Rat Brain Sections Research Articles

GABAergic Interneuron Reorganization During the Late Period May Contribute to Hippocampal Epileptogenesis.

Alterations of Hippocampal GABAergic System Contribute to Development of Spontaneous Recurrent Seizures in the Rat Lithium-Pilocarpine Model of Temporal Lobe Epilepsys Andre V, Marescaux C, Nehlig A, Fritschy JM Hippocampus 2001;11:452–468 Reorganization of excitatory and inhibitory circuits in the hippocampal formation following seizure-induced neuronal loss has been proposed to underlie the development of chronic seizures in temporal lobe epilepsy (TLE). Here, we investigated whether specific morphological alterations of the GABAergic system can be related to the onset of spontaneous recurrent seizures (SRS) in the rat lithium-pilocarpine model of TLE. Immunohistochemical staining for markers of interneurons and their projections, including parvalbumin (PV), calretinin (CR), calbindin (CB), glutamic acid decarboxylase (GAD), and type 1 GABA transporter (GAT1), was performed in brain sections of rats treated with lithium-pilocarpine and sacrificed after 24 h, during the silent phase (6 and 12 days), or after the onset of SRS (10–18 days after treatment). Semiquantitative analysis revealed a selective loss of interneurons in the stratum oriens of CA1, associated with a reduction of GAT1 staining in the stratum radiatum and stratum oriens. In contrast, interneurons in CA3 were largely preserved, although GAT1 staining was also reduced. These changes occurred within 6 days after treatment and were therefore insufficient to cause SRS. In the dentate gyrus, extensive cell loss occurred in the hilus. The pericellular innervation of granule cells by PV-positive axons was markedly reduced, although the loss of PV-interneurons was only partial. Most strikingly, the density of GABAergic axons, positive for both GAD and GAT1, was dramatically increased in the inner molecular layer. This change emerged during the silent period, but was most marked in animals with SRS. Finally, supernumerary CB-positive neurons were detected in the hilus, selectively in rats with SRS. These findings suggest that alterations of GABAergic circuits occur early after lithium-pilocarpine-induced status epilepticus and contribute to epileptogenesis. In particular, the reorganization of GABAergic axons in the dentate gyrus might contribute to synchronize hyperexcitability induced by the interneuron loss during the silent period, leading to the onset of chronic seizures.

Determination of NADH in frozen rat brain sections by laser-induced fluorescence.

Methods to assess metabolism are important analytical tools in neuroscience. The fluorophore nicotinamide adenine dinucleotide (NADH) is a parameter of cellular metabolism. NADH fluorescence was measured using a laser-based fluorescence detector with spectral and temporal filters. Distribution and intensity of NADH fluorescence were investigated in frozen brain sections. In sections containing hippocampus the intensity of NADH fluorescence was correlated to brain structures. In order to investigate the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine was injected into the dorsal raphe nucleus 4 to 240 days prior to the measurement. NADH fluorescence decreased in the affected region by 50%, indicating that no recovery in metabolic activity had occurred.

High-resolution scanner for neuroanatomical analysis

This paper describes the use of a high-resolution flatbed scanner for neuroanatomical studies. Individual neurons that had been labeled using gold intensified diaminobenzidine (DAB) could be resolved in thionin-stained rat brain sections. One of the strengths of this method is that it permits the simultaneous visualization of labeled cells and their position relative to underlying cytoarchitectonic structures, allowing for highly accurate neuroanatomical analysis. High resolution maps of complete rat brains (∼100 sections spaced at 250 μm intervals) can be obtained in 10 h. Since much of this method can be automated, the scanning of brain sections is approximately 50% faster than conventional X–Y mapping, camera lucida, or photographic procedures. The method may have many other applications, particularly in evaluating the distribution of other types of labeled cells. For example, Fos immunoreactive neurons, cells labeled with reporter genes such as β-galactosidase in brains from transgenic animals, and unique histological inclusions such as plaques found in Alzheimer's disease could all be directly imaged without a microscope. In addition, the exchange of primary data between labs via the Internet will provide additional opportunities for collaboration.

Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocin-damaged rat brain.

To reveal whether an extract of Ginkgo biloba (EGb761) may affect streptozotocin (STZ)-induced impairments in brain glucose metabolism, autoradiographies of [3H]cytochalasin-B binding to the total population of glucose transporters, [125I]insulin binding to insulin receptors, [3H]glyburide binding to sulfonylurea receptors, and radioactive in situ hybridization for GLUT3 mRNA were carried out in hippocampal brain sections of adult rats that have additionally been divided into good performers (GP) and poor performers (PP) by behavioural tests before the experiments. The STZ-induced increases in hippocampal [3H]cytochalasin-B binding to (total) glucose transporters returned to almost normal values following EGb761 treatment, regardless of the experimental animal group (GP or PP) tested. Similarly, the STZ-mediated enhancements in hippocampal insulin receptor binding of GP rats were partially compensated by the treatment with EGb761. The data suggest beneficial effects of EGb671 on impaired brain glucose metabolism, at least under the experimental conditions used in the study presented.

Neuronal nitric oxide synthase immunohistochemistry and 4,5-diaminofluorescein diacetate: tools for nitric oxide research

Nitric oxide plays an important role in many biological processes including the cardiovascular, immune, reproductive and nervous systems. There is much interest surrounding this molecule and the enzyme responsible for its synthesis, nitric oxide synthase. In order to investigate the role of nitric oxide in various biological processes it is necessary to be able to identify the released molecule itself as well as nitric oxide synthase. Detection of nitric oxide synthase was achieved by optimisation of the immunohistochemical localisation of the neuronal isoform of the enzyme in primary cultures of rat ventral mesencephala and in fixed adult rat brain sections. Furthermore, using 4,5-diaminofluorescein diacetate, we have directly visualised endogenously produced nitric oxide in mesencephalic cultures and demonstrated the potential use of this indicator for visualising nitric oxide produced in vivo.

Cation modulation of GABA A receptors in brain sections of AT and ANT rats

Changes in magnesium ion (Mg 2+) concentration may be implicated in alcohol-related behaviors through modulation of neuronal excitability by actions on ligand-gated ion channels. To study whether putative Mg 2+-binding sites differ between two rat lines, alcohol-insensitive (AT) and alcohol-sensitive (ANT) rats, selectively outbred for differential sensitivity to the motor-impairing effect of ethanol, we compared the effect of Mg 2+ on [ 35S] tert-butylbicyclophosphorothionate ([ 35S]TBPS) binding to GABA A receptors with the use of ligand autoradiographic analyses of brain sections from these rats. There were some slight differences between the rat lines in modulation of the binding in the forebrain. A low concentration of Mg 2+ (0.1 mM) inhibited basal [ 35S]TBPS binding more efficiently in the central gray matter and hippocampus in the ANT rats than in the AT rats. In the presence of γ-aminobutyric acid, the effect of a low concentration of Mg 2+ was higher in the caudate-putamen and inner layer of the cerebral cortex in the AT rats than in the ANT rats. No difference between the rat lines was found at a higher (3 mM) Mg 2+ concentration. Furosemide, a GABA A antagonist selective for cerebellar granule cell–specific α6β2/3 subunit–containing receptors, was less efficient in antagonizing the Mg 2+-induced inhibition of [ 35S]TBPS binding in the ANT rats than in the AT rats. Another divalent cation, zinc ion, was less efficient in displacing [ 35S]TBPS binding from the cerebellar granule cell layer in the ANT rats than in the AT rats, whereas a trivalent cation, lanthanum ion, produced identical modulation of the binding in the two rat lines. The results indicate that the alcohol-sensitive ANT rats have altered cerebellar granule cell–specific α6 subunit–containing GABA A receptors and seem to indicate that these receptors might be implicated in the sensitivity difference of the rat lines to ethanol and sedative drugs.

Oestradiol increases phosphorylation of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in female rat brain.

Recent studies suggest that oestrogen and progestin receptors may be activated by the neurotransmitter dopamine, as well as by their respective ligands. Because intracerebroventricular infusion of D(1), but not D(2), dopaminergic receptor agonists increases oestrous behaviour in oestradiol-primed rats, we wanted to determine if treatment with oestradiol alters the activity of D(1) receptor-associated processes in steroid receptor-containing areas in female rat brain. One D(1) receptor-associated phosphoprotein that may be influenced by oestradiol is a dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000 (DARPP-32). Because DARPP-32 is phosphorylated in response to dopamine acting via a cAMP-dependent protein kinase, it provides a useful marker to examine where in the brain a particular stimulus might be altering the activity of D(1) receptor-containing neurones. To determine if oestradiol alters the phosphorylation of DARPP-32, we stained immunocytochemically brain sections of female rats treated with behaviourally relevant doses of oestradiol or oil vehicle with an antibody that detects only the threonine 34-phosphorylated form of DARPP-32. Behaviourally effective doses of oestradiol increase the phosphorylation of DARPP-32 within the medial preoptic nucleus, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and the ventromedial nucleus of the hypothalamus, 48 h after treatment. These data suggest that oestradiol increases the activity of D(1) dopamine receptor-associated processes in oestrogen receptor-containing areas of female rat forebrain.

Recruitment of neutrophils across the blood-brain barrier: the role of E- and P-selectins.

The adult central nervous system parenchyma is resistant to inflammation, but in juvenile rats the injection of inflammatory mediators, interleukin-1 beta for example, gives rise to extensive neutrophil recruitment and neutrophil-dependent blood-brain barrier breakdown. The factors that confer this resistant phenotype are unknown. In this study, the authors demonstrate that E- and P-selectin expression is increased to a similar extent in adult and juvenile brain after the intracerebral injection of IL-1 beta. Thus, the refractory nature of the brain parenchyma cannot be attributed to an absence of selectin expression. However, in injuries where the resistant characteristic of the brain parenchyma is compromised, and neutrophil recruitment occurs, selectin blockade may be an advantage. The authors investigated the contribution that selectins make to neutrophil recruitment during acute inflammation in the brain. The authors examined neutrophil recruitment by immunohistochemistry on brain sections of juvenile rats killed four hours after the intracerebral injection of IL-1 beta and the intravenous injection of neutralizing anti-selectin monoclonal antibodies (mAb). The administration of the P-selectin blocking mAb inhibited neutrophil recruitment by 85% compared with controls. Surprisingly, E-selectin blockade had no effect on neutrophil recruitment to the brain parenchyma. Thus, P-selectin appears to play a pivotal role in mediating neutrophil recruitment to the brain parenchyma during acute inflammation.

Gliogenesis in the striatum of the adult rat: alteration in neural progenitor population after psychostimulant exposure

Cytogenesis from proliferating progenitor cells is present in the rat brain throughout adulthood, and is regulated by a variety of environmental stimuli. To determine whether adult cytogenesis occurs in the intact rat striatum and to explore the possible regulatory role of psychostimulant exposure on striatal cytogenesis, immunohistochemistry with the thymidine analog bromodeoxyuridine (BrdU), a marker of DNA synthesis that labels dividing cells and their terminal progeny, was performed on the brain sections of normal adult rats and rats treated with a psychostimulant, amphetamine (AMPH). Scattered cells that incorporated BrdU were consistently seen throughout the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum 24 h after BrdU injection. Three to four weeks after BrdU injection, approximately 10–20% of surviving newborn cells differentiated into astroglia according to their radial morphology of glia and co-expression of an astroglial marker, S100β. However, none of BrdU-positive cells were found to co-localize with a neuronal marker, neuronal nuclear antigen (NeuN). Acute injection of AMPH at a behaviorally active dose (10 mg/kg, i.p.) produced a rapid and transient decrease in the number of BrdU-labeled cells in both the dorsal (70.6% of control) and ventral (66.7% of control) striatum, but not in the subventricular zone and the hippocampal dentate gyrus. However, the fraction of differentiated astrocytes was not altered 3–4 weeks after AMPH treatment. These results indicate an existence of active gliogenesis (both proliferation and differentiation) in the adult rat striatum. Vulnerability of striatal cytogenesis to psychostimulant exposure indicates a new approach to elucidate brain mechanisms responsible for addictive properties of drugs of abuse.

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses.

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.

Regional localization and developmental profile of acetylcholinesterase-evoked increases in [(3)H]-5-fluororwillardiine binding to AMPA receptors in rat brain.

In addition to its role in hydrolyzing the neurotransmitter acetylcholine, the synaptically enriched enzyme acetylcholinesterase (AChE) has been reported to play an important role in the development and remodelling of neural processes and synapses. We have shown previously that AChE causes an increase in binding of the specific AMPA receptor ligand (S)-[(3)H]-5-fluorowillardiine ([(3)H]-FW) to rat brain membranes. In this study we have used quantitative autoradiography to investigate the regional distribution and age-dependence of AChE-evoked increases in the binding of [(3)H]-FW in rat brain. Pretreatment of rat brain sections with AChE caused a marked enhancement of [(3)H]-FW binding to many, but not all, brain areas. The increased [(3)H]-FW binding was blocked by the specific AChE inhibitor BW 284c51. The maximal potentiation of [(3)H]-FW binding occurred at different developmental age-points in different regions with a profile consistent with the peak periods for synaptogenesis in any given region. In addition to its effects on brain sections, AChE also strongly potentiated [(3)H]-FW binding to detergent solubilized AMPA receptors suggesting a direct action on the receptors themselves rather than an indirect effect on the plasma membrane. These findings suggest that modulation of AMPA receptors could provide one molecular mechanism for the previously reported effects of AChE on synapse formation, synaptic plasticity and neurodegeneration.

Magnesium potentiation of the function of native and recombinant GABA(A) receptors.

Mg2+ decreased basal and GABA-inhibited t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor ion channels in rat brain sections up to 1 mM, but increased the binding at 10 mM. The Mg2+-effect was detectable in the presence of a specific GABA site competitive antagonist. Two-electrode voltage clamp recordings of recombinant alpha1beta2gamma2S, alpha1beta2, alpha2beta2gamma2S and alpha2beta2 GABAA receptors revealed a potentiation by 0.1-1 mM Mg2+ of EC20 GABA-evoked ion currents. At 10 mM, Mg2+ decreased the currents. In the absence of GABA, Mg2+ did not evoke any currents. The results show that physiologically relevant Mg2+ concentrations affect the GABA responses on GABAA receptors in native and the main recombinant receptor subtypes, suggesting putative Mg2+ binding sites on the receptor complex.

Agonist-stimulated [ 35S]GTPγS autoradiography: optimization for high sensitivity

The receptor-stimulated accumulation of [ 35S]GTPγS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [ 35S]GTPγS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [ 35S]GTPγS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for μ-opiate receptors, 5-HT 1A receptors and α 2-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [ 35S]GTPγS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [ 35S]GTPγS incubation time (2–4 h) and guanosine 5′-diphosphate (GDP) preincubation (20–30 min), and use of [ 35S]GTPγS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [ 35S]GTPγS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [ 35S]GTPγS autoradiography to receptors, which produce low levels of [ 35S]GTPγS binding and to the measurement of changes in receptor–G protein coupling.

Cellular localization of BM88 mRNA in paraffin-embedded rat brain sections by combined immunohistochemistry and non-radioactive in situ hybridization

With the advent of gene cloning and sequencing, it has become increasingly common to identify novel genes for which no antibody is available. The best approach to study the expression and the distribution of these new genes is by in situ hybridization. One of the challenges with this method is to define the exact cellular subtype where the gene of interest is expressed. Conventional isotopic in situ hybridization methods lack precision for cellular identification because radioactive probes often result in a scattered signal. To identify the exact cellular subtype expressing BM88, we established a rapid colocalization method using non-isotopic in situ hybridization followed by chromogenic immunohistochemistry on the same tissue section. We demonstrated that BM88, which was identified from subtractive hybridization experiments between normal and ischemic tolerant brain tissue, was expressed exclusively in neurons in normal adult rat brain. Paraffin-embedded tissue was used as it resulted in better preservation of tissue and cellular morphology, thus allowing for more accurate histological localization of gene expression. It also allowed for retrospective studies on a number of archived tissue samples.

Preferential Cytoplasmic Localization of δ-Opioid Receptors in Rat Striatal Patches: Comparison with Plasmalemmal μ-Opioid Receptors

The activation of delta-opioid receptors (DORs) in the caudate-putamen nucleus (CPN) produces regionally distinct changes in motor functions, many of which are also influenced by opioids active at micro-opioid receptors (MORs). These actions most likely occur in MOR-enriched patch compartments in the CPN. To determine the functional sites for DOR activation and potential interactions involving MOR in these regions, immunoperoxidase and immunogold-silver labeling methods were applied reversibly for the ultrastructural localization of DOR and MOR in single rat brain sections containing patches of the CPN. DOR immunoreactivity was commonly seen within the cytoplasm of spiny and aspiny neurons, many of which also expressed MOR. In dendrites and spines, DOR labeling was preferentially localized to membranes of the smooth endoplasmic reticulum and spine apparatus, whereas MOR showed a prominent plasmalemmal distribution. DOR- and/or MOR-labeled spines received asymmetric, excitatory synapses, some of which showed notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids.

Allosteric modulation of [3H]-CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain.

Binding of D,L-(E)-2-amino-4-[(3)H]-propyl-5-phosphono-3-pentenoic acid ([(3)H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques. In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [(3)H]-CGP39653 binding, but competitively reversed the high affinity component of [(3)H]-CGP39653 binding inhibition by glycine, with a pK(B) value of 8.38, in line with its affinity for the glycine site (pK(i)=8.49 vs. [(3)H]-glycine). Glycine (10 microM) significantly decreased [(3)H]-CGP39653 affinity for the NMDA receptor (with no change in the B(max)), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test). In rat brain sections the addition of GV150526A (30 microM) to the incubation medium increased [(3)H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [(3)H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation. Whole brain autoradiography revealed an uneven distribution of [(3)H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [(3)H]-CGP39653 binding was increased to 216% of control (mean between areas) by 30 microM GV150526A. The enhancement, however, did not present significant regional differences. These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [(3)H]-CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L-glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [(3)H]-CGP39653 binding sites in human brain.

A modified roller method for organotypic brain cultures: free-floating slices of postnatal rat hippocampus

We describe a novel procedure for organotypic cultivation of free-floating brain sections of postnatal rats with a modified roller technique. Three hundred to 350-μm-thick sections of hippocampus are cultured for 13–15 days at 35.5°C in 10–15 ml of feeding medium in 50–100 ml bottles under constant rotation on a horizontal high-speed mini-roller (60 rpm). Histological analysis (paraffin sections, Nissl Cresyl Violet and Hematoxylin/Eosin staining) demonstrates good survival of neuronal and glial cells and complete preservation of the neuronal organization of cultivated hippocampus with minimal central necrosis. This novel protocol permits not only survival and development of long-term three-dimensional organotypic postnatal brain tissue but also allows simultaneous cultivation of any number of brain sections in one bottle (up to 50 and even more) and therefore is useful for high throughput study of neurocytotoxic and hypoxic/ischemic neuronal damage with subsequent histological, immunocytochemical, biochemical, and molecular analysis.

A GABA-neuropeptide Y (NPY) interplay in LH release

Neuropeptide Y (NPY) stimulates and γ-amino butyric acid (GABA) inhibits LH release in the rat. Since a sub-population of NPY-producing neurons in the arcuate nucleus (ARC) of the hypothalamus co-express GABA, the possibility of an interplay between NPY and GABA in the release of LH was investigated in two ways. First by employing light and electron microscopic double staining for NPY and GABA, using pre and post-immunolabeling on rat brain sections, we detected GABA in NPY immunoreactive axon terminals in the MPOA, one of the primary sites of action of these neurotransmitters/neuromodulators in the regulation of LH release. These morphological findings raised the possibility that inhibitory GABA co-released with NPY may act to restrain the excitatory effects of NPY on LH release. Muscimol (MUS, 0.44 or 1.76 nmol/rat), a GABA A receptor agonist, administered intracerebroventricularly (icv), alone failed to affect LH release, but NPY (0.47 nmol/rat icv) alone stimulated LH release in ovarian steroid-primed ovariectomized rats. On the other hand, administration of MUS blocked the NPY-induced stimulation of LH release in a dose-dependent manner. Similarly, administration of MUS abolished the excitatory effects on LH release of 1229U91, a selective NPY Y4 receptor agonist. These results support the possibility that in the event of co-release of these neurotransmitters/neuromodulators, GABA may act to restrain stimulation of LH release by NPY during the basal episodic and cyclic release of LH in vivo.

Distribution of 5-ht 5A, 5-ht 5B, 5-ht 6 and 5-HT 7 receptor mRNAs in the rat brain

The precise involvement of 5-ht 5A, 5-ht 5B, 5-ht 6 and 5-HT 7 receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht 5A mRNA was widely expressed while 5-ht 5B mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht 6 mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT 7 mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.

Characterization of 5‐HT1A,B and 5‐HT2A,C Serotonin Receptor Binding

AbstractThis unit describes assays for measuring the binding of radioligands to two major types of receptors for 5‐hydroxytryptamine (5‐HT or serotonin), 5‐HT1 and 5‐HT2 receptors, in homogenates of brain tissue or cloned into cells in culture. The specific receptor subtypes covered are 5‐HT1A, 5‐HT1B, 5‐HT2A, and 5‐HT2C. In addition, methodology for using quantitative autoradiography to measure radioligand binding to serotonin receptors in brain slices is described. Protocols are provided for characterization of both saturation and competition binding assays, and instructions for data analysis of these assays is also described. In addition, methodology is provided for the quantification (image analysis) of radioligand binding in brain tissue sections to determine receptor density, preparation of rat brain sections for quantitative autoradiography, and thionin staining of thaw‐mounted tissue sections to define certain brain regions.