Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Transforming Sensory Cues into Aversive Emotion via Septal-Habenular Pathway.

Emotions evoked by environmental cues are important for animal survival and life quality. However, neural circuits responsible for transforming sensory signals to aversive emotion and behavioral avoidance remain unclear. Here, we found that medial septum (MS) mediates aversion induced by both auditory and somatosensory stimuli. Ablation of glutamatergic or GABAergic MS neurons results in impaired or strengthened aversion, respectively. Optogenetic activation of the two cell types results in place avoidance and preference, respectively. Cell-type-specific screening reveals that glutamatergic MS projections to the lateral habenula (LHb) are responsible for the induction of aversion, which can be antagonized by GABAergic MS projections to LHb. Additionally, the sensory-induced place avoidance is facilitated by enhanced locomotion mediated by glutamatergic MS projections to the preoptic area. Thus, MS can transmit innately aversive signals via a bottom-up multimodal sensory pathway and produce concurrent emotional and motional effects, allowing animals to efficiently avoid unfavorable environments.

The ontogenetic development of neurons containing calcium-binding proteins in the septum of the guinea pig: Late onset of parvalbumin immunoreactivity versus calbindin and calretinin.

The study describes the immunoreactivity of calbindin (CB), calretinin (CR) and parvalbumin (PV), their distribution pattern and the co-distribution of CB and CR as well as CB and PV in the septum of the guinea pig during development. Immunohistochemistry was conducted on embryonic (E40, E50, E60), newborn (P0) and postnatal (P5, P10, P20, P40, P100) guinea pig brains. The presence of both CB and CR was detected at E40, while PV began to be observed at E60. Immunoreactivity for CB was constant throughout ontogeny. In contrast to CR immunoreactivity, PV immunoreactivity was higher in the postnatal stages than in the prenatal and newborn stages. Double immunostaining showed that CB co-localized with CR from E40 onwards, while with PV from P5 onwards, suggesting that CB co-operates with these proteins in the guinea pig septum during different periods of ontogeny. Our results also indicate that among the studied CaBPs, CB exhibited the highest immunoreactivity during both embryonic and postnatal development.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-µM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.

Fasting-induced changes of neuropeptide immunoreactivity in the lateral septum of male rats.

The objective of the present study was to determine the rostrocaudal distribution and the effect of reduced food intake (60% of the average daily food intake for 1-4 weeks) on the amount of leucine-enkephalin (Leu-enk), neuropeptide Y (NPY) and galanin (Gal) in the lateral septum of male rat brain. Using pre-embedding immunocytochemistry combined with densitometry on 60 microm serial vibratome sections we found that in control animals Leu-enk-immunoreactive elements showed an increasing density from rostral towards the medial part of the septum, then a gradual decrease towards the caudal direction. The distribution of NPY proved to be rather even along the examined sequence of sections with two smaller peaks roughly at the 1/3 and 2/3 of the rostrocaudal axis. Gal showed similar distribution but the peaks were shifted to more caudal direction. We also found that Leu-enk forms the most dense plexus followed by a moderate amount of NPY-positive axonal meshwork. Gal was present in the lowest amount along the lateral septal nuclei. The effect of reduced food intake was marked and differential in the case of the three examined peptides. During the first 2 weeks of reduced food intake NPY-immunoreactivity was upregulated as compared to the control, then it was reduced close to the control value by the 4th week. The changes in Gal immunoreactivity showed similar pattern. The average relative density of Leu-enk-immunoreactive elements immediately decreased as a result of reduced food intake for 1 week and it gradually decreased by the end of the 4th week. These results indicate that reduced food intake affects the expression of NPY, Gal and Leu-enk not only in the relevant hypothalamic neuroendocrine centres, but also in the lateral septal area.

Neonatal handling and reproductive function in female rats.

Neonatal handling induces anovulatory estrous cycles and decreases sexual receptivity in female rats. The synchronous secretion of hormones from the gonads (estradiol (E2) and progesterone (P)), pituitary (luteinizing (LH) and follicle-stimulating (FSH) hormones) and hypothalamus (LH-releasing hormone (LHRH)) are essential for the reproductive functions in female rats. The present study aimed to describe the plasma levels of E2 and P throughout the estrous cycle and LH, FSH and prolactin (PRL) in the afternoon of the proestrus, and the LHRH content in the medial preoptic area (MPOA), median eminence (ME) and medial septal area (MSA) in the proestrus, in the neonatal handled rats. Wistar pup rats were handled for 1 min during the first 10 days after delivery (neonatal handled group) or left undisturbed (nonhandled group). When they reached adulthood, blood samples were collected through a jugular cannula and the MPOA, ME and MSA were microdissected. Plasma levels of the hormones and the content of LHRH were determined by RIA. The number of oocytes counted in the morning of the estrus day in the handled rats was significantly lower than in the nonhandled ones. Neonatal handling reduces E2 levels only on the proestrus day while P levels decreased in metestrus and estrus. Handled females also showed reduced plasma levels of LH, FSH and PRL in the afternoon of the proestrus. The LHRH content in the MPOA was significantly higher than in the nonhandled group. The reduced secretion of E2, LH, FSH and LHRH on the proestrus day may explain the anovulatory estrous cycle in neonatal handled rats. The reduced secretion of PRL in the proestrus may be related to the decreased sexual receptiveness in handled females. In conclusion, early-life environmental stimulation can induce long-lasting effects on the hypothalamus-pituitary-gonad axis.

Phase segregation of medial septal GABAergic neurons during hippocampal theta activity.

Septo-hippocampal GABAergic neurons immunoreactive for parvalbumin are thought to play a crucial role in the generation of hippocampal theta oscillations associated with a specific stage of memory formation. Here we use in vivo juxtacellular recording and filling in the medial septum followed by immunocytochemical identification of the recorded cells containing parvalbumin to determine their firing pattern, phase relationship with hippocampal theta, morphology, and to thereby reveal their involvement in the generation of hippocampal theta activity. We have demonstrated that GABAergic medial septal neurons form two distinct populations exhibiting highly regular bursting activity that is tightly coupled to either the trough (178 degrees ) or the peak (330 degrees ) of hippocampal theta waves. Additionally, different types of bursting as well as nonbursting activity patterns were also observed. The morphological reconstruction of theta-bursting neurons revealed extensive axon arbors of these cells with numerous local collaterals establishing symmetrical synapses; thus, synchrony among the septal pacemaker units may be brought about by their recurrent collateral interactions. Long projecting axons could also be found running dorsally toward the hippocampus and ventrally in the direction of basal forebrain regions. We conclude that GABAergic neurons in the medial septum, which are known to selectively innervate hippocampal interneurons, are in a position to induce rhythmic disinhibition in the hippocampus and other theta-related subcortical areas at two different phases of hippocampal theta.

Distribution of vesicular glutamate transporter-2 messenger ribonucleic Acid and protein in the septum-hypothalamus of the rat.

The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.

Catecholaminergic innervation of the septum in the frog: a combined immunohistochemical and tract-tracing study.

In the present study, we have investigated the distribution and the origin of the catecholaminergic innervation of the septal region in the frog Rana perezi. Immunohistochemistry for dopamine and two enzymes required for the synthesis of catecholamines, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) revealed a complex pattern of catecholaminergic (CA) innervation in the anuran septum. Dopaminergic fibers were primarily present in the dorsal portion of the lateral septum, whereas noradrenergic (DBH immunoreactive) fibers predominated in the medial septum/diagonal band complex. Catecholaminergic cell bodies were never observed within the septum. To determine the origin of this innervation, applications of dextran amines, both under in vivo and in vitro conditions, into the septum were combined with immunohistochemistry for TH. Results from these experiments demonstrated that four catecholaminergic cell groups project to the septum: (1) the group related to the zona incerta in the ventral thalamus, (2) the posterior tubercle/mesencephalic group, (3) the locus coeruleus, and (4) the nucleus of the solitary tract. While the two first groups provide dopaminergic innervation to the septum, the locus coeruleus provides the major noradrenergic projection. Noradrenergic fibers most likely arise also in the nucleus of the solitary tract. The results obtained in Rana perezi are readily comparable to those in mammals suggesting that the role of catecholamines in the septum is well conserved through phylogeny and that the CA innervation of the amphibian septum may be involved in functional circuits similar to those in mammals.

Alterations in septohippocampal cholinergic neurons resulting from interleukin-2 gene knockout.

Interleukin-2 (IL-2) has potent effects on acetylcholine (ACh) release from septohippocampal cholinergic neurons and trophic effects on fetal septal and hippocampal neuronal cultures. Previous work from our lab showed that the absence of endogenous IL-2 leads to impaired hippocampal neurodevelopment and related behaviors. We sought to extend this work by testing the hypotheses that the loss of IL-2 would result in reductions in cholinergic septohippocampal neuron cell number and the density of cholinergic axons found in the hippocampus of IL-2 knockout mice. Stereological cell counting and imaging techniques were used to compare C57BL/6-IL-2(-/-) knockout and C57BL/6-IL-2(+/+) wild-type mice for differences in choline acetyltransferase (ChAT)-positive somata in the medial septum and vertical limb of the diagonal band of Broca (MS/vDB) and acetylcholine esterase (AChE)-labeled cholinergic axons in hippocampal projection fields. IL-2 knockout mice had significantly lower numbers (26%) of MS/vDB ChAT-positive cell bodies than wild-type mice; however, there were no differences in striatal ChAT-positive neurons. Although AChE-positive axon density in CA1, CA3b, the internal, and external blades of the dentate gyrus did not differ between the knockout and wild-type mice, the distance across the granular cell layer of the external blade of the dentate gyrus was reduced significantly in IL-2 knockout mice. Further research is needed to determine whether these outcomes in IL-2 knockout mice may be due to the absence of central and/or peripheral IL-2 during brain development or neurodegeneration secondary to autoimmunity.

Estrogen, progesterone, and pregnancy termination alter neural activity in brain regions that control maternal behavior in rats.

Estrogen stimulates maternal behavior in rats, but does so most potently when its administration is temporally coupled with the termination of pregnancy. In contrast, this effect of estrogen is blocked when subjects are administered a large dose of progesterone concurrent with estrogen. The current study was performed to examine the neural circuitry influenced by these treatments and pup presentation during the hormonally-mediated onset and inhibition of maternal behavior. In experiment I, estrogen induced c-Fos immunoreactivity (Fos-IR) in the medial preoptic area (MPOA) in virgin rats, but was much more effective when administered to pregnancy-terminated rats, suggesting that pregnancy termination increases MPOA's susceptibility to the physiological effects of estrogen. In experiment II, administering progesterone concurrently with estrogen in pregnancy-terminated rats strongly inhibited estrogen-stimulated Fos-IR in the MPOA, indicating that the physiological effects of estrogen on the MPOA are blocked if high progesterone levels are maintained. In experiment III, pregnancy-terminated subjects were administered estrogen, progesterone, or both hormones and presented with pups for 2 h. Approximately half of the subjects administered estrogen alone showed maternal behavior, but subjects receiving the other treatments were not maternal. In the MPOA, ventral bed nucleus of the stria terminalis (BSTv), and dorsal and intermediate lateral septum (LSd,i), maternal subjects showed the highest levels of Fos-IR, whereas subjects treated with progesterone alone or progesterone in combination with estrogen showed low levels of Fos-IR. These experiments suggest that estrogen could promote maternal behavior by enhancing pup-stimulated activity in the MPOA, BSTv, and LSd,i, regions believed to constitute the neural circuit that promotes maternal behavior, whereas progesterone could inhibit maternal behavior by inhibiting neural activity in some of these regions.

Co-distribution of Fos- and mu opioid receptor immunoreactivity within the rat septopreoptic area and hypothalamus during acute glucose deprivation: effects of the mu receptor antagonist CTOP.

Mu opioid receptors occur throughout the brain, but central sites where ligand neuromodulatory effects occur during glucopenia have not been identified. The present studies investigated whether septal, preoptic, and hypothalamic neurons that express immunoreactivity for this receptor are transcriptionally activated in response to the glucose antimetabolite, 2-deoxy-D-glucose (2DG), and if intracerebroventricular (icv) administration of the selective mu receptor antagonist, CTOP, modifies this functional response to glucose substrate imbalance. Neurons labeled for mu receptor-immunoreactivity (-ir) were observed in the lateral septal nucleus (LS), medial septum (MS), anterior division of the stria terminalis (BSTa), median preoptic nucleus (MEPO), medial preoptic nucleus (MPN), parastrial nucleus (PS), anterior hypothalamic periventricular nucleus (PVa), and lateral hypothalamic area (LPO). 2DG injection (400 mg/kg i.p.) resulted in co-labeling of mu receptor-positive neurons in the LS, MS, BSTa, MEPO, PVa, and LPO for nuclear Fos-ir. Icv delivery of CTOP decreased mean numbers of co-labeled neurons in the LS, MS, BSTa, and MEPO. These results provide evidence for transactivational effects of glucopenia on mu opioid receptor-expressing neurons within the septum, preoptic area, and hypothalamus, and suggest that the functional status of these receptors within discrete septopreoptic sites may be critical for maximal glucoprivic induction of the Fos stimulus-transcription cascade within local cells. These results thus support the view that the neural loci described above may serve as substrates for regulatory effects of mu opioid receptor ligands on central compensatory activities during acute glucose deprivation.

Electron microscopic evidence for different myelination of rat septohippocampal fibres.

Rat septohippocampal fibres are known to originate from GABAergic parvalbumin-containing, fast-firing, fast-conducting neurons and from cholinergic slow-firing, slow-conducting neurons. In the present electron microscopic study, based on immunocytochemical demonstration of parvalbumin and choline acetyltransferase in transverse and horizontal septal sections, it was shown that parvalbumin-immunoreactive fibres are myelinated, but the vast majority of cholinergic fibres are not. As revealed, especially in horizontal sections, the cholinergic axons show considerably finer calibres than parvalbumin-containing ones. These results confirm and extend our previous light microscopic findings. It can be concluded that differences in conduction velocities, presence or absence of myelin sheaths and differences axonal diameters are correlated in the septohippocampal pathway.

Potassium (K+) channel expression in basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCN) are depleted early in the course of Alzheimer's disease (AD). BFCN voltage-gated K(+) channels regulate acetylcholine release and may play a role in BFCN neurodegeneration. Neuronal voltage-gated K(+) channels are heterotetrameric assemblies of K(v) and accessory subunits. Currently, there is no available information about the K(v) proteins expressed in BFCN. Immunohistochemical techniques were used to investigate the expression of specific K(v) subunits in rat brain BFCN. Our results showed that BFCN express both K(v)3.1 and K(v)2.1 subunits. However, the K(v)2.1 subunit showed a wider distribution in noncholinergic neurons than the K(v)3.1 subunit. K(v)3.1 and K(v)2.1 immunostaining was noticeable not only in neuronal cell bodies but also in the dendritic ramifications of these neurons. Insofar as the K(v)3.1 subunit has been classically associated with "fast-spiking neurons" and BFCN have low firing rates and long-duration action potentials, K(v)3.1 subunits may have functions other than facilitating high-frequency firing in BFCN.

Monoaminergic activities of limbic regions are elevated during aggression: influence of sympathetic social signaling.

A visual social signal inhibiting aggression is coincident with limiting serotonergic and noradrenergic activity in subiculum, hippocampus, nucleus accumbens, medial amygdala, but not lateral amygdala, septum, and hypothalamus. Darkening of postorbital skin in the lizard Anolis carolinensis is stimulated by sympathetic activation of beta(2)-adrenergic receptors via adrenal catecholamines, and occurs more rapidly in dominant males during social interaction. Eyespot darkening functions as a social signal limiting aggressive interaction. To assess the effect of this social signal on telencephalic activity of monoamines, males were painted postorbitally with green or black paint, and exposed to a mirror. Serotonergic and noradrenergic turnover, as estimated by ratios of catabolite to transmitter, were elevated in the subiculum, hippocampus, nucleus accumbens, and medial amygdala of animals in which the eyespots were masked by green paint. Conversely, dopaminergic activity in these brain regions was lower in males with hidden eyespots (painted green). Hiding the eyespot evoked significantly increased aggressive activity toward the mirror image. Furthermore, changes in monoaminergic turnover were coincident with altered aggressive behavior, suggesting a relationship between them. Changes of monoaminergic activity were not observed in the septum, lateral amygdala, or hypothalamus, when males with eyespots permanently marked (black) were compared with those with eyespots hidden (painted green). Stimulated (serotonergic and noradrenergic) or inhibited (dopaminergic) activity due to social signal and aggression are confined to regions of the brain similarly activated during social stress, and do not constitute a generalized activation of monoaminergic systems.

Differential expression of small heat shock protein 27 in the rat hippocampus and septum after fimbria-fornix lesion.

mRNA, Western analysis and immunohistochemistry were used to study the expression of the small heat shock protein (HSP) 27 in the rat septum and hippocampus following fimbria-fornix lesions, a model of neurodegeneration and regeneration. (HSP) 27 mRNA level was increased 2.5-fold in the medial septum 3 days after lesion and this increase persisted for 10 days. In the hippocampus, after an initial 15-fold increase at 3 days post-injury, HSP27 mRNA returned to basal levels 10 days after the lesion. Three and 10 days after lesion, HSP27 protein levels were increased in the septum (4.5 and 5-fold, respectively) and hippocampus (65 and 10-fold, respectively). The morphology of the HSP27 positive cells was indistinguishable from that of GFAP-immunoreactive cells. In addition, in the septum of injured rats, occasional neurons were heavily labelled with anti-HSP27 antibodies. Thus, up-regulation of HSP27, particularly in glial cells, may be a component of glial input in the processes on degeneration/regeneration which occur in this model.

Treatment with estrogen and progesterone affects relative levels of brain-derived neurotrophic factor mRNA and protein in different regions of the adult rat brain.

Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine the effects of acute estrogen and progesterone replacement on relative levels of brain-derived neurotrophic factor (BDNF) mRNA and protein in different regions of the adult rat brain. Adult ovariectomized animals were killed 53 h after receiving estrogen (E53), 53 h after receiving estrogen and 5 h after receiving progesterone (E53P), or 72 h after receiving estrogen and 24 h after receiving progesterone (E72P). Ovariectomized controls were killed 53 and 72 h after receiving vehicle. Tissues from the hippocampus, pyriform cortex, olfactory bulbs, septum, and nucleus basalis/ventral pallidum were dissected. Tissues from the right hemisphere were processed for quantitative RT-PCR analysis of BDNF mRNA, and tissues from the left hemisphere were processed for the detection and quantification of BDNF protein by ELISA. The results demonstrate significant increases in BDNF mRNA in the pyriform cortex of E53- and E53P-treated animals, as well as an increase in BDNF protein in the pyriform cortex of E72P-treated animals, relative to controls. Significant increases in BDNF mRNA were likewise detected in the hippocampus of E53- and E72P-treated animals, but were accompanied by a significant decrease in BDNF protein in the hippocampus of E53P- and E72P-treated animals relative to controls. No significant changes in BDNF mRNA or protein were detected in the olfactory bulbs, frontal cortex, or nucleus basalis/ventral pallidum following hormone treatment; however, an increase in BDNF protein was detected in the septum of E53-treated animals. This may indicate an increase in the retrograde transport of BDNF from the hippocampus to the septum, which could help account for the decrease in BDNF protein detected in the hippocampus following hormone treatment. These findings demonstrate that hormone replacement significantly affects relative levels of BDNF mRNA and protein within specific regions of the brain. These effects may, in turn, contribute to the effects of estrogen replacement on hippocampal connectivity and cognitive processes that have recently been reported.

Expression of the vesicular acetylcholine transporter, proteins involved in exocytosis, and functional calcium signaling in varicosities and soma of a murine septal cell line.

The expression and localization of the vesicular acetylcholine transporter in a septal cell line, SN56, were investigated. Immunoprecipitation and immunoblot analysis of postnuclear supernatants indicated that this cell line expresses reasonable amounts of the transporter. Immunofluorescence and confocal microscopy experiments showed that the vesicular transporter is present in varicosities and also in the cell body of differentiated cells. Varicosities have the potential to be functional sites of transmitter release because they responded to depolarization with calcium influx through voltage-gated calcium channels and expressed the synaptic proteins synaptotagmin, SV2, synaptophysin, and a subunit of P/Q calcium channels. In the soma of SN56 cells, the transporter immunoreactivity was similar to that for synaptotagmin, and it colocalized with synaptophysin, but it did not colocalize with SV2. Labeling for SV2 appeared prominently in a defined perinuclear structure, whereas the two former proteins were widely distributed in the soma, where several endocytic compartments could be identified with the vital dye FM4-64. These data suggest that distinct synaptic vesicle proteins exist in different subcellular compartments, and consequently they may follow distinct pathways in neurites before reaching sites of transmitter storage and release in SN56 cells.