Diagonal Band Of Broca Neurons Research Articles

Amyloid β Protein Modulates Glutamate-Mediated Neurotransmission in the Rat Basal Forebrain: Involvement of Presynaptic Neuronal Nicotinic Acetylcholine and Metabotropic Glutamate Receptors

Amyloid beta (Abeta) protein, a 39-43 amino acid peptide deposited in brains of individuals with Alzheimer's disease (AD), has been shown to interact directly with a number of receptor targets including neuronal nicotinic acetylcholine receptors (nAChRs) and glutamate receptors. In this study, we investigated the synaptic effects of Abeta(1-42) on glutamate-mediated neurotransmission in the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Glutamatergic miniature EPSCs (mEPSCs) were recorded using whole-cell patch-clamp recordings from identified cholinergic DBB neurons in rat forebrain slices. In 54% of DBB neurons, bath application of Abeta(1-42) (100 nM), but not Abeta(42-1) (inverse fragment), significantly increased the frequency of mEPSCs without affecting amplitude or kinetic parameters (rise or decay time). In 32% of DBB neurons, bath application of Abeta(1-42) significantly decreased only the frequency but not amplitude of mEPSCs. Application of dihydro-beta-erythroidine (DHbetaE) (an antagonist for the alpha4beta2 subtype of nAChRs) but not alpha-bungarotoxin (an antagonist for the alpha7 subtype of nAChRs) blocked Abeta(1-42)-mediated increases in mEPSC frequency. The Abeta(1-42)-mediated increase in glutamatergic transmission is thus presynaptic and mediated via non-alpha7 AChRs. In contrast, Abeta(1-42)-mediated decreases in mEPSC frequency could not be antagonized by either DHbetaE or alpha-bungarotoxin. However, the Abeta(1-42)-evoked depression in mEPSC frequency was antagonized by (RS)-alpha-methyl-4-carboxyphenyglycine, a nonselective group I/II metabotropic glutamate receptor antagonist. These observations provide further insight into the mechanisms whereby Abeta affects synaptic function in the brain and may be relevant in the context of synaptic failure observed in AD.

Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.

Human amylin (hAmylin), a 37-amino acid pancreatic peptide, and amyloid beta protein (A beta), a 39-43 amino acid peptide, abundantly deposited in the brains of Alzheimer's patients, induce neurotoxicity in hippocampal and cortical cultures. Although the mechanism of this neurotoxicity is unknown, both peptides are capable of modulating ion channel function that may result in a disruption of cellular homeostasis. In this study, we examined the effects of hAmylin on whole cell currents in chemically identified neurons from the rat basal forebrain and the interactions of hAmylin-induced responses with those of A beta. Whole cell patch-clamp recordings were performed on enzymatically dissociated neurons of the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Bath application of hAmylin (1 nM to 5 microM) resulted in a dose-dependent reduction in whole cell currents in a voltage range between -30 and +30 mV. Single-cell RT-PCR analysis reveal that all DBB neurons responding to hAmylin or A beta were cholinergic. Using specific ion channel blockers, we identified hAmylin and A beta effects on whole cell currents to be mediated, in part, by calcium-dependent conductances. Human amylin also depressed the transient outward (IA) and the delayed rectifier (IK) potassium currents. The hAmylin effects on whole cell currents could be occluded by A beta and vice versa. Human amylin and A beta responses could be blocked with AC187 (50 nM to 1 microM), a specific antagonist for the amylin receptor. The present study indicates that hAmylin, like A beta, is capable of modulating ion channel function in cholinergic basal forebrain neurons. Furthermore, the two peptides may share a common mechanism of action. The ability of an amylin antagonist to block the responses evoked by hAmylin and A beta may provide a novel therapeutic approach for Alzheimer's disease.

Nociceptin/orphanin FQ modulation of ionic conductances in rat basal forebrain neurons.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous opioid-like heptadecapeptide that plays an important role in a variety of physiological functions. N/OFQ and its receptor opioid receptor-like orphan receptor-1 are abundant in the diagonal band of Broca (DBB), a basal forebrain nucleus where the loss of cholinergic neurons is linked to memory and spatial learning deficits. In the whole animal, central injections of N/OFQ have been shown to disrupt spatial learning. In this study, we investigated the basis for these behavioral observations by examining the cellular effects of N/OFQ on chemically identified DBB neurons. Whole cell patch-clamp recordings were performed on enzymatically dissociated DBB neurons. Under voltage-clamp conditions, bath application of N/OFQ (10 pM-1 microM) resulted in a dose-dependent depression of whole cell currents. Single cell reverse transcription-polymerase chain reaction analysis identified cholinergic and fewer GABAergic cells to be N/OFQ-responsive. [Nphe(1)]nociceptin-(1-13)-NH(2) and CompB (J-113397) antagonized the N/OFQ response, but both compounds also displayed partial agonist activity. Using a combination of channel blockers we determined that the effects of N/OFQ were mediated via a suite of Ca(2+) (N- and L-type) and Ca(2+)-dependent K(+) (iberiotoxin-sensitive) conductances. In addition, biophysical analysis of voltage subtraction protocols revealed that N/OFQ reduces transient outward and the delayed rectifier K(+) currents. Because N-type and L-type Ca(2+) channels are important in the context of neurotransmitter release, our observations indicate that N/OFQ inhibition of Ca(2+)-dependent conductances in cholinergic neurons would be expected to result in depression of acetylcholine release, which may explain the behavioral actions of N/OFQ in the brain.

Novel excitatory actions of galanin on rat cholinergic basal forebrain neurons: implications for its role in Alzheimer's disease.

Galanin, a 29-amino-acid neuropeptide, is generally viewed as an inhibitory neuromodulator in a variety of central systems. Galanin expression is upregulated in the cholinergic basal forebrain nuclei in Alzheimer's disease (AD) and is postulated to play an important role in memory and cognitive function. In this study, application of galanin to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. Galanin reduces a suite of potassium currents, including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, application of galanin evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that galanin actions were specific to cholinergic, but not GABAergic DBB neurons The notion that galanin plays a deleterious role in AD is based, in part, on galanin hyperinnervation of cholinergic cells in the basal forebrain of AD patients, its ability to depress acetylcholine release and its inhibitory actions at other CNS sites. However, our results suggest that by virtue of its excitatory actions on cholinergic neurons, galanin may in fact play a compensatory role by augmenting the release of acetylcholine from remaining cholinergic basal forebrain neurons. This action might serve to delay the progression of AD pathology linked to a reduction in central cholinergic tone.

Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons.

The deposition of amyloid beta-protein (Abeta) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta(25-35) or Abeta(1-40) to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology.

Baroreceptor sensitivity of rat supraoptic vasopressin neurons involves noncholinergic neurons in the DBB.

Previous studies suggest that cholinergic neurons in the diagonal band of Broca (DBB) participate in the baroreceptor-mediated inhibition of phasic vasopressin neurons in the supraoptic nucleus (SON). To test this hypothesis, extracellular recordings were obtained from putative vasopressin SON neurons of anesthetized rats injected with the cholinergic immunotoxin 192 IgG-saporin (0.8 microg/microl) in the DBB. Baroreceptor sensitivity of neurons was tested with brief phenylephrine-induced (10 microg/10 microl iv) increases in blood pressure of at least 40 mmHg. In rats injected with vehicle or unconjugated saporin, 19 of 21 and 18 of 20 phasic neurons, respectively, were inhibited by increased blood pressure. In rats injected with 192 IgG-saporin, which significantly reduced the number of choline acetyltransferase (ChAT)-positive DBB neurons, 33 of 36 phasic neurons were inhibited. Normal rats and rats with DBB saporin injections received rhodamine bead injections into the perinuclear zone (PNZ) to retrogradely label DBB neurons, and their brains were stained for ChAT. ChAT-positive DBB neurons were not retrogradely labeled from the PNZ. Together, these results indicate that the pathway relaying baroreceptor information to the SON involves noncholinergic DBB neurons.

Growth cones exhibit enhanced cell–cell adhesion after neurotransmitter release

Evoked release of acetylcholine and subsequent cell–cell adhesive contacts between growth cones and acetylcholine sensing neurons were observed using cultured neurons dissociated from the diagonal band of Broca of the rat. Stimulation to the soma of the diagonal band of Broca neurons evoked release of acetylcholine from the growth cones. The release of acetylcholine was monitored using whole-cell patch-clamp recording from acetylcholine receptor-rich superior cervical ganglion neuron positioned on the growth cone as a sensor of acetylcholine release. By measuring changes in fluorescence from the growth cone using Ca 2+-sensitive dye while voltage-clamping the superior cervical ganglion neuron, transient intracellular Ca 2+ concentration increase and acetylcholine release from growth cone were recorded simultaneously. Video-enhanced differential interference contrast imaging of the growth cones demonstrated tether formation between the growth cone and superior cervical ganglion cell soma when the superior cervical ganglion cell soma was moved away from the growth cone after acetylcholine release, suggesting formation of adhesive contacts between the growth cone and the sensor neuron. Adhesive contacts between growth cones and sensor neurons were also detected when a high K + solution or α-latrotoxin was applied to the growth cone. Adhesions were also observed between growth cones and latex beads, when growth cones were exposed to high K + solution. The properties of the adhesive contacts at the growth cone were studied by optically manipulating a latex bead attached to the growth cone surface. These results suggest that growth cones exhibit cell–cell adhesion after neurotransmitter release.

GABAA receptor modulation by protein tyrosine kinase in the rat diagonal band of Broca

The diagonal band of Broca (DBB) is involved in a wide array of physiological functions which are, in part, mediated by activation of GABAA receptors. DBB is enriched in GABA and protein tyrosine kinase (PTK) immunoreactivity. Whole-cell patch-clamp recording were performed from acutely dissociated DBB neurons to investigate the involvement of PTK in GABAA receptor function. The activation of GABAA receptor by the selective agonist, muscimol (5 μM) was dependent on the presence of intracellular ATP. Omission of ATP in the intracellular medium resulted in a fast decrement of the response whereas inclusion of sodium orthovanadate (100 μM), a non-specific phosphatase inhibitor, augmented the response and inhibited `run down' of the response. Genistein (100 μM) and tyrphostin B-44 (–), specific inhibitors of PTK, attenuated the response to muscimol. The muscimol response was not affected by daidzein (100 μM; an inactive analogue of genistein) nor by tetraethylammonium bromide (1 mM). These observations suggest that phosphorylation is important for the activation and long term maintenance of GABAA receptor function. PTK phosphorylation, which has been previously identified as an important event in signal transduction, may modulate GABA mediated neurotransmission in the forebrain.

Vasopressin receptor subtypes differentially modulate calcium-activated potassium currents in the horizontal limb of the diagonal band of Broca

The actions of vasopressin on acutely dissociated neurons within the rat horizontal limb of the diagonal band of Broca were examined using the whole-cell patch-clamp technique. Vasopressin elicited two distinct responses in 45 of 62 neurons. In one group of cells, 300 nM vasopressin decreased voltage-activated outward currents (26/45 cells) whereas in a second group, vasopressin increased outward currents (19/45 cells). The vasopressin-mediated decrease in outward currents was blocked by 1 μM Manning compound, a V 1 receptor antagonist, suggesting that this response was mediated via V 1 receptors. In contrast, the vasopressin-induced increase in outward current was blocked by 1 μM d(CH 2) 5) 1,D-Ile 2,Ile 4,Arg 8,Ala 9, a V 2 receptor antagonist, indicating that V 2 receptor activation underlies this second response. When cells were perfused with 0 Ca 2+/50 μM Cd 2+, application of vasopressin did not cause any change in voltage-activated outward currents, suggesting that vasopressin modulates a calcium-dependent conductance. In the presence of 25 nM charybdotoxin, an I C channel antagonist, vasopressin application did not influence outward currents, indicating that vasopressin modulates I C. Currents through voltage-gated calcium channels which are responsible for activation of I C were unaffected by vasopressin, suggesting a direct effect of vasopressin, on I C channels. These observations indicate a differential modulation of I C channels by vasopressin via V 1 and V 2 receptors in the horizontal limb of the diagonal band of Broca. Our data also demonstrate the ionic mechanisms whereby vasopressin may act at V 1 or V 2 receptors to influence the excitability of the horizontal limb of the diagonal band of Broca neurons.

Synthesis, storage and release of acetylcholine at and from growth cones of rat central cholinergic neurons in culture

Neurons from the nucleus diagonal band of Broca (DBB) from new born rats protrude neuronal processes and growth cones in culture. Cytochemical observations with the light and electron microscope indicate that growth cones of these neurons take up choline, synthesize acetylcholine (ACh) and store ACh in the vesicles. Electrical stimuli at the soma of DBB neurons evoked inward currents in ACh-sensitive neurons attached to DBB growth cones. These currents were suppressed by TTX, a Ca 2+ channel blocker (Cd 2+), and an ACh nicotinic antagonist (C 6). These results suggest that ACh is synthesized, stored and released from the growth cones of DBB neurons prior to synapse formation.

In vivo intracellular recordings of medial septal and diagonal band of Broca neurons: relationships with theta rhythm.

Transmembrane potentials from medial septal and diagonal band of Broca (MS-DBB) neurons and hippocampal field activity were recorded in curarized and urethanized rats. MS-DBB cells were studied during large amplitude irregular activity and during hippocampal theta rhythm, elicited by either sensory (i.e. stroking the fur on the animal's back) or electrical stimulation of the reticularis pontis oralis nucleus (RPO). Three types of cells were described according to their firing pattern and the characteristics of their "intracellular theta" rhythm. Type A neurons displayed continuous rhythmic oscillations in the membrane potential (Vm) of approximately 17 mV. These oscillations generated rhythmic high-frequency spike trains which were phase-locked with hippocampal theta rhythm. Type A cells revealed intracellular theta rhythm even in the absence of hippocampal theta rhythm, suggesting that the activity of this type of cell was the most important in hippocampal theta genesis. Type B cells were characterized by marked postspike after hyperpolarization and intracellular theta oscillations of smaller amplitude than in type A cells. Type C cells revealed a postspike afterdepolarization and a lower amplitude, intracellular theta rhythm only in the presence of hippocampal theta rhythm. Type C neurons could fire slow spikes at depolarizing (46% of cells) or hyperpolarizing (15% of cells) Vms. Type B and C cells were intracellularly stained with Lucifer yellow. Although type B and C neurons revealed dissimilar electrophysiological properties, they had comparable morphological shapes. RPO electrical stimulation generated hippocampal theta rhythm and intracellular theta rhythm in types A and B cells but not in type C cells, and increased the spike rate in type C neurons. Electrical stimulation of the fornix only evoked synaptic responses in type B and C neurons, with antidromic responses being elicited in 12% of type C cells. These results indicate that probably most of the type A rhythmic cells did not receive direct hippocampal feedback and that at least some type C cells were projecting neurons. The present findings demonstrate that theta rhythm oscillations in the Vm of MS-DBB neurons elicit different rhythmic discharge patterns.

Axotomized medial septal-diagonal band neurons express Jun-like immunoreactivity

The expression of the transcription factors Fos and Jun was studied in rat brain after transection of the fornix-fimbria (FF) using polyclonal antibodies to these proteins and immunocytochemical detection methods. FF-transection lead to a massive induction of Jun-like immunoreactivity (JLI) in neurons in the medial septal nucleus and in the vertical limb of the diagonal band of Broca, within 48 hours and lasting up to 14 days after lesion. Fos was not induced in these neurons after FF-transection. These results indicate that axotomized medial septal and diagonal band of Broca neurons selectively and rapidly express JLI. The role of Jun expression in axonal regeneration or neuronal death is discussed.

Ibotenate Lesions of the Diagonal Band of Broca Attenuate Baroreceptor Sensitivity of Rat Supraoptic Vasopressin Neurons

Previous electrophysiological studies in the rat suggest that neurons in the diagonal band of Broca participate in baroreceptor-induced suppression of the spontaneous activity of vasopressin-secreting neurons in the hypothalamic supraoptic nucleus. In order to test this hypothesis, extracellular recordings were obtained from phasically-active vasopressin neurons in the supraoptic nucleus of anesthetized rats injected at least 3 days previously with ibotenic acid (1.25 μg/250 nl) in the diagonal band of Broca, the medial and lateral septum, or the median preoptic nucleus. In normal rats, brief increases in blood pressure produced by injections of metaraminol (10 μg/10 μl iv) that were sufficient to activate peripheral baroreceptors, suppressed the activity of a majority (21 tested, 19 suppressed) of phasically-active vasopressin-secreting neurons. In rats with ibotenic acid lesions of the diagonal band of Broca, the number of phasically-active neurons that were baroreceptor-sensitive was significantly reduced (21 tested, 8 suppressed) while lesions of the medial and lateral septum (17 tested, 16 suppressed) or the median preoptic nucleus (21 tested, 20 suppressed) had no effect. The results support the hypothesis that diagonal band of Broca neurons participate in a central pathway mediating the inhibitory effects of peripheral baroreceptor stimulation on the activity of vasopressin-secreting neurons in the rat supraoptic nucleus.

Cardiovascular input to hypothalamic neurosecretory neurons

In vivo extracellular recordings from rat supraoptic and paraventricular magnocellular neurosecretory cells (MNCs) indicate that putative vasopressin-secreting MNCs may be identified by an abrupt and brief cessation in firing consequent to a transient drug-induced rise in arterial pressure sufficient to activate arterial baroreceptors. In the diagonal band of Broca (DBB), a population of neurons projecting towards the supraoptic nucleus are activated during this drug-induced hypertension. Electrical stimulation in DBB selectively depresses supraoptic vasopressin-secreting MNCs. Intracellular recordings in perfused hypothalamic expiants confirm a DBB-evoked bicuculline-sensitive and chloride-dependent postsynaptic inhibition, similar to that associated with the application of γ-aminobutyric acid (GABA) in approximately half of supraoptic MNCs. Since bicuculline also selectively blocks baroreceptor-induced inhibition in supraoptic MNCs, it is proposed that the depressant baroreflex input to vasopressin-secreting MNCs involves a population of DBB neurons and GABAergic interneurons located close to MNCs. An excitatory and selective input to vasopressin-secreting MNCs follows chemoreceptor activation, possibly mediated by the A1 noradrenergic cell group in the ventrolateral medulla. Another excitatory input to both vasopressin- and oxytocin-secreting MNCs is triggered by circulating angiotensin II and appears to be relayed centrally through an angiotensinergic projection from the subfornical organ.

Contrasting properties of medial septal neurons projecting to hippocampus or interpeduncular nucleus in the rat

The properties of neurons of the medial septal nucleus and of the nucleus of the diagonal band of Broca which project to hippocampus or to interpeduncular nucleus were compared in rats anesthetized with urethane. Neurons projecting to the interpeduncular nucleus had a slower conduction velocity and a lower spontaneous discharge rate. In contrast, their responses to various putative neurotransmitters (glutamate, GABA, acetylcholine) were similar. In a few cases, neurons projecting to both structures (i.e., with branched axons) were observed. Both septohippocampal and septointerpeduncular pathways are known to be partly cholinergic. Our results show that they originate from two independent populations of medial septal-nucleus of the diagonal band of Broca neurons with different physiologic properties.

Diagonal band neurons may mediate arterial baroreceptor input to hypothalamic vasopressin-secreting neurons

Diagonal band of Broca (DBB) neurons projecting to the hypothalamic supraoptic neurosecretory neurons were antidromically identified in pentobarbital-anesthetized male Long-Evans rats. Six of 12 DBB neurons tested for response to baroreceptor activation showed a consistent increase in firing. We propose that the established baroreceptor-induced inhibition of hypothalamic vasopressinergic neurons is mediated through DBB neurons.

Effects of intraventricular norepinephrine on preoptic-anterior hypothalamic electrical activity in the freely-moving rat: modulation by ovarian steroid hormones

In freely-moving female rats the effects of intraventricular infusion of norepinephrine (NE) on multiunit activity (MUA) were examined in the Diagonal Band of Broca (DBB), medial preoptic area (MPOA) and anterior hypothalamic area (AHA), regions containing neurons producing luteinizing hormone-releasing hormone (LHRH). NE was infused at a dosage known to depress plasma LH levels in ovariectomized (OVX) rats and elevated plasma LH concentrations in OVX-estrogen-progesterone-primed (EBP) animals. It was found that in adult OVX rats MUA in the brain areas listed above was almost invariably inhibited by NE (20 of 23 cases or 87%; the other three cases showed no change in electrical activity). However, after priming with estrogen and progesterone only about one-third of the OVX-EBP rats gave an inhibitory response, with another one-third showing no change in MUA and the final one-third of the cases actually giving an excitatory MUA response to NE — the DBB neurons being the most positive in this regard. Thus it appears that responsiveness of LHRH and/or adjacent neurons to the modulatory action of NE may itself be modulated by the influence of gonadal steroids.