Summary of a National Institute of Mental Health workshop: developing animal models of anxiety disorders.

RATIONALE: There exists a wide range of animal models and measures designed to assess anxiety or fearfulness. However, the relationship between these models and clinical anxiety symptoms and syndromes is unclear. The National Institute of Mental Health convened a workshop to discuss the relationship between existing behavioral models of anxiety and the clinical profile of anxiety disorders. A second goal of this workshop was to outline various approaches towards modeling components of anxiety disorders. OBJECTIVES: To briefly describe epidemiological and behavioral manifestations of clinical anxiety syndromes and how they relate to commonly employed animal models of anxiety. To describe approaches and considerations for developing, improving, and adapting anxiety models to better understand the neurobiology of anxiety. METHODS: Clinicians, psychiatrists and clinical and basic neuroscientists presented data exemplifying different approaches towards understanding anxiety and the role of animal models. Panel members outlined what they considered to be critical issues in developing and employing animal models of anxiety. RESULTS: This review summarizes the discussions and conclusions of the workshop including recommendations for improving upon existing models and strategies for developing novel models. CONCLUSIONS: The probability of developing comprehensive animal models that accurately reflect the relative influences of factors contributing to anxiety disorder syndromes is quite low. However, ample opportunity remains to better define and extend existing models and behavioral measures related to specific processes that may be disrupted in anxiety disorders and to develop new models that consider the impact of combined factors in determining anxious behaviors.

The mouse calretinin gene promoter region: structural and functional components.

The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.

Calretinin expression in the chick brainstem auditory nuclei develops and is maintained independently of cochlear nerve input.

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR-IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR-IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR-IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR-IR levels at 3 hours, 1 day, or 3-4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound-evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching.

Calretinin mRNA and immunoreactivity in the medullary reticular formation of the rat: colocalization with glutamate receptors.

Calretinin-positive cells were identified in the medullary reticular formation of the rat by both immunohistochemistry and in situ hybridization histochemistry. In addition, double immunocytochemical labeling was used to examine the degree of colocalization of calretinin with GluR2/R3, GluR4 and GluR5-7 glutamate receptor subtypes. Results indicated regional variation in calretinin expression across reticular formation regions with the exception of the largest cells which were mostly calretinin-positive. Calretinin mRNA was particularly abundant in the parvocellular reticular nucleus. Most calretinin-immunoreactive cells also expressed at least one of the glutamate receptor subtypes examined with the exception of the smallest calretinin-positive cells of the parvocellular reticular formation which were generally not immunoreactive for any of the glutamate receptors examined. Calretinin immunoreactivity was colocalized with immunoreactivity for all three glutamate receptor subtypes examined in most of the large cells of the reticular formation. Immunoreactivity for the GluR4 antibody was least abundant in the reticular formation and GluR4 immunoreactive cells were least likely to co-express calretinin. These results suggest that calretinin and glutamate receptor antibodies may be used to identify specific subsets of reticular formation neurons.

Cellular expression of MAP 2 kinase in rat brain.

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density. Thus, MAP 2 kinase expression was particularly high in regions such as the locus coeruleus, the piriform cortex, the dentate gyrus granule cell layer, pyramidal cells of the hippocampus, the mitral cells of the olfactory bulb, and the large motor neurons of the V and VII nerves. This apparent ubiquitous distribution suggests an important role of MAP 2 kinase in the cellular functions in most cells of the adult brain.

Antibody recognition of calcium-binding proteins depends on their calcium-binding status.

Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.

Alteration in levels of expression of brain calbindin D-28k and calretinin mRNA in genetically epilepsy-prone rats.

Variations in the concentration of free calcium in neurons is believed to play a major role in regulating neuronal excitability. Because calcium-binding proteins such as calbindin D-28k and calretinin help to regulate intracellular calcium, we investigated the possibility that the expression of these proteins may be affected in genetically epilepsy-prone rats (GEPRs). The mRNA levels of both proteins were compared across several brain regions using in situ hybridization histochemistry and Northern blot analysis with semiquantitation by optical density measures in autoradiograms from two GEPR strains that differ in the severity of audiogenic seizures (GEPR9 and GEPR3) and from Sprague-Dawley rats. Results revealed a lower level of expression in calbindin D-28k mRNA in the in the caudate putamen-accumbens nuclei in GEPR3 (-30%) and GEPR9 (-60%) relative to controls. The calbindin D-28k mRNA level was also lower in the reuniens nucleus of the thalamus (-41% in GEPR3; -34% in GEPR9). The calretinin mRNA level was lower in the substantia nigra compacta of both GEPR rat strains (-31% in GEPR3 and -34% in GEPR9 relative to controls). No changes in mRNA were detected in other brain regions expressing calbindin D-28k or calretinin mRNA. These results indicate that the expression of these related calcium-binding proteins is altered in the GEPRs before the induction of seizures. This initial defect could alter either the calcium-buffering capacity or regulation of calcium-mediated processes by these proteins and thus play a role in the molecular cascade of events inducing the genetic susceptibility to, and the generalization of, seizures in these rat strains.

Distribution of calretinin, calbindin D28k, and parvalbumin in subcellular fractions of rat cerebellum: effects of calcium.

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

Localization of Ca(2+)-dependent conformational changes of calretinin by limited tryptic proteolysis.

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

Quadruple colocalization of calretinin, calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in fibers within the villi of the rat intestine.

Double-labeling immunofluorescent histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.

Effects of unilateral cochlea ablation on the distribution of calretinin mRNA and immunoreactivity in the guinea pig ventral cochlear nucleus.

The predominantly neuronal, calcium-binding protein calretinin is highly expressed in the guinea pig auditory system. Within the ventral cochlear nucleus (VCN), calretinin-positive auditory nerve fibers terminate on many calretinin-containing bushy, octopus, and multipolar cells. The abundance of calretinin in the cochlear nucleus provides an ideal system for examining the effects of altered neuronal input on the expression of this calcium-binding protein. The present experiments examined the effects of unilateral cochlea ablation on calretinin immunoreactivity and mRNA levels in the VCN. Calretinin mRNA was labeled by in situ hybridization histochemistry using a radioactive oligonucleotide probe and was quantified by optical density measures on autoradiograms. Survival times of 1, 7, and 56 days postlesion were examined. The results revealed a consistent increase in calretinin mRNA in the rostral portion of the ipsilateral anterior VCN 1 day postlesion but no effect on calretinin mRNA in this region at 7 and 56 days postlesion. The intensity of immunohistochemical label was also increased at 1 and 7 days after surgery. In contrast, calretinin mRNA was not affected 1 day postlesion in the ipsilateral posterior VCN but was decreased at both 7 and 56 days postlesion. The decrease in calretinin mRNA in the posterior VCN at longer survival times was accompanied by decreased immunolabeling of fibers projecting from VCN cells to the superior olivary complex. These results suggest that calretinin gene expression is regulated in part by auditory nerve activity in some cochlear neurons but that additional factors related to the unique cellular milieu also control calretinin expression.

Ca(2+)-dependent and independent interactions of calretinin with hydrophobic resins.

The ability of rat calretinin to bind to hydrophobic resins in a Ca(2+)-dependent manner was examined. Both native calretinin present in cerebellum extract and purified recombinant calretinin bound similarly to hydrophobic resins such as phenyl-, hexyl-, octyl-, and W7-agarose. Hydrophobic interactions of calretinin were partially Ca(2+)-dependent since 1/3 of bound protein was released from the resins by EGTA under varied conditions. Some calretinin tryptic fragments bound to octyl-agarose in a manner similar to uncleaved calretinin, while others bound to the resin in a Ca(2+)-independent manner. These and other results suggest that calretinin has several hydrophobic regions of varied strength and sensitivity to Ca2+. It is proposed that the local changes in hydrophobicity induced by Ca2+ binding might be relevant for calretinin functions.

Expression and rapid purification of recombinant rat calretinin: similarity to native rat calretinin.

Rat calretinin coding region was subcloned into a prokaryotic expression vector (pGEX). The glutathione-S-transferase:calretinin fusion protein produced in Escherichia coli was purified on a glutathione-Sepharose affinity column. Recombinant rat calretinin was cleaved on the column by thrombin, eluted, and purified to homogeneity using DEAE-cellulose chromatography. Recombinant and native rat calretinin performed the same on DEAE columns, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Western blots, and 45Ca overlay on nitrocellulose blots. The recombinant calretinin migrated similarly to the more basic (pI 5.3) of two forms of native calretinin demonstrated by two-dimensional SDS-PAGE. Calcium binding equilibria revealed identical apparent binding affinity and capacity. Difference(s) between native and recombinant did not affect the binding of calcium to calretinin or antibody recognition. Thus recombinant calretinin may be useful in the elucidation of possible cellular targets of native calretinin.

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes.

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensen's cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.

Calretinin distribution in the thalamus of the rat: immunohistochemical and in situ hybridization histochemical analyses.

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei. A subpopulation of calretinin cell bodies was also identified in the reticular nucleus. The mediorostral lateral posterior nucleus, subparafascicular, lateral geniculate and habenular nuclei also contained calretinin messenger RNA probe label. In contrast, no positive cells were found in the anterior, ventral or posterior thalamic nuclei. The distribution of calretinin cells did not correspond directly with that of other histochemical markers. Thus, the in situ hybridization histochemical and immunohistochemical results revealed calretinin as a unique identifying marker for distinct sets of thalamic neurons.

Identification of neuron subpopulations in the rat vestibular ganglion by calbindin-D 28K, calretinin and neurofilament proteins immunoreactivity.

Immunocytochemical and morphometric analyses were combined to demonstrate the presence of neuron subpopulations in the rat vestibular ganglion. Monoclonal antibodies reacting with neurofilament proteins (NF), calbindin-D 28K (CaBP) and calretinin (CaR) were used. Three subpopulations were identified: (1) CaBP- and CaR-positive neurons were the largest neurons (16%) and they were also highly NF-immunoreactive; (2) exclusively NF-positive neurons; (3) unlabelled neurons, representing about two-thirds of the population.

6-Hydroxydopamine induced impairment of Pavlovian conditioning in the rabbit.

This study employed bilateral, intraventricular injections of 6-hydroxydopamine (6-HDA) to examine the effects of monoamine depletion on Pavlovian conditioning of the rabbit's nictitating membrane response. 6-HDA produced dose-dependent and highly correlated decreases in the rate of acquisition of conditioned responses and in the telencephalic content of 5-HT, DA, and NE. At the highest dose of 6-HDA (1340 micrograms), 5-HT, DA, and NE were reduced by 42, 48, and 89%, respectively, and the number of trials required to achieve criterion acquisition was increased by 123%. Control experiments established that the highest dose of 6-HDA: 1) had no effect on the unconditioned nictitating membrane reflex; 2) had no effect on the threshold of the conditioned stimulus for eliciting conditioned responses; and 3) produced only a small, less than 5%, decrease in nonassociative responding. It was concluded that decreases in 5-HT, DA, and NE can impair associative learning without altering sensory or motor function.

Calretinin, a neuronal calcium binding protein, inhibits phosphorylation of a 39 kDa synaptic membrane protein from rat brain cerebral cortex.

The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin. At the same concentration, calretinin produced a slight increase in the phosphorylation of several other synaptic membrane proteins which appeared additive with the stimulation produced by either calmodulin or phosphatidylserine in the presence of calcium.

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear.

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, at Mr = 29 k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.

Immunohistochemical localization of calretinin in the rat hindbrain.

The localization of calretinin in the rat hindbrain was examined immunohistochemically with antiserum against calretinin purified from the guinea pig brain. Calretinin immunoreactivity was found within neuronal elements. The distribution of calretinin-immunoreactive cell bodies and fibers is presented in schematic drawings and summarized in a table. Major calretinin-immunoreactive neurons were found in the lateral and medial geniculate nuclei, substantia nigra, ventral tegmental area, interpeduncular nucleus, periaqueductal gray, mesencephalic trigeminal nucleus, superior and inferior colliculi, pontine nuclei, parabrachial nucleus, dorsal and laterodorsal tegmental nuclei, cochlear nuclei, vestibular nuclei, medullary reticular nuclei, nucleus of the solitary tract, area postrema, substantia gelatinosa of the spinal trigeminal nucleus, and cerebellum. These results show that distinct calretinin-immunoreactive neurons are widely distributed in the rat hindbrain.