Intracellular responses of olfactory bulb granule cells to stimulating the horizontal diagonal band nucleus.

The effects of centrifugal afferents on membrane potentials of identified granule cell layer using evoked field potential profiles, and trans-synaptic activation via antidromic stimulation of output cell axon collaterals. Intracellular recordings maintained for 4-30 min showed complex spontaneous spike discharges and allowed characterization of the cell's input resistance, and on some occasions its morphology following intracellular injection of Lucifer Yellow. Stimulation in the nucleus of the horizontal limb of the diagonal band, but not surrounding regions, produced hyperpolarizing responses in 13 of 27 cells in the granule cell layer; four of these were morphologically identified as granule cells of two types, in five the responses had reversal potentials more negative than the resting potential, and six were identified as granule cells by monosynaptic activation from output axon collaterals. A different set of three cells in the granule cell layer responded with depolarization. The results are consistent with the inhibition of tonic activity of granule cells by the nucleus of the horizontal limb of the diagonal band, leading to disinhibition of mitral and tufted cells via dendrodendritic synapses of granule cells on mitral/tufted cell secondary dendrites.     

Projections of medullary and pontine noradrenergic neurons to the horizontal limb of the nucleus of diagonal band in the rat.

Recent investigations in the rat have implicated a noradrenergic innervation to the horizontal nucleus of the diagonal band of Broca as a critical link in a neural circuit that conveys baroreceptor information centrally to inhibit the firing of vasopressin-secreting neurons in the hypothalamic supraoptic nucleus. In this study we used small intra-diagonal band injections of a retrograde tracer, rhodamine latex microspheres, in combination with tyrosine hydroxylase histochemistry to identify brainstem noradrenergic cells contributing to this innervation. In three cases where tracer injections were limited to the horizontal limb of the diagonal band, we observed 20-50 double-labelled neurons ipsilaterally in the dorsal part of the locus coeruleus (A6) and the caudal nucleus tractus solitarius (A2), and bilaterally in the caudal ventrolateral medulla (A1). Double-labelled neurons were also noted in the ventral tegmental area (dopaminergic A10 cell group). Although all major brainstem noradrenergic cell groups contribute fibers to the horizontal limb of the nucleus of diagonal band, data from physiological studies suggest that the noradrenergic A2 neurons in the nucleus tractus solitarius are the most likely pathway through which it receives this baroreceptor information.     

Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca

We examined modulation of ionic currents by Zn2+ in acutely dissociated neurons from the rat's horizontal limb of the diagonal band of Broca using the whole-cell patch-clamp technique. Application of 50 microM Zn2+ increased the peak amplitude of the transiently activated potassium current, I(A) (at + 30 mV), from 2.20+/-0.08 to 2.57+/-0.11 nA (n = 27). This response was reversible and could be repeated in 0 Ca2+/1 microM tetrodotoxin (n = 15). Zn2+ shifted the inactivation curve to the right, resulting in a shift in the half-inactivation voltage from 76.4+/-2.2 to -53.4+/-2.0 mV (n = 11), with no effect on the voltage dependence of activation gating (n = 15). There was no significant difference in the time to peak under control conditions (7.43+/-0.35 ms, n = 14) and in the presence of Zn2+ (8.20+/-0.57 ms, n = 14). Similarly, the time constant of decay of I(A) (tau(d)) at + 30 mV showed no difference (control: 38.68+/-3.68 ms, n = 15; Zn2+: 38.48+/-2.85 ms, n = 15). I(A) was blocked by 0.5-1 mM 4-aminopyridine. In contrast to its effects on I(A), Zn2+ reduced the amplitude of the delayed rectifier potassium current (I(K)). The reduction of outward K+ currents was reproducible when cells were perfused with 1 microM tetrodotoxin in a 0 Ca2+ external solution. The amplitude of the steady-state outward currents at +30 mV under these conditions was reduced from 6.40+/-0.23 (control) to 5.76+/-0.18 nA in the presence of Zn2+ (n = 16). The amplitudes of peak sodium currents (INa) were not significantly influenced (n = 10), whereas barium currents (I(Ba)) passing through calcium channels were potently modulated. Zn2+ reversibly reduced I(Ba) at -10 mV by approximately 85% from -2.06+/-0.14 nA under control conditions to -0.30+/-0.10 nA in the presence of Zn2+ (n = 14). Further analyses of Zn2+ effects on specific calcium channels reveals that it suppresses all types of high-voltage-activated Ca2+ currents. Under current-clamp conditions, application of Zn2+ resulted in an increase in excitability and loss of accommodation (n = 13), which appears to be mediated through its effects on Ca2+-dependent conductances.     

Electrical stimulation of the horizontal limb of the diagonal band of broca modulates population EPSPs in piriform cortex.

Electrical stimulation of the horizontal limb of the diagonal band of Broca (HDB) was coupled with recording of evoked potentials in the piriform cortex. Stimulation of the HDB caused an enhancement of the late, disynaptic component of the evoked potential elicited by stimulation of the lateral olfactory tract but caused a suppression of the synaptic potential elicited by stimulation of the posterior piriform cortex. The muscarinic antagonist scopolamine blocked both effects of HDB stimulation. The enhancement of disynaptic potentials could be due to cholinergic depolarization of pyramidal cells, whereas the suppression of potentials evoked by posterior piriform stimulation could be due to presynaptic inhibition of intrinsic fiber synaptic transmission by acetylcholine.     

GABA and Glutamate receptors in the horizontal limb of diagonal band of Broca (hDB): effects on cardiovascular regulation

The horizontal limb of diagonal band of Broca (hDB) is a part of the limbic system. It has been shown that microinjection of L-glutamate into the hDB elicited cardiovascular depressive responses in anesthetized rats and pressor effect in unanesthetized rats. But the role of glutamate receptor subtypes has not yet been investigated. In addition the role of the GABAergic system of the hDB in cardiovascular responses is not known. Therefore, we examined the cardiovascular responses elicited by glutamate and GABA receptors in the hDB by using their agonists and antagonists. Drugs (50 nl) were microinjected into the hDB of anaesthetized rats. Blood pressure and heart rate were recorded before and throughout each experiment. The average changes in the mean arterial pressure and heart rate at different intervals were compared both within each case group and between the case and control groups using repeated measures of ANOVA. Microinjection of GABA_A receptor antagonist, bicuculline methiodide (BMI, 1 mM) increased both the mean arterial pressure and heart rate, and muscimole, a GABA_A agonist (500 pmol) caused a significant decrease in the mean arterial pressure and heart rate. Microinjection of L-glutamate (0.25 M) into the hDB resulted in a maximum decrease of the mean arterial pressure of 24.4±3.7 mmHg and heart rate of 25.2±3.08 beats/min. Injection of AP5, an antagonist of glutamate NMDA receptor (1 and 2.5 mM), and CNQX, an antagonist of glutamate AMPA receptor (0.5 and 1 mM) caused small, nonsignificant changes of the heart rate and the blood pressure. Either AP5 or CNQX when coinjected with glutamate abolished the depressor effect of glutamate, suggesting that simultaneous activation of both glutamate receptors is necessary for the effect of glutamate to emerge. The depressor effect of the glutaminergic system of the hDB on the cardiovascular system was similar to the previous studies. For the first time, the effects of CNQX, AP5, BMI, and muscimole have been shown in this study.     

Respiratory patterning of the rat olfactory bulb unit activity: nasal versus tracheal breathing

To assess the role of nasal/tracheal (N/T) breathing in the respiratory patterning of the olfactory bulb (OB) neurons, the activity of 21 units was recorded in 6 anesthetized rats set with a cannula enabling reversible tracheotomy: the rats could inhalate either through nasal pathways or through trachea directly. Shift from tracheal to nasal breathing induced respiratory patterning in 7 units. The changes were steady, reversible and reproducible. The present data, matched with previous ones, indicate that tracheotomy and anesthesia decrease the occurrence of respiratory patterning in OB neurons. The experiments also suggest that peripheral as well as central structures may be a source of respiratory modulation in the olfactory bulb.     

Morphological characterization of cholinergic neurons in the horizontal limb of the diagonal band of Broca in the basal forebrain of the rat

Summary A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was used to identify cholinergic neurons in the nucleus of the horizontal limb of the diagonal band of Broca at the light and electron microscopic levels. ChAT-labelled somata were fusiform, triangular or round in shape and varied considerably in size. Depending on the type of the cell, one to four dendrites emerged from the soma, but an axon could rarely be seen. The nuclei of most cells were round or oval, showed invaginations and displayed prominent nucleoli. The karyoplasm of the larger fusiform and triangular neurons contained abundant organelles including parallel arrays of granular endoplasmic reticulum. The synaptic input to labelled perikarya and proximal dendrites was sparse. It consisted chiefly of asymmetrical synaptic contacts, sometimes with postjunctional densities, but a few symmetrical synapses were also noted. ChAT-positive axon terminals were not identified which suggests that axon collaterals are rare within the nucleus of the horizontal limb of the diagonal band of Broca.     

Effect of olfactory bulb pathology on olfactory function in normal aging

Decline of olfactory function is frequently observed in aging and is an early symptom of neurodegenerative diseases. As the olfactory bulb (OB) is one of the first regions involved by pathology and may represent an early disease stage, we specifically aimed to evaluate the contribution of OB pathology to olfactory decline in cognitively normal aged individuals without parkinsonism or dementia. This clinicopathological study correlates OB tau, amyloid β (Aβ) and α‐synuclein (αSyn) pathology densities and whole brain pathology load to olfactory identification function as measured with the University of Pennsylvania Smell Identification Test (UPSIT) and clinical data measured proximate to death in a large autopsy study including 138 cases considered non‐demented controls during life. Tau pathology was frequently observed in the OB (95% of cases), while both Aβ (27% of cases) and αSyn (20% of cases) OB pathologies were less commonly observed. A weak correlation was only observed between OB tau and olfactory performance, but when controlled for age, neither OB tau, Aβ or αSyn significantly predict olfactory performance. Moreover, whole brain tau and αSyn pathology loads predicted olfactory performance; however, only αSyn pathology loads survived age correction. In conclusion, OB tau pathology is frequently observed in normally aging individuals and increases with age but does not appear to independently contribute to age‐related olfactory impairment suggesting that further involvement of the brain seems necessary to contribute to age‐related olfactory decline.     

A centrifugal respiratory modulation of olfactory bulb unit activity: a study on acute rat preparation

Summary In behaving rats, unit activity in the mitral and granule cell layers of the olfactory bulb (OB) can be modulated by respiration. In order to determine whether central influences could take part in this phenomenon, respiratory rhythm and the activity of OB units were recorded in the present experiment and analyzed temporally in 18 anaesthetized tracheotomized rats. In spite of the interrupted nasal airflow, 30 of the 80 cells recorded in the mitral and granule cell layers, still displayed a significant respiratory patterning of their activity. Maximal neuronal discharges were time-locked with different phases of the respiratory cycle, most often synchronized with the end of expiration. This is in contrast with previous observations in intact animals. Possible underlying mechanisms are discussed.     

Cholinergic and peptidergic projections from the medial septum and the nucleus of the diagonal band of Broca to dorsal hippocampus, cingulate cortex and olfactory bulb: a combined wheatgerm agglutinin-apohorseradish peroxidase-gold immunohistochemical study

We have examined the distribution pattern and the density of various neuropeptide, neurotransmitter and enzyme containing neurons in the rat medial septum and the nucleus of the diagonal band of Broca to assess their possible involvement in the septohippocampal, septocortical and septobulbar pathways. Immunohistochemical methods were combined with the retrograde transport of a protein-gold complex injected in the hippocampus, the cingulate cortex or the olfactory bulb. Cholinergic neurons were the most numerous. Galanin-positive neurons were about two or three times less numerous than cholinergic cells. Both these cell types had a similar location though the choline acetyl transferase-like immunoreactive cells extended more caudally in the horizontal limb of the nucleus of the diagonal band of Broca. Immunoreactive cells for other neuroactive substances were few (calcitonin gene-related peptide, luteinizing hormone releasing hormone. [Met]enkephalin-arg-gly-leu) or occasional (dynorphin B, vasoactive intestinal polypeptide, somatostatin, neurotensin, cholecystokinin, neuropeptide Y and substance P). No immunoreactive cells for bombesin, alpha atrial natriuretic factor, corticotropin releasing factor, 5-hydroxytryptamine, melanocyte stimulating hormone, oxytocin, prolactin, tyrosine hydroxylase or arg-vasopressin were present. Choline acetyltransferase- and galanin-like immunoreactive cells densely participate to septal efferents. Cholinergic neurons constituted the bulk of septal efferent neurons. Galanin-positive cells were 22% of septohippocampal, 8% of septocortical, and 9% of septobulbar neurons. Galanin containing septohippocampal neurons were found in the medial septum and the nucleus of the diagonal band of Broca; galanin-positive septobulbar and septocortical cells were limited to the nucleus of the diagonal band of Broca. Occasional double-labellings were noticed with some peptides other than galanin. Luteinizing hormone-releasing hormone, calcitonin gene-related peptide and enkephalin were the most often observed; some other projecting cells stained for vasoactive intestinal polypeptide or dynorphin B. Luteinizing hormone-releasing hormone, calcitonin gene-related peptide and enkephalin were observed in septohippocampal neurons; luteinizing hormone-releasing hormone and vasoactive intestinal peptide were observed in septocortical neurons and calcitonin gene-related peptide, luteinizing hormone-releasing hormone and dynorphin B were observed in septo-bulbar cells. These results show that, in addition to acetylcholine, galanin is a major cellular neuroactive substance in septal projections to the hippocampus, the cingulate cortex and the olfactory bulb. The presence of septal projecting neurons immunoreactive for other peptides shows that a variety of distinct peptides may also participate, but in a smaller number, to septal efferent pathways.     

Food-anticipatory activity persists after olfactory bulb ablation in the rat

Restricted daily food access acts as an entraining stimulus (zeitgeber) for a circadian clock, the feeding-entrainable oscillator (FEO). There are many properties of a daily meal that could potentially convey timing information to the FEO. Olfactory cues associated with feeding are one such property. In order to rule out olfaction as a necessary entraining stimulus, olfactory bulbectomized and sham-operated male Sprague--Dawley rats had access to food for 2 h each day. Food bin approach behavior was monitored as an index of food-anticipatory activity (FAA). Both groups entrained to the daily meal with an increase in feeder approach time several hours before meal onset. There were no significant differences in the timing or the amount of FAA between groups. Furthermore, FAA was maintained during 3 days of food deprivation in both groups. In accordance with previous studies, the results show that olfactory cues are not necessary for the entrainment of FAA.     

Electron microscopy of single cells in the olfactory bulb using Golgi impregnation

Summary A technique for studying the same cell stained by Golgi impregnation with both light and electron microscopy is described. The application of the method, termed Golgi-E.M., to the rat olfactory bulb and its contribution to a wider organizational study of this region is considered. While providing important confirmation of many of the intercellular relationships found by examination of conventional electron microscopic material, it has also allowed certain special features of middle tufted cells to be noted, including reciprocal, axo-dendritic, dendro-axonic synaptic contacts. The advantages and drawbacks of the technique are briefly stated and the role to which it is best suited in organizational study is set out; it is clear that, while unsuitable at present for routine study, this material can provide both important correlations of light and electron microscopy and certain specific types of information, in conjunction with more conventional methods.     

Differential distribution of diagonal band afferents to subnuclei of the interpeduncular nucleus in rats

Previous research has demonstrated a subnuclear organization within the interpeduncular nucleus (IPN) based upon cytoarchitecture, synaptology, and the distribution of biogenic amines and peptides. To determine whether individual subnuclei of IPN can be further differentiated with regard to their afferents, we investigated the distribution of inputs from the nucleus of the diagonal band. Autoradiographic analyses demonstrated a diagonal band projection to IPN which is not homogeneously distributed among individual subnuclei. The greatest density of silver grains was located over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. These findings confirm the existence of a projection from the nucleus of the diagonal band to the IPN, and support the concept that individual subnuclei within the IPN may be further differentiated on the basis of their afferent input.     

Discrimination among odorants by single neurons of the rat olfactory bulb

1. Intracellular and extracellular recordings were made from rat olfactory bulb mitral and tufted cells during odor stimulation and during electrical stimulation of the olfactory nerve. Neurons were identified by horseradish peroxidase injections and/or antidromic activation. The presentation of multiple concentrations of at least one odorant in a cyclic artificial sniff paradigm, as reported previously (10), allowed the study of odor responses. This approach was extended to multiple odorants to compare their concentration-response profiles. This procedure avoids the problems of interpretation resulting from nonequivalence of the effective concentrations of different odorants used as stimuli that have characterized previous studies of odor quality effects. Comparisons of intracellular events and responses to electrical stimulation with the odor-induced spike train activity allow us to begin to delineate the local circuitry involved in generating odor-induced responses. 2. The concentration-response profiles of the 72 cells in the present study are comparable to those previously reported for output neurons of the olfactory bulb, showing ordered changes in the temporal patterning of spike activity with step changes in odor concentration. However, eight of the neurons exhibited inhibitory responses to lower concentrations, but excitation, at similar latency, to higher concentrations of the same odorant. These data emphasize that to study pattern changes induced by changing odor quality the influence of stimulus intensity must also be carefully examined. The data also provide evidence that the temporal pattern evoked by an odorant is probably not in itself the code for odor quality recognition. 3. Complete concentration-response profiles, including subthreshold concentrations, to more than one odorant show that, although responses to the different odorant can evolve systematically with concentration, the responses to different odorants can evolve through very different patterns. For example, in some cells, the response patterns to different odors were complementary in form. These results demonstrate that the patterned responses of olfactory bulb neurons can reflect changes in odor quality as well as intensity. 4. Intracellular recording was employed to compare the temporal patterning of spikes during odor stimulation with membrane potential changes. In some cases, the spike pattern was closely correlated with apparent postsynaptic potentials. However, there were several clear exceptions. In five cells, a prominent hyperpolarization, seen in the first sniff of a series of 10 consecutive sniffs, was associated with pauses in spike activity. In the following