Carrie T. Drake

Ultrastructural localization of estrogen receptor β immunoreactivity in the rat hippocampal formation

Several lines of evidence indicate that estrogen affects hippocampal synaptic plasticity through rapid nongenomic mechanisms, possibly by binding to plasma membrane estrogen receptors (ERs). We have previously shown that ERα immunoreactivity (ir) is in select interneuron nuclei and in several extranuclear locations, including dendritic spines and axon terminals, within the rat hippocampal formation (Milner et al., [ 2001 ] J Comp Neurol 429:355). The present study sought to determine the cellular and subcellular locations of ERβ‐ir. Coronal hippocampal sections from diestrus rats were immunolabeled with antibodies to ERβ and examined by light and electron microscopy. By light microscopy, ERβ‐ir was primarily in the perikarya and proximal dendrites of pyramidal and granule cells. ERβ‐ir was also in a few nonprincipal cells and scattered nuclei in the ventral subiculum and CA3 region. Ultrastructural analysis revealed ERβ‐ir at several extranuclear sites in all hippocampal subregions. ERβ‐ir was affiliated with cytoplasmic organelles, especially endomembranes and mitochondria, and with plasma membranes primarily of principal cell perikarya and proximal dendrites. ERβ‐ir was in dendritic spines, many arising from pyramidal and granule cell dendrites. In both dendritic shafts and spines, ERβ‐ir was near the perisynaptic zone adjacent to synapses formed by unlabeled terminals. ERβ‐ir was in preterminal axons and axon terminals, associated with clusters of small, synaptic vesicles. ERβ‐labeled terminals formed both asymmetric and symmetric synapses with dendrites. ERβ‐ir also was detected in glial profiles. The cellular and subcellular localization of ERβ‐ir was generally similar to that of ERα, except that ERβ was more extensively found at extranuclear sites. These results suggest that ERβ may serve primarily as a nongenomic transducer of estrogen actions in the hippocampal formation. J. Comp. Neurol. 491:81–95, 2005.

Some Forms of cAMP-Mediated Long-Lasting Potentiation Are Associated with Release of BDNF and Nuclear Translocation of Phospho-MAP Kinase

Long-lasting forms of synaptic plasticity like the late phase of LTP (L-LTP) typically require an elevation of cAMP, the recruitment of the cAMP-dependent protein kinase (PKA), and ultimately the activation of transcription and translation; some forms also require brain-derived neurotrophic factor (BDNF). Both cAMP and BDNF can activate mitogen-activated protein kinase (MAPK/ERK), which also plays a role in LTP. However, little is known about the mechanisms whereby cAMP, BDNF, and MAPK interact. We find that increases in cAMP can rapidly activate the BDNF receptor TrkB and induce BDNF-dependent long-lasting potentiation at the Schaffer collateral-CA1 synapse in hippocampus. Surprisingly, in these BDNF-dependent forms of potentiation, which are also MAPK dependent, TrkB activation is not critical for the activation of MAPK but instead appears to modulate the subcellular distribution and nuclear translocation of the activated MAPK.

Ultrastructural Localization of Full-Length trkB Immunoreactivity in Rat Hippocampus Suggests Multiple Roles in Modulating Activity-Dependent Synaptic Plasticity

Neurotrophins acting at the trkB receptor have been shown to be important modulators of activity-dependent plasticity in the hippocampus, but the mechanisms underlying these effects are not yet well understood. To identify the cellular and subcellular targets of trkB ligands in the adult rat hippocampal formation, full-length trkB receptor immunoreactivity (trkB-IR) was localized using electron microscopy. trkB-IR was present in the glutamatergic pyramidal and granule cells. Labeling in these neurons appeared as discrete clusters and was primarily in axons, excitatory-type axon terminals, and dendritic spines and to a lesser extent in somata and dendritic shafts. trkB-IR was commonly found on the plasma membrane of dendritic spines, whereas in other subcellular regions trkB-IR was often intracellular. Labeling was strikingly dense within axon initial segments, suggesting extensive receptor trafficking. trkB-IR was not confined to pyramidal and granule cells. Dense trkB-IR was found in occasional interneuron axon initial segments, some axon terminals forming inhibitory-type synapses onto somata and dendritic shafts, and excitatory-type terminals likely to originate extrahippocampally. This suggests that trkB is contained in some GABAergic interneurons, neuromodulatory (e.g., cholinergic, dopaminergic, and noradrenergic) afferents, and/or glutamatergic afferents. These data indicate that full-length trkB receptor activation may modulate glutamatergic pathways of the trisynaptic circuit both presynaptically at axon terminals and initial segments and postsynaptically at dendritic spines and shafts. Signaling via catalytic trkB may also presynaptically affect inhibitory and modulatory neurons. A pan-trkB antibody labeled the same neuronal populations as the full-length-specific trkB antiserum, but the labels differed in density at various subcellular sites. These findings provide an ultrastructural foundation for further examining the mechanisms through which neurotrophins acting at trkB receptors contribute to synaptic plasticity.

The role of neuronal signaling in controlling cerebral blood flow

Well-regulated blood flow within the brain is vital to normal function. The brains requirement for sufficient blood flow is ensured by a tight link between neural activity and blood flow. The link between regional synaptic activity and regional cerebral blood flow, termed functional hyperemia, is the basis for several modern imaging techniques that have revolutionized the study of human brain activity. Here, we review the mechanisms of functional hyperemia and their implications for interpreting the blood oxygen level-dependent (BOLD) contrast signal used in functional magnetic resonance imaging (fMRI).

Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS

The present study examined immunohistochemically the CNS distributions of a splice variant of the mu-opioid receptor, MOR-1D, in both rats and mice. In MOR-1D, exon 4 of MOR-1 is replaced by two additional exons that code for seven amino acids. Using rabbit antisera, we compared immunohistochemically the regional distribution of a C-terminal epitope of MOR-1D to that of a C-terminal epitope from MOR-1 and a C-terminal epitope from another splice variant, MOR-1C. The general distribution of MOR-1D-like immunoreactivity was similar in both mouse and rat. MOR-1D-like immunoreactivity was seen in the dentate gyrus and in the mossy fibers of the hippocampal formation, the nucleus of the solitary tract and the area postrema, the inferior olivary nucleus, the nucleus ambiguous, the spinal trigeminal nucleus and the spinal cord. MOR-1D-like immunoreactivity was not observed in some regions containing dense MOR-1-like immunoreactivity, such as the striatum or the locus coeruleus. In regions containing MOR-1, MOR-1C and MOR-1D, the pattern of each variant was unique.MOR-1D and MOR-1C are splice variants of the cloned mu-opioid receptor MOR-1. Although they differ only at the tip of the carboxy terminus, they show marked differences in their regional distributions, as determined immunohistochemically by epitopes in their unique carboxy termini. Since the splice variants are derived from the same gene, these differences in regional distribution imply region-specific messenger RNA processing.

Methylphenidate Administration to Juvenile Rats Alters Brain Areas Involved in Cognition, Motivated Behaviors, Appetite, and Stress

Thousands of children receive methylphenidate (MPH; Ritalin) for attention deficit/hyperactivity disorder (ADHD), yet the long-term neurochemical consequences of MPH treatment are unknown. To mimic clinical Ritalin treatment in children, male rats were injected with MPH (5 mg/kg) or vehicle twice daily from postnatal day 7 (PND7)–PND35. At the end of administration (PND35) or in adulthood (PND135), brain sections from littermate pairs were immunocytochemically labeled for neurotransmitters and cytological markers in 16 regions implicated in MPH effects and/or ADHD etiology. At PND35, the medial prefrontal cortex (mPFC) of rats given MPH showed 55% greater immunoreactivity (-ir) for the catecholamine marker tyrosine hydroxylase (TH), 60% more Nissl-stained cells, and 40% less norepinephrine transporter (NET)-ir density. In hippocampal dentate gyrus, MPH-receiving rats showed a 51% decrease in NET-ir density and a 61% expanded distribution of the new-cell marker PSA-NCAM (polysialylated form of neural cell adhesion molecule). In medial striatum, TH-ir decreased by 21%, and in hypothalamus neuropeptide Y-ir increased by 10% in MPH-exposed rats. At PND135, MPH-exposed rats exhibited decreased anxiety in the elevated plus-maze and a trend for decreased TH-ir in the mPFC. Neither PND35 nor PND135 rats showed major structural differences with MPH exposure. These findings suggest that developmental exposure to high therapeutic doses of MPH has short-term effects on select neurotransmitters in brain regions involved in motivated behaviors, cognition, appetite, and stress. Although the observed neuroanatomical changes largely resolve with time, chronic modulation of young brains with MPH may exert effects on brain neurochemistry that modify some behaviors even in adulthood.

Opioid systems in the dentate gyrus.

Opiate drugs alter cognitive performance and influence hippocampal excitability, including long-term potentiation (LTP) and seizure activity. The dentate gyrus (DG) contains two major opioid peptides, enkephalins and dynorphins, which have opposing effects on excitability. Enkephalins preferentially bind to delta- and mu-opioid receptors (DORs and MORs) while dynorphins preferentially bind to kappa-opioid receptors (KORs). Opioid receptors can also be activated by exogenous opiate drugs such as the MOR agonist morphine. Enkephalins are contained in the mossy fiber pathway, in the lateral perforant path (PP) and in scattered GABAergic interneurons. MORs and DORs are predominantly in distinct subpopulations of GABAergic interneurons known to inhibit granule cells, and are present at low levels within granule cells. MOR and DOR agonists increase excitability and facilitate LTP in the molecular layer. Anatomical and physiological evidence is consistent with somatodendritic and axon terminal targeting of both MORs and DORs. Dynorphins are in the granule cells, most abundantly in mossy fibers but also in dendrites. KORs have been localized to granule cell mossy fibers, supramammillary afferents to granule cells, and PP terminals. KOR agonists, including endogenous dynorphins, diminish the induction of LTP. Recent evidence indicates that opiates and opioids also modulate other processes in the hippocampal formation, including adult neurogenesis, the actions of gonadal hormones, and development of neonatal transmitter systems.

Extranuclear estrogen receptor beta immunoreactivity is on doublecortin-containing cells in the adult and neonatal rat dentate gyrus.

In adult female rats, estrogen receptor (ER) activation, particularly of ERbeta, promotes hippocampal neurogenesis. We previously reported that extranuclear ERbeta immunoreactivity (ir) in adult rats is on cellular profiles in or near the granule cell layer, which is the location of newly generated cells. During development, cells in or near the granule cell layer transiently express high levels of estrogen binding and nuclear ERs. Thus, we sought to determine if extranuclear ERbeta is in newly generated cells in adult and neonatal rat dentate gyrus. Sections from the dentate gyrus of adult proestrus or postnatal day 7 and 14 female rats were dual-labeled for ERbeta and the new-cell marker doublecortin (DCX) and examined by electron microscopy. DCX-containing neurons were found in the subgranular hilus in adult rats and were more widespread throughout the granule cell layer and hilus of neonatal rats. In both adults and neonatal rats, ERbeta immunoreactivity was found in a subset of DCX-labeled neurons. Electron microscopic examination of the adult dentate gyrus revealed that most perikarya with DCX-ir had the morphological characteristics of granule cells, although a few resembled interneurons. Dendrites with DCX-ir also were observed. In both adults and neonates, DCX-labeled neuronal perikarya and dendrites contained ERbeta-ir; ERbeta-ir usually was aggregated near the plasma membrane, mitochondria or endoplasmic reticula. ERbeta-ir was in glial profiles that apposed DCX-labeled perikarya and dendrites. These findings are consistent with data showing that estrogens can exert non-genomic effects directly and indirectly on newly generated cells in neonatal and adult rat dentate gyrus.

Kappa opioid receptor-like immunoreactivity in guinea pig brain: Ultrastructural localization in presynaptic terminals in hippocampal formation

Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR‐like immunoreactivity (‐LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity‐purified KT antibody was confirmed by Western blotting, enzyme‐linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT‐LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT‐LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT‐labeled processes were also present in stratum lacunosum‐moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT‐LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense‐core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT‐LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation.

Kappa opioid receptors in rat spinal cord vary across the estrous cycle

Kappa opioid receptors (KORs) were immunocytochemically localized in the lumbosacral spinal cord of female rats in different stages of the estrous cycle to examine the influence of hormonal status on receptor density. KOR labeling was primarily in fine processes and a few neuronal cell bodies in the superficial dorsal horn and the dorsolateral funiculus. Quantitative light microscopic densitometry of the superficial dorsal horn revealed that rats in diestrus had significantly lower KOR densities than those in proestrus or estrus. This suggests that female reproductive hormones regulate spinal KOR levels, which may contribute to variations in analgesic effectiveness of KOR agonists across the estrous cycle.