transmission by Dopamine in Rat Basal Forebrain Nuclei: Activation of Presynaptic D1-like Dopaminergic Receptors

The effects of dopamine (DA) on inhibitory transmission onto identified magnocellular neurons were examined in rat basal forebrain slices using whole-cell recording. IPSCs evoked by focal stimulation within basal forebrain nuclei were reversibly blocked by 10 microM bicuculline and had a decay time constant of 20.1 +/- 0.77 msec in the presence of 6-cyano-7-nitroquinoxalline-2,3-dione (5 mM). Bath application of DA reduced the amplitude of IPSCs up to 71.1 +/- 1.49% in a concentration-dependent manner between 0.003 and 1 mM (the IC50 value being 6.6 microM), without any effect on the holding current at -70 mV. DA (10 microM) reduced the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), without affecting their mean amplitude, rise time, and decay time constant. Furthermore, the DA-induced effect on mIPSCs remained unaffected by 100 microM cadmium, suggesting a presynaptic mechanism independent of calcium influx. SKF 81297, a D1-like agonist, mimicked DA-induced effect on evoked IPSCs (IC50, 10.9 microM), whereas R(-)-TNPA or (-)-quinpirole, D2-like agonists (30 microM), had little or no effect on the amplitude of evoked IPSCs. R(+)-SCH 23390, a D1-like antagonist, antagonized the DA-induced effect on IPSCs (K(B) 0.82 microM), whereas S(-)-eticlopride, a D2-like antagonist, showed slight antagonism (K(B) 7.8 microM). Forskolin (10 microM) reduced the amplitude of evoked IPSCs to approximately 58% of the control and occluded the inhibitory effect of DA. These findings indicate that DA reduces inhibitory transmission onto magnocellular basal forebrain neurons by activating presynaptic D1-like receptors.

Dopamine D1-like receptor-mediated presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones.

1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3.81 +/- 0.09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM). 2. Bath-applied dopamine (DA) reduced evoked EPSC amplitude by up to 54.2 +/- 2.3% with an IC50 of 19.9 microM in normal Krebs solution (2.5 mM Ca2+, 1.2 mM Mg2+) without effect on postsynaptic holding current. 3. DA (30 microM) reduced the mean frequency of spontaneous miniature EPSCs recorded in 0.5 microM tetrodotoxin without affecting their mean amplitude, rise time or decay time constant. This effect was diminished by 100 microM Cd2+. 4. The effect of DA on evoked EPSCs was mimicked by the D1-like receptor agonist, SKF 81297 (IC50 25.6 microM), but not by the D2-like receptor agonist R(-)-TNPA (30 microM) or (-)-quinpirole (30 microM), and was antagonized by the D1-like receptor antagonist R(+)-SCH 23390 (estimated dissociation constant KB = 1.7 microM) but not by the D2-like receptor antagonist S(-)-eticlopride (10 microM). 5. Forskolin (10 microM) reduced evoked EPSCs to approximately 60% of the control amplitude, and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D1-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

Muscarinic inhibition of glutamatergic transmissions onto rat magnocellular basal forebrain neurons in a thin-slice preparation.

We have examined excitatory and inhibitory transmission in visually identified rat magnocellular basal forebrain neurons using whole-cell patch-clamp recordings in a thin-slice preparation of the rat brain. In most cells, spontaneous excitatory and inhibitory synaptic activities could be recorded from their resting membrane potential. Following focal stimulation within the basal forebrain nucleus or directly onto visualized neighbouring neurons, postsynaptic currents were elicited in magnocellular basal forebrain cells held at -70 mb (a value close to their resting membrane potential). The synaptic responses were complex, consisting either mainly of excitatory postsynaptic currents (EPSCs), or inhibitory postsynaptic currents (IPSCs), or an EPSC-IPSC sequence. The EPSC component was consistent with the activation of AMPA/KA receptors, as it could be selectively blocked by CNQX. The IPSC component resulted in the activation of GAGAA receptors, and could be blocked by bicuculline. Since GABA-mediated trasmissions were not frequently recorded, we focused on the glutamate-mediated transmission. Studies using specific calcium channel blockers suggested that both omega-conotoxin GVIA-sensitive and omega-agatoxin VIA-sensitive calcium channels contribute to the glutamatergic transmission onto magnocellular basal forebrain neurons. Carbachol (0.3-30 microM) had no observable effect on holding current, but produced a dose-dependent inhibition of the amplitude of evoked EPSCs. This cholinergic modulation was mediated by muscarinic receptors, as it could be antagonized by atropine. The inhibitory effect of carbachol on the amplitude of EPSCs could be significantly antagonized by 100 nM methoctramine, an M2-receptor antagonist. In contrast, only a small degree of antagonism could be obtained with pirenzepine, and M1-muscarinic receptor antagonist, when present at relatively high concentration of 1 microM. Moreover, the action of carbachol was presynaptic, since the frequency of miniature postsynaptic currents was reduced without affecting their amplitude. In conclusion, the present findings indicate that glutamate-mediated transmission onto magnocellular basal forebrain neurons appeared to involve both N- and P/Q-type calcium channels, and that muscarinic modulation of glutamatergic transmission to MBF neurons is mediated by a presynaptic M2-muscarinic receptor subtypes.

Detection and modulation of acetylcholine release from neurites of rat basal forebrain cells in culture.

1. Nicotinic acetylcholine (ACh) receptor-rich patches prepared from rat myotubes were used as focal ACh detectors to record the release of ACh from magnocellular basal forebrain (MBF) neurones from 11- to 14-day-old postnatal rats maintained in dissociated cell culture. 2. An action potential generated by intracellularly stimulating the MBF cell soma through a patch electrode induced a brief (mean tau(decay), 6.3 ms) short latency (1.35-5.1 ms; median 3.1 ms) burst of nicotinic channel openings in the detector patch when the latter was positioned at discrete loci along the MBF neurites. Detected ACh concentrations ranged from approximately 480 nM to > 50 microM. Concentrations increased markedly during the first 14 days in vitro and were inversely related to response latency. 3. Sites of release were generally confined to the more proximal neurites within 100 microm of the cell body and were invariably associated with the presence of small (2-3 microm diameter) phase-dark puncta located at discrete intervals along the length of the neurites or at points where short collaterals branched from the main process. Release was never detected from the cell soma except under extreme non-physiological conditions but could occasionally be elicited from growth cones at the ends of the shorter thicker neurites in the absence of a target cell. 4. Evoked release was abolished by tetrodotoxin (0.5 microM) and by superfusing with low Ca(2+)-high Mg(2+)-containing solutions (0.25 mM Ca(2+), 5 mM Mg(2+)). Myotube patch responses were antagonized by d-tubocurarine (3 microM). 5. Muscarine (10 microM) inhibited release by 70 +/- 3% (n = 12 cells). This effect was antagonized by 100 nM methoctramine but not by 100 nM pirenzepine, indicating that it was mediated by M(2) muscarinic ACh receptors. 6. These results indicate that ACh release from the processes of magnocellular cholinergic basal forebrain neurones arises from highly specialized and discrete sites, and that it can be inhibited through activation of muscarinic receptors. It is suggested that the latter results from inhibition of presynaptic Ca(2+) channels and that it might be responsible for feedback autoinhibition of ACh release from cortical afferents of nucleus basalis neurones in vivo.

212 Presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones by dopamine

Dopamine (DA) D-1 and D-2 receptor agonists and antagonists were characterized in receptor binding and adenylate cyclase assays with respect to affinity, selectivity and efficacy. The ability of the ligands to interact with the discriminative stimulus effects of d-amphetamine (AMPH) was then assessed. The D-2 agonists, quinpirole, pergolide and CH 29-717, substituted completely for AMPH while neither partial (SKF 38393 and SKF 75670) nor full D-1 receptor agonists (SKF 89626 and SKF 81297) substituted. On the other hand, the selective D-1 and D-2 antagonists all blocked AMPH. The substitution for AMPH by pergolide was blocked by raclopride but not by SCH 23390, indicating D-2 mediation. In contrast, the motor effects of pergolide were blocked by both raclopride and SCH 23390, indicating mixed D-1/D-2 receptor involvement. These results suggest that D-1 and D-2 are equally involved in the expression of functional effects in the DAergic motor systems. Conversely, D-2 receptors may play a primary role in the DA systems involved in the AMPH cue; furthermore, the D-1 and D-2 receptors in these systems are relatively uncoupled.

Differential Oscillatory Properties of Cholinergic and Non‐cholinergic Nucleus Basalis Neurons in Guinea Pig Brain Slice

Evidence has suggested that the nucleus basalis magnocellularis has the potential to influence the functional state of the cerebral cortex through topographically organized, widespread projections of the cholinergic cells in that nucleus. It has also been shown that, in addition to the cholinergic neurons, other non-cholinergic magnocellular basal forebrain neurons, some of which have been identified as gamma-aminobutyric acid-ergic, project into the cerebral cortex and thus may also participate in the modulation of its activity. We have performed a comparative study of the intrinsic rhythmic properties of immunohistochemically and morphologically characterized choline acetyltransferase (ChAT)-positive and ChAT-negative cells of the nucleus basalis by means of intracellular recordings in guinea pig brain slices. Our results demonstrate that relatively large, multipolar cholinergic and non-cholinergic neurons each display differential voltage-dependent properties that allow them to discharge rhythmically in spike bursts and spike clusters, respectively, at low frequencies (< 10 Hz). Cholinergic cells display bursts of 2-4 action potentials (at approximately 200 Hz) riding on low-threshold spikes recurring at a low frequency (< 5 Hz) when depolarized from a membrane potential more negative than -55 mV and display low-frequency (< 10-15 Hz) tonic firing when depolarized from a more positive level. In contrast, non-cholinergic cells fire in a unique mode, displaying non-adapting clusters of spikes interspersed with rhythmic subthreshold membrane-potential oscillations when depolarized from levels less negative than -55 mV. The spike clusters repeat rhythmically at relatively low frequencies (2-10 Hz). The intracluster spiking frequency is relatively high and coincides approximately with that of the intervening membrane-potential oscillations (approximately 20-70 Hz). The cluster frequency of the non-cholinergic cells corresponds, in the same manner as the burst frequency of the cholinergic cells, to a delta (1-4 Hz) or theta (4-10 Hz) range of activity, whereas the intra-cluster and tonic spike frequencies of the non-cholinergic cells correspond to high beta to gamma ranges of electroencephalographic activity (19-30 Hz and 30-60 Hz, respectively). We propose that the different modes of oscillatory firing by the cholinergic and non-cholinergic basal forebrain cell populations could collectively contribute to the rhythmic modulation of slow and fast rhythms within the cerebral cortex.

Nerve growth factor-like immunoreactive profiles in the primate basal forebrain and hippocampal formation.

The distribution of nerve growth factor (NGF), the prototypic neurotropin, within the basal forebrain and hippocampal formation of young adult monkeys and aged humans was characterized with an affinity purified polyclonal beta-NGF antibody raised against mouse beta-NGF. In the basal forebrain of both primates, a granular NGF-like immunoreactive (ir) reaction product was observed within neurons of the medial septum, nucleus of the diagonal band, and nucleus basalis of Meynert. NGF-like immunoreactivity exclusively colocalized within p75 NGF receptor (NGFR) containing basal forebrain neurons. The intensity of NGF immunolabeling varied between cell bodies. Many NGF-ir perikarya were highly immunoreactive. In other basal forebrain neurons, NGF-like immunoreactivity was either undetectable or minimally expressed. In the hippocampus of both species, NGF-like immunoreactivity was mainly localized within the hilus of the dentate gyrus and within CA3 and CA2 hippocampal subfields. A marked diminution in NGF-like staining was seen in CA1. Within the hippocampal formation, NGF-like immunoreactivity was heaviest within the neuropil of stratum radiatum, intermediate in stratum oriens, and lightest in stratum pyramidal. NGF-like immunoreactivity was not found within the granule or pyramidal cells of the dentate gyrus and hippocampal formation, respectively. These findings demonstrate the presence of an NGF-like antigen in association with monkey and human magnocellular basal forebrain neurons and within their hippocampal target sites. This lends support to the hypothesis that NGF is internalized from sources located within target regions of the primate cholinergic basal forebrain neurons and is retrogradely transported to these cell bodies where the NGF trophic effect likely occurs.

Evidence for presynaptic inhibition of the olfactory commissural pathway by cholinergic agonists and stimulation of the nucleus of the diagonal band

We have investigated the role of the projection from the magnocellular basal forebrain to the olfactory bulb in regulating synaptic transmission in the commissural connection between the two olfactory bulbs. Commissural fibers arise in the contralateral anterior olfactory nucleus, travel in the anterior wing of the anterior commissure (AC), and terminate in the granule cell layer of the olfactory bulb. Electrical stimulation of the commissure causes synaptic activation of granule cells in the granule cell layer of the bulb; the resulting field potential is a reliable indicator of this synaptic current. Microinjections of cholinergic agonists, but not of identical, or larger, quantities of vehicle, reduced the amplitude of this AC field potential. Systemic injection of scopolamine reversed this depression and returned the AC response amplitude to control levels. Irreversible AChE inhibition also reduced the amplitude of the AC response, and muscarinic blockade reversed this effect. Cholinergic terminals in the olfactory bulb arise entirely from the axons of magnocellular basal forebrain neurons in the nucleus of the diagonal band (NDB). Electrical stimulation of NDB, which should release ACh, as well as other transmitters, depressed the AC response. Brief trains of NDB shocks caused a moderate decrease in the AC response that lasted 1-2 sec. Longer shock trains, which caused marked potentiation of the NDB field potential, caused a profound, prolonged (> 20 sec) inhibition of the AC response. Antidromic tests demonstrated that NDB stimulation significantly decreased the excitability of AC terminals. This and other characteristics of the inhibition strongly suggest that the decrease in amplitude of the field potential response to AC stimulation caused by cholinergic agonists and stimulation of NDB is due to presynaptic inhibition leading to reduced release of transmitter from AC terminals. These results suggest that one function of the basal forebrain projection to the olfactory bulb is inhibition of the commissural connection between the two olfactory bulbs. As NDB has been implicated in theta pacemaker input to the olfactory bulb, phasic NDB inhibition of centrifugal afferents to the bulb could function to coordinate signal processing temporally in the olfactory system. Temporal coordination may be particularly important to olfactory circuit function, as this system lacks the point-to-point topographical organization characteristic of other sensory systems.

Galanin and NADPH-diaphorase coexistence in cholinergic neurons of the rat basal forebrain

The distribution of the enzyme NADPH-diaphorase in the rat basal forebrain was examined in relation to the neuropeptide galanin and the neurotransmitter synthetic enzyme choline acetyltransferase. Immunoperoxidase staining permitted camera lucida mapping of galanin and choline acetyltransferase distributions in serial sections through the basal forebrain for comparison with adjacent sections prepared for NADPH-diaphorase histochemistry. Photographs of sections subjected to indirect immunofluorescence for galanin and choline acetyltransferase were compared to photographs of the same sections taken after NADPH-diaphorase histochemistry. This permitted the direct investigation of co-localization within the cholinergic basal forebrain. The distributions of choline acetyltransferase- and galanin-immunoreactive neurons in the basal forebrain agreed with previous descriptions. NADPH-diaphorase histochemistry selectively stained a population of magnocellular basal forebrain neurons with a distribution similar to that observed with galanin immunohistochemistry. Double and triple staining experiments indicated that NADPH-diaphorase labels a majority of the magnocellular cholinergic neurons in the medial septum and diagonal band nuclei. Most of these neurons also contain galanin immunoreactivity. However, small populations of galanin-positive/diaphorase-negative or diaphorase-positive/galanin-negative cholinergic neurons were also observed. In the more caudal portions of the cholinergic basal forebrain, very few galanin or NADPH-diaphorase-positive neurons were observed. Thus, galanin and NADPH-diaphorase coexist in the majority of cholinergic basal forebrain neurons in the regions innervating limbic structures. The neocortically projecting cholinergic cells in the caudal basal forebrain appear to lack these other neurochemical markers.

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: Response to fimbria‐fornix transection

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.

Galanin-like immunoreactivity is present in human substantia innominata and in senile plaques in Alzheimer's disease

Galanin immunoreactivity has been shown to be present in cholinergic magnocellular basal forebrain neurons in both rat and monkey brain. In the present study galanin immunoreactivity in human substantia innominata was found in both the magnocellular neurons of the nucleus basalis of Meynert and in a dorsomedial group of intensely immunoreactive parvocellular neurons. Scattered galanin-immunoreactive fibers, but no immunoreactive neurons, were found in normal human cerebral cortex. Galanin-immunoreactive neurites were found in a small number of senile plaques in the hippocampus and temporal isocortex of Alzheimer's disease patients. The existence of galanin immunoreactivity in human magnocellular basal forebrain neurons which are depleted in Alzheimer's disease suggests that galanin may be similarly affected.

Neurophysiology of magnocellular forebrain inputs to the olfactory bulb in the rat: frequency potentiation of field potentials and inhibition of output neurons

Basal forebrain nuclei send projections, including cholinergic fibers, to forebrain cortical targets. These systems have been associated with several important functions, but their physiological actions are poorly understood. We have studied the neurophysiological characteristics of one of these systems, the projection from the nucleus of the horizontal limb of the diagonal band (HDB) to the main olfactory bulb (MOB) in the rat. Single shocks to HDB produce modest field potentials in MOB with no detectable effect on the discharge characteristics of the principal output neurons of the MOB, the mitral cells. By contrast, continuous stimulation at 10 Hz for several seconds causes dramatic changes in the HDB field potential and mitral cell firing. During this period of stimulation, there is an initial facilitation of the field potential followed by a period of moderately reduced response amplitude that lasts a few seconds. This brief period of depression is succeeded by a sudden and marked potentiation of response amplitude and duration. This potentiated response can be maintained indefinitely by stimulation at lower frequencies than those required to initiate the potentiation effect. Coincident with the onset of the potentiated response, the spontaneous activity of the mitral cells is completely inhibited. Both the potentiation and mitral cell inhibition can be maintained indefinitely by continued stimulation at frequencies as low as 6 Hz. These observations demonstrate that magnocellular basal forebrain neurons exert powerful regulatory actions on specific neuronal populations in cortical targets.