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The progressive development of seizures in rats by amygdala kindling, which models temporal lobe epilepsy, allows the study of molecular regulators of enduring synaptic changes. Neurotrophins play important roles in synaptic plasticity and neuroprotection. Activin, a member of the transforming growth factor-beta superfamily of growth and differentiation factors, has recently been added to the list of candidate synaptic regulators. We mapped the induction of activin betaA mRNA in amygdala and cortex at several stages of seizure development. Strong induction, measured 2 hours after the first stage 2 (partial) seizure, appeared in neurons of the ipsilateral amygdala (confined to the lateral, basal, and posterior cortical nuclei) and insular, piriform, orbital, and infralimbic cortices. Activin betaA mRNA induction, after the first stage 5 (generalized) seizure, had spread to the contralateral amygdala (same nuclear distribution) and cortex, and the induced labeling covered much of the convexity of neocortex as well as piriform, perirhinal, and entorhinal cortices in a nearly bilaterally symmetrical pattern. This pattern had filled in by the sixth stage 5 seizure. Induced labeling in cortical neurons was confined mainly to layer II. A similar temporal and spatial pattern of increased mRNA expression of brain-derived neurotrophic factor (BDNF) was found in the amygdala and cortex. Activin betaA and BDNF expression patterns were similar at 1, 2, and 6 hours after the last seizure, subsiding at 24 hours; in contrast, c-fos mRNA induction appeared only at 1 hour throughout cortex and then subsided. In double-label studies, activin betaA mRNA-positive neurons were also BDNF mRNA positive, and they did not colocalize with GAD67 mRNA (a marker of gamma-aminobutyric acidergic neurons). The data suggest that activin and BDNF transcriptional activities accurately mark excitatory neurons participating in seizure-induced synaptic alterations and may contribute to the enduring changes that underlie the kindled state., (Published 2004 Wiley-Liss, Inc.)

Studies have demonstrated neuronal expression of class I major histocompatibility complex (MHC) mRNA and protein in normal and developing brain and in response to injury or viral infection. We report neuronal expression of class I MHC mRNA in hypothalamic paraventricular nucleus (PVN) neurons in rats following systemic infection with Trypanosoma brucei brucei parasites (chronic) and in response to intravenous 1 mg/kg lipopolysaccharide administration (acute peripheral) and in striatal neurons following intrastriatal 5 microg lipopolysaccharide injection (acute central). These results demonstrate that neurons can be a source of immune signaling molecules and establish class I MHC as part of the neuronal component of immune responses.

Interleukin-1beta (IL-1beta) is a pro-inflammatory cytokine that appears in brain and cerebrospinal fluid following peripheral immune challenges and central infections or injury. We examined the consequences of i.c.v. infusion of IL-1beta on mRNA expression of several immune markers and on recruitment of peripheral leukocytes. Awake rats were infused with IL-1beta (100 ng/rat) into the lateral ventricle, and 0.5, 2, 4, 8, 12, or 24 h later, animals were killed and their fresh-frozen brains processed for in situ hybridization and immunohistochemistry. Widespread vascular expression of inhibitory factor kappa(B)alpha (Ikappa(B)alpha, marker of nuclear factor kappa(B)alpha transcriptional activity) and inducible cyclooxygenase (COX-2) mRNAs at 0.5-2 h was credited to movement of IL-1beta along ventricular, subarachnoid, and perivascular pathways to target endothelia that express type 1 IL-1 receptor mRNA. Induction of monocyte chemoattractant protein-1 mRNA and intercellular adhesion molecule-1 (ICAM-1) immunostaining on endothelia began at 0.5-2 h. Leukocytes (neutrophils and monocytes, recognized by morphology and CD45 and ED1 immunostaining) appeared in meninges and blood vessels at 2-4 h and diffusely penetrated the parenchyma at 8-24 h. The leukocytes strongly expressed IL-1beta and inducible nitric oxide synthase mRNAs. Beginning at 4-12 h, astrocytes (glial acidic fibrillary protein mRNA and protein and c-fos mRNA) and microglia (ionized calcium-binding adaptor molecule 1 mRNA and protein) showed widespread activation. Other rats received i.v. IL-1beta (6 microg/kg). Their brains showed induction of Ikappa(B)alpha and COX-2 mRNAs in the vasculature at 2 h but none of the other sequelae. In summary, our data indicate that IL-1beta in the cerebrospinal fluid reaches its target receptors on the endothelia via perivascular volume transmission, up-regulates ICAM-1, and triggers a targeted leukocyte emigration and widespread glial activation stimulated perhaps by pro-inflammatory molecules expressed by leukocytes. The dramatic difference between i.c.v. and i.v. routes of administration underscores the potency of IL-1beta within the brain to dynamically affect the cellular trafficking component of 'immune privilege'.

Clinical studies demonstrate that the antidepressant efficacy of St John's wort (Hypericum) is comparable to that of tricyclic antidepressants such as imipramine. Onset of efficacy of these drugs occurs after several weeks of treatment. Therefore, we used in situhybridization histochemistry to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, Hypericum extract, and hypericin (an active constituent of St John's wort) on the expression of genes that may be involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Imipramine (15 mg kg(-1)), Hypericum (500 mg kg(-1)), and hypericin (0.2 mg kg(-1)) given daily by gavage for 8 weeks but not for 2 weeks significantly decreased levels of corticotropin-releasing hormone (CRH) mRNA by 16-22% in the hypothalamic paraventricular nucleus (PVN) and serotonin 5-HT(1A) receptor mRNA by 11-17% in the hippocampus. Only imipramine decreased tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (by 23%), and only at 8 weeks. The similar delayed effects of the three compounds on gene transcription suggests a shared action on the centers that control HPA axis activity. A second study was performed to assess the effects of long-term imipramine and Hypericum administration on stress-induced changes in gene transcription in stress-responsive circuits. Repeated immobilization stress (2 h daily for 7 days) increased mRNA levels of CRH in the PVN, proopiomelanocortin (POMC) in the anterior pituitary, glutamic acid decarboxylase (GAD 65/67) in the bed nucleus of the stria terminalis (BST), cyclic AMP response element binding protein (CREB) in the hippocampus, and TH in the locus coeruleus. It decreased mRNA levels of 5-HT(1A) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Long-term pre-treatment with either imipramine or Hypericum reduced to control levels the stress-induced increases in gene transcription of GAD in the BST, CREB in the hippocampus, and POMC in the pituitary. The stress-induced increases in mRNA levels of CRH in the PVN and TH in the locus coeruleus were reduced by imipramine but not by Hypericum. The stress-induced decreases in BDNF and 5-HT(1A)mRNA levels were not prevented by either drug. Taken together, these data show: (1) that Hypericum and hypericin have delayed effects on HPA axis control centers similar to those of imipramine; and (2) that select stress-induced changes in gene transcription in particular brain areas can be prevented by long-term treatment with either the prototypic tricyclic antidepressant imipramine or the herbiceutical St John's wort. However, imipramine appears to be more effective in blocking stress effects on the HPA axis than the plant extract.

The brain's response to a direct immune challenge was examined by in situ hybridization histochemistry. Lipopolysaccharide (bacterial endotoxin) injected acutely into rat striatum induced mRNA expression for inhibitory factor kappaBalpha, interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6, IL-12 p35, inducible nitric oxide synthase, IL-1 receptor antagonist, and the type 1 IL-1 receptor. Expression patterns were evaluated at select time points ranging from 15 min to 3 days post-injection. Rats injected with vehicle alone were used to control for mechanical effects. Following lipopolysaccharide administration, a wave of mRNA induction within brain parenchyma radiated outward from the injection site, generally peaking in intensity at the 16-h time point. The individual profiles of cytokine mRNA induction patterns reveal that the brain's immune response to local inflammatory stimulation is quite elaborate and in many ways resembles the progression of cytokine induction customary of localized inflammation in peripheral tissues.

In situ hybridization histochemistry was used to show the distribution of messenger RNA for central cannabinoid CB 1 receptors in dorsal root ganglia of the rat. CB1 messenger RNA was highly expressed in neuronal subpopulations of rat dorsal root ganglia. The phenotypes of neurons that express messenger RNA for CB1 were subsequently examined by combining a 35S-labeled ribonucleotide probe for CB1 messenger RNA with digoxigenin-labeled riboprobes for preprotachykinin A (substance P precursor), alpha-calcitonin gene-related peptide and preprosomatostatin (somatostatin precursor) messenger RNAs. Qualitative examination revealed expression of CBI messenger RNA predominantly in medium-and large-sized cells distributed throughout the dorsal root ganglia. The majority of neurons expressing substance P messenger RNA were CB1 messenger RNA negative and smaller in size than the CB1 messenger RNA-positive cells. Only 13% of substance P messenger RNA-positive cells expressed CB1 messenger RNA. A similar degree of co-localization was observed with alpha-calcitonin gene-related peptide: 10% of cells expressing messenger RNA for this neuropeptide were CB1 messenger RNA positive. Co-localization of CB1 and somatostatin messenger RNAs was observed in less than 0.5% of somatostatin messenger RNA-positive cells. The data suggest that subpopulations of neurons in rat dorsal root ganglia are capable of synthesizing cannabinoid receptors and inserting them on terminals in the superficial dorsal horn. These findings provide anatomical evidence for cannabinoid modulation of primary afferent transmission. Although an anatomical basis for cannabinoid-mediated suppression of release of neurogenic peptides from nociceptive primary afferents is provided, our results demonstrate that the majority of CB messenger RNA-positive neurons in the dorsal root ganglia contain transmitters and/or neuromodulators other than the neuropeptides examined herein.

Brain cells responsive to a peripheral immune challenge, identified by in situ hybridization of c-fos mRNA following intravenous administration of the proinflammatory cytokine interleukin-1beta (IL-1) or sterile saline, were investigated at 0.5, 1, and 3 hours postinjection in rats. Doses of IL-1 ranged from 0.05 to 10 microg/kg; induction of c-fos mRNA occurred at > or = 0.5 pg/kg. The majority of IL-1-induced c-fos mRNA-positive cells were non-neuronal cells located in barrier regions of the brain. The cells became radiolabeled in two separate but related spatiotemporal patterns. The first pattern, occurring at 0.5 hour, was characterized by c-fos mRNA labeling of cells of the outer meninges (mainly arachnoid), blood vessels (arteries, veins, and capillaries), and choroid plexus. This activation pattern disappeared at 1 hour. At 3 hours, a second activation pattern appeared in cells located just inside the now quiescent barrier cells. In addition, the circumventricular organs each showed characteristic spatiotemporal labeling patterns resulting from successive activation of specific cell types, with a general spread of activation directed away from the circumventricular organs over time. At 3 hours post IL-1, c-fos and glial fibrillary acidic protein (GFAP) mRNAs showed colocalization in the arcuate nucleus/median eminence/glia limitans region. We propose that the first wave of activation is elicited by blood-borne immune signals, but the second wave is caused by molecules generated within the first set of activated cells. The transduced signal appears to propagate to neighboring receptive cells by extracellular diffusion. In this manner, blood-brain barrier cells can transduce peripheral IL-1 signals in widespread areas of the brain, although the circumventricular organs may be the most effective loci for signal transduction.

Inducible cyclooxygenase 2 (COX 2) converts arachidonic acid to prostaglandins, which are thought to mediate various peripheral lipopolysaccharide (LPS)-induced central effects, including generation of fever and activation of the hypothalamic-pituitary-adrenal axis. To localize prostaglandin production in the brain following peripheral LPS administration, COX 2 mRNA expression was examined by in situ hybridization histochemistry in rats injected intraperitoneally (i.p.) or intravenously (i.v.) with various doses of LPS or saline. Constitutive expression of COX 2 mRNA was found in neurons of cortex, hippocampus, and amygdala, but not in cells of the blood vessels. COX 2 mRNA levels were not altered in saline-injected animals as compared to non-injected controls. In LPS-injected animals, no consistent changes of neuronal COX 2 mRNA expression were observed. COX 2 mRNA expression appeared ex novo at 0.5-h post-injection in cells closely associated with blood vessels, however, ex novo labeling of the number of labeled cells increased to a peak at 2 h and subsided gradually to basal levels by 24 h. Initially, labeling was observed in cells comprising major surface-lying blood vessels and meninges. Later, vascular and perivascular cells associated with smaller penetrating blood vessels were labeled. This pattern of COX 2 mRNA induction is independent of the route and dose of the LPS injection. The induced COX 2 mRNA producing cells are identified as endothelial and leptomeningeal cells. Changes in COX 2 mRNA expression were not observed in circumventricular organs. These results suggest that peripheral LPS induces a rapid increase in COX 2 production throughout the vasculatures of the brain, which could affect the neuronal activity of widespread brain regions by elevating the levels of prostaglandins.

The inflammatory cytokine interleukin-1 has been implicated as a mediator of many centrally controlled responses, such as fever and increased activity of the hypothalamic-pituitary adrenal axis, after systemic infections. To identify the neuroanatomical loci of brain interleukin-1-producing cells during infection, we investigated interleukin-1beta messenger RNA expression by in situ hybridization histochemistry using a 500 nt ribonucleotide probe applied on brain sections from rats injected intraperitoneally with 2.5 mg/kg bacterial lipopolysaccharide or saline. In control animals, interleukin-1beta messenger RNA was not detectable. In the brains of lipopolysaccharide-injected animals, two temporally and spatially distinct waves of interleukin-1beta messenger RNA induction were observed. First, cell labelling appeared at 0.5 h, peaked at 2 h, and declined at 4-8 h. The labelled cells were concentrated in circumventricular organs--organum vasculosum of the lamina terminalis, subfornical organ, median eminence, and area postrema--and in choroid plexus, meninges, and blood vessels. Second, at 8-12 h, scattered small cells became labelled throughout the entire brain parenchyma; the labelling subsided by 24 h. Labelling was not observed in any neurons. In the pituitary, lipopolysaccharide induced strong interleukin-1beta messenger RNA expression initially in the anterior lobe at 0.5-1 h, and later in the neural lobe at 1-2 h, and subsiding thereafter. The results show that at early time points, peripheral lipopolysaccharide induces interleukin-1beta message production at the blood brain barrier and in circumventricular organs where the blood brain barrier is leaky. After a time delay of 6-10 h, however, interleukin-1beta messenger RNA is primarily expressed by non-neuronal cells of the brain in the brain parenchyma. These results suggest that the source of initial brain IL-1 activity after peripheral lipopolysaccharide injection derives from cells of the blood-brain barrier and the circumventricular organs, and the sustained interleukin-1 activity in the central nervous system thereafter is derived from glia.

The possible role that the hypothalamic arcuate nucleus might play in mediating the increase in paraventricular nucleus corticotropin-releasing hormone mRNA levels following adrenalectomy was investigated in two series of experiments. In the first series in situ hybridization histochemistry was used to quantify levels of eight accurate nucleus neuropeptide and neurotransmitter mRNAs in neurons that potentially relay adrenal steroid feedback to the paraventricular nucleus. In the second series of experiments, arcuate neuropeptidergic projections to the hypothalamic paraventricular nucleus were characterized using retrograde tracing in combination with in situ hybridization histochemistry. Despite an increase in paraventricular nucleus corticotropin-releasing hormone (60%) and pituitary proopiomelanocortin mRNA levels (sixfold), arcuate mRNA levels for proopiomelanocortin, neuropeptide Y, somatostatin, galanin, dynorphin, tyrosine hydroxylase, glutamate decarboxylase, and the glucocorticoid receptor were unchanged 14 days following adrenalectomy. Neuropeptidergic characterization of arcuatoparaventricular projections was achieved by injection of the retrograde tracer fluorogold into the paraventricular nucleus; retrogradely labeled neurons were characterized with polyclonal antisera against fluorogold in combination with oligonucleotide probes directed against neuropeptide Y, proopiomelanocortin, or somatostatin. Out of these three arcuate neuropeptide Y mRNA was contained in 18% of the fluorogold-positive neurons in the arcuate, proopiomelanocortin mRNA was contained in 8%, and somatostatin mRNA was contained in 6%. Overall, the results from both experiments suggest that the arcuatoparaventricular neuropeptide Y, proopiomelanocortin, and somatostatin projections are not sensitive to a chronic (14 day) lack of adrenal steroids. These projections as well as the other arcuate neurotransmitter and neuropeptide systems appear not to contribute to the persistent elevations in paraventricular nucleus corticotropin-releasing hormone mRNA levels or pituitary proopiomelanocortin mRNA levels found in 14 day adrenalectomized rats.

The distribution and density of cannabinoid receptor binding and messenger RNA expression in aged human brain were examined in several forebrain and basal ganglia structures. In vitro binding of [3H]CP-55,940, a synthetic cannabinoid, was examined by autoradiography in fresh frozen brain sections from normal aged humans (n = 3), patients who died with Alzheimer's disease (n = 5) and patients who died with other forms of cortical pathology (n = 5). In the structures examined--hippocampal formation, neocortex, basal ganglia and parts of the brainstem--receptor binding showed a characteristic pattern of high densities in the dentate gyrus molecular layer, globus pallidus and substantia nigra pars reticulata, moderate densities in the hippocampus, neocortex, amygdala and striatum, and low densities in the white matter and brainstem. In situ hybridization histochemistry of human cannabinoid receptor, a ribonucleotide probe for the human cannabinoid receptor messenger RNA, showed a pattern of extremely dense transcript levels in subpopulations of cells in the hippocampus and cortex, moderate levels in hippocampal pyramidal neurons and neurons of the striatum, amygdala and hypothalamus, and no signal over dentate gyrus granule cells and most of the cells of the thalamus and upper brainstem, including the substantia nigra. In Alzheimer's brains, compared to normal brains, [3H]CP-55,940 binding was reduced by 37-45% in all of the subfields of the hippocampal formation and by 49% in the caudate. Lesser reductions (20-24%) occurred in the substantia nigra and globus pallidus, internal segment. Other neocortical and basal ganglia structures were not different from control levels. Levels of messenger RNA expression did not differ between Alzheimer's and control brains, but there were regionally discrete statistically significant losses of the intensely expressing cells in the hippocampus. The reductions in binding did not correlate with or localize to areas showing histopathology, estimated either on the basis of overall tissue quality or silver staining of neuritic plaques and neurofibrillary tangles. Reduced [3H]55,940 binding was associated with increasing age and with other forms of cortical pathology, suggesting that receptor losses are related to the generalized aging and/or disease process and are not selectively associated with the pathology characteristic of Alzheimer's disease, nor with overall decrements in levels of cannabinoid receptor gene expression.

Electroconvulsive shock (ECS) is a highly effective therapy for the treatment of major depression, but its mechanisms of action are not known. We report that repeated ECS in rats produces enduring changes in two clinically relevant stress-responsive brain systems: (a) the hypothalamic-pituitary-adrenal axis regulated by corticotropin-releasing hormone (CRH) in the paraventricular nucleus; and (b) the NE system in the locus coeruleus regulated by tyrosine hydroxylase (TH). CRH and TH mRNA levels in these brain regions were assessed by in situ hybridization histochemistry. A single interaural ECS elevated TH but not CRH mRNA measured 24 h later. Repeated daily treatments (3, 7, or 14) elevated both mRNAs, maximally with 7, correlating with the time course of clinical efficacy. The elevations persisted for 3 (CRH) or 8 wk (TH) after the ECS. No other therapeutic treatment is known to produce such long-lasting changes in central nervous system gene expression. The time course of events (delayed onset, long duration) implicate CRH as a principal mediator of the antidepressant effects of ECS. The locus coeruleus-NE system may be important in initiating the central nervous system response.

This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.

A recent study demonstrated that hypothalamic lesions induced by goldthioglucose (GTG) in mice produced an increase in neuronal immunoreactivity for neuropeptide Y (NPY) in the hypothalamic arcuate nucleus. Since NPY is a potent stimulator of feeding, this increase represented a potential explanation for the hyperphagia seen after GTG lesions. To examine whether or not this increase in NPY immunoreactivity was accompanied by an increase in the mRNA for NPY, in situ hybridization histochemistry for NPY mRNA in control and in lesioned mice was performed. A 47% increase in NPY mRNA levels in the arcuate nucleus was observed in lesioned mice compared with controls, suggesting that an increased expression of the gene for NPY contributes to elevations in hypothalamic NPY after lesioning. This elevation in NPY may, in turn, relate to mechanisms provoking hyperphagia.

The acute and long-term effects of a single injection of psychomotor stimulants (amphetamine (1.5 mg/kg i.p.), cocaine (30 mg/kg i.p.) and GBR 12909 (10 mg/kg i.p.)) were studied with in situ hybridization histochemistry to assess alterations in the mRNA expression of enkephalin, dynorphin and substance P in the striatum. The greatest alterations on mRNA levels of enkephalin, dynorphin and substance P were observed 2 h following the first administration of each drug compared to that observed following a second challenge injection 14 days later. Of the drugs tested, the dopamine uptake inhibitory agents cocaine and GBR 12909 acutely elevated mRNA levels of all three neuropeptides, while amphetamine elevated mRNA levels of substance P only. A second challenge administration of the stimulants 14 days subsequent to the initial single injection re-elevated the mRNA level of substance P. An overall tolerance is speculated to account for diminution of the enkephalin and dynorphin responses to a challenge injection while a relative sensitization is suggested for the enkephalin response due to a reduction in the baseline level of expression produced by the first injection. The data also show that there are regional variation within the striatum following systemic administration of psychomotor stimulants, with greater elevations in the sensorimotor dorsolateral striatum than in the ventromedial 'limbic' nucleus accumbens region.

Various classes of antidepressant drugs with distinct pharmacologic actions are differentially effective in the treatment of classic melancholic depression--characterized by pathological hyperarousal and atypical depression--associated with lethargy, hypersomnia, and hyperphagia. All antidepressant agents exert their therapeutic efficacy only after prolonged administration. In situ hybridization histochemistry was used to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) administration of 3 different classes of activating antidepressant drugs which tend to be preferentially effective in treating atypical depressions, on the expression of central nervous system genes thought to be dysregulated in major depression. Daily administration (5 mg/kg, i.p.) of the selective 5-hydroxytryptophan (5-HT) reuptake inhibitor fluoxetine, the selective alpha 2-adrenergic receptor antagonist idazoxan, and the nonspecific monoamine oxidase A and B inhibitor phenelzine increased tyrosine hydroxylase mRNA levels by 70-150% in the locus coeruleus after 2 weeks of drug and by 71-115% after 8 weeks. The 3 drugs decreased corticotropin-releasing hormone mRNA levels by 30-48% in the paraventricular nucleus of the hypothalamus. The decreases occurred at 8 weeks but not at 2 weeks. No consistent change in steroid hormone receptor mRNA levels was seen in the hippocampus with the 3 drugs, but fluoxetine and idazoxan increased the level of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, respectively, after 8 weeks of drug administration. Proopiomelanocortin (POMC) mRNA levels in the anterior pituitary and plasma adrenocorticotropic-hormone (ACTH) levels were not altered after 2 or 8 weeks of drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The hippocampus appears to be an important modulator of the negative feedback effects of glucocorticoids on the hypothalamic-pituitary-adrenal axis. It is not known if hippocampal subfields CA1-4 or the dentate gyrus differentially alter gene expression of corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) of the hypothalamus. We, therefore, examined the effects of selective destruction of dentate gyrus granule cells, which send excitatory glutaminergic inputs to subfields CA4, CA3 and CA2, on CRH expression in the PVN. To determine the possible involvement of steroid receptors in the regulation of CRH expression, we examined the effects of intrahippocampal colchicine on gene expression of the mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptors in hippocampal CA fields and dentate gyrus. Colchicine produced a selective loss of dentate gyrus granule cells without affecting pyramidal cells in CA1-4 as early as 1 day after injection; granule cells were completely destroyed after 3 days. CRH mRNA levels were reduced by 38-48% in the PVN 2-14 days after colchicine. MR mRNA levels were decreased in dorsal and ventral CA fields 1-7 days after colchicine. GR mRNA levels were relatively unchanged, showing a slight decrease only in dorsal CA fields on days 2-7. Unexpectedly, CRH was transiently expressed in dorsal and ventral CA fields 1-3 days after colchicine. In the same time period, mRNA levels of inositol 1,4,5-trisphosphate kinase were decreased, suggesting that increases in neural metabolic activity, indicated by this marker, are not responsible for the transient CRH effect. The results suggest that the dentate gyrus is important for maintenance of steroid hormone receptor mRNA levels in the hippocampus and CRH expression in the hypothalamic PVN, and that CRH gene expression is differentially regulated in the hypothalamus and hippocampus.

The locus ceruleus-norepinephrine system is one of the principal effectors of the stress response. Acute stress induces norepinephrine synthesis and release, and noradrenergic cells compensate by increasing the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. Here we use in situ hybridization histochemistry to show the effects of acute and chronic intermittent stress on the expression of tyrosine hydroxylase mRNA in the rat locus ceruleus. Restraint stress increased tyrosine hydroxylase mRNA in the locus ceruleus but not in dopaminergic nuclei such as the substantia nigra or ventral tegmental area. One hour of footshock or restraint caused a rapid increase in locus ceruleus tyrosine hydroxylase mRNA which returned to basal levels within 24 h. Chronic intermittent stress (1 hour of restraint or footshock per day for 14 days) produced no change in tyrosine hydroxylase mRNA. Neither adrenalectomy nor dexamethasone replacement significantly affected mRNA expression. These findings indicate that acute stress can increase the expression of tyrosine hydroxylase mRNA in the locus ceruleus but that adaptation occurs to repeated stress, and that the expression of tyrosine hydroxylase mRNA in the locus ceruleus is independent of direct glucocorticoid modulation.

Hypothalamic neuropeptides play a role in appetite and weight regulation. Food restriction for 2 weeks and food deprivation for 4 days were used as models to characterize the effects of weight loss on hypothalamic peptide gene expression in male and female rats. We used in situ hybridization to examine the mRNA levels of hypothalamic peptides which stimulate and inhibit food intake and found selective effects primarily in the arcuate nucleus. Neuropeptide Y (NPY) mRNA was increased and pro-opiomelanocortin (POMC) and galanin (GAL) mRNA were decreased in the hypothalamic arcuate nucleus and corticotropin-releasing hormone (CRH) mRNA was decreased in the hypothalamic paraventricular nucleus in male and female food-restricted and food-deprived rats. Food restriction produced larger changes in peptide mRNA expression than did food deprivation. Changes in NPY, POMC and CRH gene expression induced by food restriction were greater in male than female rats. Elevated NPY and reduced CRH gene expression may be a compensatory physiological response to restore food intake in food-restricted and food-deprived animals. The discrete changes in NPY, POMC, GAL and CRH gene expression in food-restricted and food-deprived animals suggest the involvement of these peptides in abnormal appetitive behavior and weight loss associated with human eating disorders.

1. We have described a general ribonucleotide probe in situ hybridization methodology for localization of mRNA in frozen, unfixed tissue sections of brain. 2. The most important steps in obtaining consistent and reproducible autoradiographs with ribonucleotide probes were tissue acetylation and application of the radiolabeled probe to tissue sections under unsealed, glass coverslips. 3. Variability of the hybridization signal in tissue sections has been minimized to achieve a high degree of reproducibility within a given experiment as determined by densitometric analysis of rat glucocorticoid and mineralocorticoid receptor mRNA hybridization autoradiographs. 4. Tissue quality has been optimized for high-resolution anatomical localization of mRNA species by nuclear track emulsion. 5. The protocol is amenable to rapid, batchwise processing of tissue samples.