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Introduction: The septal area provides a rich innervation to the hippocampus regulating hippocampal excitability to different behavioral states and modulating theta rhythmogenesis. However, little is known about the neurodevelopmental consequences of its alterations during postnatal development. The activity of the septohippocampal system is driven and/or modulated by ascending inputs, including those arising from the nucleus incertus (NI), many of which contain the neuropeptide, relaxin-3 (RLN3). Methods: We examined at the molecular and cellular level the ontogeny of RLN3 innervation of the septal area in postnatal rat brains. Results: Up until P13–15 there were only scattered fibers in the septal area, but a dense plexus had appeared by P17 that was extended and consolidated throughout the septal complex by P20. There was a decrease in the level of colocalization of RLN3 and synaptophysin between P15 and P20 that was reversed between P20 and adulthood. Biotinylated 3-kD dextran amine injections into the septum, revealed retrograde labeling present in the brainstem at P10-P13, but a decrease in anterograde fibers in the NI between P10–20. Simultaneously, a differentiation process began during P10–17, resulting in fewer NI neurons double-labeled for serotonin and RLN3. Discussion: The onset of the RLN3 innervation of the septum complex between P17–20 is correlated with the onset of hippocampal theta rhythm and several learning processes associated with hippocampal function. Together, these data highlight the relevance and need for further analysis of this stage for normal and pathological septohippocampal development. [ABSTRACT FROM AUTHOR]

IntroductionThe septal area provides a rich innervation to the hippocampus regulating hippocampal excitability to different behavioral states and modulating theta rhythmogenesis. However, little is known about the neurodevelopmental consequences of its alterations during postnatal development. The activity of the septohippocampal system is driven and/or modulated by ascending inputs, including those arising from the nucleus incertus (NI), many of which contain the neuropeptide, relaxin-3 (RLN3).MethodsWe examined at the molecular and cellular level the ontogeny of RLN3 innervation of the septal area in postnatal rat brains.ResultsUp until P13–15 there were only scattered fibers in the septal area, but a dense plexus had appeared by P17 that was extended and consolidated throughout the septal complex by P20. There was a decrease in the level of colocalization of RLN3 and synaptophysin between P15 and P20 that was reversed between P20 and adulthood. Biotinylated 3-kD dextran amine injections into the septum, revealed retrograde labeling present in the brainstem at P10-P13, but a decrease in anterograde fibers in the NI between P10–20. Simultaneously, a differentiation process began during P10–17, resulting in fewer NI neurons double-labeled for serotonin and RLN3.DiscussionThe onset of the RLN3 innervation of the septum complex between P17–20 is correlated with the onset of hippocampal theta rhythm and several learning processes associated with hippocampal function. Together, these data highlight the relevance and need for further analysis of this stage for normal and pathological septohippocampal development.

The basal forebrain provides cholinergic inputs to primary visual cortex (V1) that play a key modulatory role on visual function. While basal forebrain afferents terminate in the infragranular layers of V1, acetylcholine is delivered to more superficial layers through volume transmission. Nevertheless, direct synaptic contact in deep layers 5 and 6 may provide a more immediate effect on V1 modulation. Using helper viruses with cell type specific promoters to target retrograde infection of pseudotyped and genetically modified rabies virus evidence was found for direct synaptic input onto V1 inhibitory neurons. These inputs were similar in number to geniculocortical inputs and, therefore, considered robust. In contrast, while clear evidence for dorsal lateral geniculate nucleus input to V1 excitatory neurons was found, there was no evidence of direct synaptic input from the basal forebrain. These results suggest a direct and more immediate influence of the basal forebrain on local V1 inhibition.

The prethalamic eminence (PThE), a diencephalic caudal neighbor of the telencephalon and alar hypothalamus, is frequently described in mammals and birds as a transient embryonic structure, undetectable in the adult brain. Based on descriptive developmental analysis of Tbr1 gene brain expression in chick embryos, we previously reported that three migratory cellular streams exit the PThE rostralward, targeting multiple sites in the hypothalamus, subpallium and septocommissural area, where eminential cells form distinct nuclei or disperse populations. These conclusions needed experimental corroboration. In this work, we used the homotopic quail-chick chimeric grafting procedure at stages HH10/HH11 to demonstrate by fate-mapping the three predicted tangential migration streams. Some chimeric brains were processed for Tbr1 in situ hybridization, for correlation with our previous approach. Evidence supporting all three postulated migration streams is presented. The results suggested a slight heterochrony among the juxtapeduncular (first), the peripeduncular (next), and the eminentio-septal (last) streams, each of which followed differential routes. A possible effect of such heterochrony on the differential selection of medial to lateral habenular hodologic targets by the migrated neurons is discussed. [ABSTRACT FROM AUTHOR]

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area. [ABSTRACT FROM AUTHOR]

Exercise has been shown to improve cognitive functioning in a range of species, presumably through an increase in neurotrophins throughout the brain, but in particular the hippocampus. The current study assessed the ability of exercise to restore septohippocampal cholinergic functioning in the pyrithiamine-induced thiamine deficiency (PTD) rat model of the amnestic disorder Korsakoff Syndrome. After voluntary wheel running or sedentary control conditions (stationary wheel attached to the home cage), PTD and control rats were behaviorally tested with concurrent in vivo microdialysis, at one of two time points: 24-h or 2-weeks post-exercise. It was found that only after the 2-week adaption period did exercise lead to an interrelated sequence of events in PTD rats that included: (1) restored spatial working memory; (2) rescued behaviorally-stimulated hippocampal acetylcholine efflux; and (3) within the medial septum/diagonal band, the re-emergence of the cholinergic (choline acetyltransferase [ChAT +]) phenotype, with the greatest change occurring in the ChAT +/nestin + neurons. Furthermore, in control rats, exercise followed by a 2-week adaption period improved hippocampal acetylcholine efflux and increased the number of neurons co-expressing the ChAT and nestin phenotype. These findings demonstrate a novel mechanism by which exercise can modulate the mature cholinergic/nestin neuronal phenotype leading to improved neurotransmitter function as well as enhanced learning and memory. [ABSTRACT FROM AUTHOR]

Ts65Dn mice have been developed as a model for Down syndrome (DS). Because of its involvement in complex behaviors, including sexual and aggressive behaviors, we investigated the nitric oxide (NO) system in specific brain regions of these mutant mice (TS) after isolation-induced aggression. Male TS mice displayed significantly higher aggression than wild type (WT) mice and the comparison of the NO system, both with immunohistochemical and histochemical methods, resulted in robust differences between TS and WT mice in the hypothalamic paraventricular nucleus, in the nucleus of the diagonal band and in the medial septum, but not in the striatum of TS mice. In conclusion, we document alterations in the neuronal NO system of the TS mouse model of DS, suggesting a correlation of the behavioral aggressiveness with deficient NO production.

We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), differentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. We also investigated the involvement of the lateral accumbens core and the dorsal caudate putamen. The anterior cingulate 1 (Cg1) region served as a negative control. Contrary to our expectations, we found that all regions of the accumbens corridor showed increased expression of the early growth response protein 1 (EGR1, Zif268) in rats 2 h after reacquisition of CPP for cocaine after a history of cocaine CPP acquisition and extinction. Previous counterconditioning with dyadic social interaction inhibited both the reacquisition of cocaine CPP and the activation of the whole accumbens corridor. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less affected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders.

We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), differentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. We also investigated the involvement of the lateral accumbens core and the dorsal caudate putamen. The anterior cingulate 1 (Cg1) region served as a negative control. Contrary to our expectations, we found that all regions of the accumbens corridor showed increased expression of the early growth response protein 1 (EGR1, Zif268) in rats 2 h after reacquisition of CPP for cocaine after a history of cocaine CPP acquisition and extinction. Previous counterconditioning with dyadic social interaction inhibited both the reacquisition of cocaine CPP and the activation of the whole accumbens corridor. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less affected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders. [ABSTRACT FROM AUTHOR]

ABSTRACT Down syndrome (DS), trisomy 21, is a multifaceted condition marked by intellectual disability and early presentation of Alzheimer's disease (AD) neuropathological lesions including degeneration of the basal forebrain cholinergic neuron (BFCN) system. Although DS is diagnosable during gestation, there is no treatment option for expectant mothers or DS individuals. Using the Ts65Dn mouse model of DS that displays age-related degeneration of the BFCN system, we investigated the effects of maternal choline supplementation on the BFCN system in adult Ts65Dn mice and disomic (2N) littermates at 4.3-7.5 months of age. Ts65Dn dams were maintained on a choline-supplemented diet (5.1 g/kg choline chloride) or a control, unsupplemented diet with adequate amounts of choline (1 g/kg choline chloride) from conception until weaning of offspring; post weaning, offspring were fed the control diet. Mice were transcardially perfused with paraformaldehyde, and brains were sectioned and immunolabeled for choline acetyltransferase (ChAT) or p75-neurotrophin receptor (p75NTR). BFCN number and size, the area of the regions, and the intensity of hippocampal labeling were determined. Ts65Dn-unsupplemented mice displayed region- and immunolabel-dependent increased BFCN number, larger areas, smaller BFCNs, and overall increased hippocampal ChAT intensity compared with 2N unsupplemented mice. These effects were partially normalized by maternal choline supplementation. Taken together, the results suggest a developmental imbalance in the Ts65Dn BFCN system. Early maternal-diet choline supplementation attenuates some of the genotype-dependent alterations in the BFCN system, suggesting this naturally occurring nutrient as a treatment option for pregnant mothers with knowledge that their offspring is trisomy 21. J. Comp. Neurol. 522:1390-1410, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: This study was focused on determining the possible role of corticotropin-releasing hormone (CRH) on play fighting in juvenile golden hamsters. As no specific neural sites have been proposed, we looked for changes in CRH innervations at the peak of play-fighting activity on postnatal day 35 (P-35) from a week before on P-28. We noted that the increase in play-fighting activity between these two dates was associated with a 100% increase of the density of CRH fibers within the lateral septum. We, then, tested the possible role of CRH receptors on play fighting within the lateral septum through microinjections of α-helical CRH, a CRH receptor antagonist (either 0, 30, or 300 ng), directly into the area. The treatments inhibited play-fighting attacks and pins as well as reduced the duration of time that the resident hamsters spent in contact with the intruders, though locomotor activity remained unaffected. The possible source of CRH release in the lateral septum was addressed by quantification of CRH neurons also labeled with a marker of cellular activity, c-Fos, after consummation of play fighting. CRH neurons in the horizontal part of the diagonal band, an area reciprocally connected with the lateral septum, showed a 75% increase in double labeling with c-Fos as compared to controls. Together, these data show that CRH receptors in the lateral septum have a general role on play fighting, not just facilitating its consummation, but also likely enhancing appetitive aspects as well. In addition, this effect is associated with enhanced CRH availability in the area and enhanced neuronal activity within interconnected areas. [Copyright &y& Elsevier]

Abstract: The effect of estrogen on the number and size of cholinergic neurons in the basal forebrain was examined in surgically menopausal young and middle-aged cynomolgus monkeys. Young and middle-aged female monkeys were ovariectomized and treated with conjugated equine estrogens (Premarin) at doses that are equivalent to those currently prescribed to postmenopausal women. In the medial septum/diagonal band (MS/DB), no effect of treatment with Premarin was observed in the cholinergic neurons in either ovariectomized young or middle-aged monkeys. However, the number and size of cholinergic neurons in the MS/DB of middle-aged monkeys was greater than that in the young monkeys. In the nucleus basalis of Meynert (NBM) of middle-aged monkeys, the number of cholinergic neurons in the intermediate region (Ch4i) was greater in Premarin-treated monkeys as compared to controls and numbers of neurons in this region were greater at higher levels of estrogen. No effects of estrogen were observed in other NBM regions in the middle-aged monkeys and the size of cholinergic neurons was unaffected by Premarin. These findings suggest that treatment with Premarin has selective beneficial effects on cholinergic neurons in the basal forebrain but that these effects are both age and region specific. [Copyright &y& Elsevier]

Abstract: Age-related and aged memory deficit changes in Nestin-immunoreactive (Nestin-IR) neurons were studied following recent evidence of distinct Nestin-IR neurons within adult rat basal forebrain. Morris water maze task assessed spatial learning capacity of 3- and 24-month rats (aged-impaired and aged-unimpaired groups). Nestin-IR neuron distributional and morphological features were investigated by immunohistochemistry and positive neuronal number calculation. Nestin-IR neuron number declined with aging, especially aged-impaired. Significant negative correlations existed between average escape latencies and Nestin-IR neuron number in medial septum-diagonal band of Broca (MS-DBB). Correlations of rostral portion [medial septum (MS) and vertical limb diagonal band (vDB)] were higher than caudal portion [horizontal limb diagonal band (hDB)]. Aged-impaired showed reduced complexity of Nestin-IR neuron dendrite arborization and dendritic length. Nestin-IR astrocyte-like cells appeared scattered among Nestin-IR neurons on some aged-impaired slices. In conclusion, aged-impaired rats showed worse cognitive spatial performance and less Nestin-IR neuronal number compared to aged-unimpaired. Nestin-IR neuronal loss and morphological changes are some pathological characteristics of rat aged basal forebrain and may be important in neurobiological mechanisms of brain aging and aged memory deficit. [Copyright &y& Elsevier]

Abstract: 5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [3H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S1), S2, S3; 360 pulses, 3 Hz, 2 ms, 26–28 mA). In all septal regions the Ca2+-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [3H] was inhibited by the 5-HT1B agonist CP-93,129 and the α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the μ- and κ-opioid receptor agonists (d-Ala2, N-Me-Phe4, glycinol5)-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the δ-opioid receptor agonist (d-Pen2, d-Pen5)-enkephalin (DPDPE), the dopamine D2 receptor agonist quinpirole and the adenosine A1 receptor agonist N6-cyclopentyladenosine were all ineffective; the GABAB receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S1. It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT1B autoreceptors, as well as by α2-, μ- and κ-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals. [Copyright &y& Elsevier]

Abstract: The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ∼355,000 and the numbers of ChAT-immuno-positive (+) as ∼22,000, GAD+ ∼119,000 and PAG+ ∼316,000, corresponding to ∼5%, ∼35% and ∼90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites. [Copyright &y& Elsevier]

Abstract: Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT−/GAD− cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca2+ channel subunits. Cav3.2 was detected in 31/34 cholinergic neurons and Cav3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Cav3.3 mRNA expression, although BF tissue expression was observed. In young rats (2–4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT−/GAD−) possessed fast TTX-R sodium currents resembling those mediated by Na+ channel subunit Nav1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT−/GAD− neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function. [Copyright &y& Elsevier]

Ts65Dn mice have been developed as a model for Down syndrome (DS). Because of its involvement in complex behaviors, including sexual and aggressive behaviors, we investigated the nitric oxide (NO) system in specific brain regions of these mutant mice (TS) after isolation-induced aggression. Male TS mice displayed significantly higher aggression than wild type (WT) mice and the comparison of the NO system, both with immunohistochemical and histochemical methods, resulted in robust differences between TS and WT mice in the hypothalamic paraventricular nucleus, in the nucleus of the diagonal band and in the medial septum, but not in the striatum of TS mice. In conclusion, we document alterations in the neuronal NO system of the TS mouse model of DS, suggesting a correlation of the behavioral aggressiveness with deficient NO production. [Copyright &y& Elsevier]

The projection from the medial septal-diagonal band complex (MSDB) to the hippocampal formation is generally considered to be primarily an uncrossed projection. The present study examined the number of MSDB neurons which project to the contralateral hippocampus, the position of these cells, their identity as either cholinergic or GABAergic, and the question of bilateral collateral projections. The fluorescent dyes Fluoro-Gold or diamidino yellow were injected into 5 equally-spaced sites along the septotemporal extent of the right hippocampus of adult female Sprague-Dawley rats. In some animals True Blue (5%) was also injected in a similar fashion into the left hippocampus. Five days later the brains were processed for fluorescence microscopic examination. The number of retrogradely labeled cells on each side was counted in every fifth section (200 μm interval) through the MSDB and the percentage of contralaterally projecting neurons was calculated. Averaging across all sections and all brains, it was found that approximately 15% of the medial septal neurons and 17% of the diagonal band neurons project to the contralateral hippocampus. Most of the labeled neurons, both ipsilateral and contralateral, were present in the caudal half of the MSDB. Within the medial septum contralaterally placed cells were distributed uniformly throughout the mediolateral extent and some of these stained immunocytochemically for choline acetyltransferase or glutamic acid decarboxylase. In brains in which diamidino yellow was injected into the right hippocampus and True Blue into the left numerous neurons were observed to contain both dyes, thus indicating projections to both hippocampi. [ABSTRACT FROM AUTHOR]