R. Linke

Differential projection patterns of superior and inferior collicular neurons onto posterior paralaminar nuclei of the thalamus surrounding the medial geniculate body in the rat

The thalamic nuclei at the medial border of the medial geniculate body (i.e. the suprageniculate nucleus, the medial division of the medial geniculate nucleus, the posterior intralaminar nucleus and the peripeduncular nucleus) which relay sensory information to the amygdala are thought to receive convergent input from multiple sites. In order to delineate the organization of these multimodal thalamic nuclei, the locations of superior and inferior collicular neurons projecting to these nuclei were studied by means of retrograde transport methods.

Modulation of mRNA expression of the neurotrophins of the nerve growth factor family and their receptors in the septum and hippocampus of rats after transient postnatal thyroxine treatment I. Expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin 4 mRNA

Abstract Early postnatal application of thyroid hormones to rats results in morphological changes of the septo-hippocampal cholinergic and the hippocampal mossy fiber systems. Modulation in the expression of either neurotrophins and/or their receptors is postulated to be involved in these effects. In a recent study, we showed that, after thyroxine application, the mRNA expression of neurotrophins of the nerve-growth-factor (NGF) family is significantly upregulated both in septum and hippocampus. To test whether the neurotrophin receptors (the low-affinity neurotrophin receptor p75 and the specific high-affinity receptors trkA, trkB, and trkC) were also affected by hormone administration, newborn rats were treated daily with subcutaneous injections of thyroxine until postnatal day 12 (P12) at latest. Control animals received corresponding injections of saline. The pups were sacrificed at defined intervals from P9 to P14. The septal areas and the hippocampi were analyzed using the reverse-transcription polymerase chain reaction (RT-PCR) method for quantification of p75, trkA, trkB, and trkC mRNA levels. Analysis of variance over the total investigation period revealed no significant general increases of the gene expressions of either neurotrophin receptor, neither in the septum nor in the hippocampus, although previous results have shown marked changes in neurotrophin levels. On particular postnatal days, significant upregulation could be observed in hippocampus for trkB and trkC. From these and recent data, we conclude that modulation of neurotrophin expression rather than neurotrophin-receptor expression contributes to the morphological modifications within the hippocampal mossy fiber system and the septo-hippocampal cholinergic system.

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic system in mice. I. Cholinergic and GABAergic markers.

In the present study, variations of cholinergic and GABAergic markers in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) and the hippocampus of eight different inbred mouse strains were investigated. By means of immunocytochemistry against the acetylcholine‐synthesizing enzyme choline acetyltransferase (ChAT), the cholinergic neurons were visualized and the number of ChAT‐positive neuronal profiles in the MS/vDB was counted. Cholinergic and GABAergic septo‐hippocampal projection neurons were detected with a combined retrograde tracing and immunocytochemical approach. In order to quantify the cholinergic innervation of various hippocampal subregions, we estimated the density of acetylcholinesterase (AChE)‐containing fibers as visualized by AChE histochemistry. Additionally, the densities of muscarinic receptors (mainly the subtypes M1 and M2) in different hippocampal areas of seven inbred strains were measured by means of quantitative receptor autoradiography.

Organization of projections to temporal cortex originating in the thalamic posterior intralaminar nucleus of the rat

Abstract Thalamic nuclei surrounding the medial geniculate body, among which the posterior intralaminar nucleus (PIN) is one of the largest, have great importance in fear-potentiated emotional behavior. Due to limited knowledge of the distribution of the cortical projections of the PIN, the connections between the temporal neocortex and the PIN were investigated by means of axonal transport of Phaseolus vulgaris leucoagglutinin or Miniruby. After iontophoretic injections of either tracer, anterogradely labeled terminals showed a broad, but not a diffuse, distribution in temporal and adjacent cortices (perirhinal, secondary auditory, visceral, secondary somatosensory, agranular insular cortices). A common projection to all areas was found in the upper layer I except for perirhinal cortex, where this projection was confined to the basal layer I. In selected cortical fields (ectorhinal, perirhinal, visceral cortices), an additional projection to layers III/IV was found. The corticofugal projection to the PIN originated from pyramidal neurons in layer V and – in some regions – in layer VI. The present results demonstrate a distinct and selective projection of the PIN to several areas of the temporal neocortex, which may activate inter- and intra-areal cortical circuits during processing of auditory stimuli.

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic systems in mice: II. Morpho-behavioral correlations

We investigated the contribution of the septo‐hippocampal cholinergic and GABAergic system to spatial and nonspatial aspects of learning and memory that had previously been found to correlate with the extent of the hippocampal intra‐ and infrapyramidal mossy fiber projection in different inbred mouse strains. The following cholinergic and GABAergic markers were measured in the septi and hippocampi of male mice: the number of cholinergic and parvalbumin‐containing neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB), the number of septo‐hippocampal cholinergic and GABAergic projection neurons, the density of cholinergic fibers in different hippocampal subfields, and the density of muscarinic receptors (predominantly M1 and M2) in the hippocampus. In addition, animals were behaviorally tested for spatially dependent and activity‐dependent variables in a water maze and spatial and nonspatial working and reference memory in different experimental set‐ups in an eight‐arm radial maze.

Trefoil factor family (TFF) expression in the mouse brain and pituitary: changes in the developing cerebellum

Trefoil factor family (TFF) peptides, besides their prominent expression in mucous epithelia, are also synthesized in the central nervous system. Previously TFF1 expression was observed in mouse brain astrocytes, while oxytocinergic neurons of the hypothalamo-pituitary axis are recognized sites of TFF3 synthesis. Here, the expression of TFF1, TFF2, and TFF3 was systematically studied using reverse transcription-polymerase chain reaction (RT-PCR) analysis of dissected adult mouse brain regions including the pituitary. Additionally, the developmental profile of TFF expression in murine cerebral cortex and cerebellum was monitored. Overall, the expression patterns of the three TFF genes differed. The TFF1 and TFF2 profiles shared some similarities, whereas the TFF3 expression pattern was completely different. TFF1 was nearly uniformly, but weakly expressed in all brain regions tested. The TFF1 and TFF2 expression patterns differed characteristically in the pituitary where abundant TFF2 transcription was detected in the anterior and not the posterior lobe and the expression level in males was higher than in females. In contrast, TFF3 expression was limited to the hippocampus, the temporal cortex, and the cerebellum, the latter being surprisingly the major site of expression. Here, TFF3 mRNA appeared to be restricted mainly to neurons and not glial cells. Cerebellar TFF3 expression is clearly developmentally regulated (maximum at P15), indicating a role for TFF3 during postnatal cerebellar development.

Long-lasting sensitization towards morphine in motoric and limbic areas as determined by c-fos expression in rat brain

Chronic application of morphine leads to the development of tolerance towards several of its effects, e.g., analgesia or respiratory depression. Simultaneously, however, sensitization arises which becomes apparent in behavioral tests as increased locomotion or increased self-application. A human correlate for the latter may be the increasing craving for opioids in addicts. To identify brain areas involved in these long-lasting processes, we studied the expression of the transcription factor c-fos by in situ hybridization in rat brain as a marker for changes in gene expression after single or repeated morphine applications in the animals. The only c-fos signal that exceeded background after a single dose of morphine (50 mg/kg) was a diffuse expression in the lateral septum. In contrast, repeated dosage twice daily for 10 days and ascending from 10 to 50 mg/kg resulted in a sharply delineated morphine-induced c-fos synthesis in the dorsomedial and lateral striatum, lateral septum, medial mammillary nuclei, anterior thalamus and, in part masked by a high background due to injection stress, in the cingulate cortex. Most of these areas belong to the limbic system or are closely associated with it. The c-fos response was inducible by morphine in pretreated animals for up to 8 weeks after finishing the repeated application scheme. Retrograde tracing studies revealed that the dorsomedial part of the striatum, which was strongly labeled with the c-fos probe, received inputs from limbic as well as from motoric parts of the thalamus and cortex. Therefore, the sensitization of morphine-induced c-fos expression in parts of the striatum seems to correlate with the locomotor effects of repeated morphine application, whereas the observed sensitization in several limbic brain areas might reflect emotional phenomena like increased self-administration in rats or drug craving in humans.

Two Wistar rat lines selectively bred for anxiety-related behavior show opposite reactions in elevated plus maze and fear-sensitized acoustic startle tests.

Two Wistar rat lines selectively bred for high (HAB), and low anxiety-related behavior (LAB) on the elevated plus maze were tested for the fear-sensitized acoustic startle response. The study of male rats from the F9 generation revealed a higher anxiety level of HAB rats on the elevated plus maze. However, the LAB rats displayed a higher baseline and fear-sensitized acoustic startle response compared to HAB rats, although the two rat lines did not differ in freezing duration during the interstimulus intervals in the startle experiment (neither before, nor after, footshocks). Counts of neurons immunoreactive for corticotropin-releasing factor and neuropeptide Y in amygdaloid nuclei did not reveal any differences between the two lines, which is in marked contrast to findings in the Roman rat lines (Yilmazer-Hanke, D. M. et al. [2002]). The data indicate that opposite types of anxiety/fear responses are elicited in HAB/LAB rats in the elevated plus maze and fear-sensitized startle tests. Moreover, the animals displayed a differential fear response in the startle experiment, as assessed by measuring the fear-sensitized startle response and freezing.

Behavioral effects and pattern of brain c-fos mRNA induced by 2,5-dihydro-2,4,5-trimethylthiazoline, a component of fox feces odor in GAD67-GFP knock-in C57BL/6 mice.

Predator odors, which are non-intrusive and naturalistic stressors of high ethological relevance, were used to study the neurobiology of innate fear in rodents. The present study investigates behavioral effects and the induction of c-fos mRNA in adult male predator naive mice caused by acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of the fox feces odor. On the behavioral level, TMT potently increased unconditioned freezing and decreased non-defensive grooming behavior. With quantitative real time PCR we established a strong TMT-induced activation in the bed nucleus of the stria terminalis (BNST) (eight-fold increase, p<0.016) and in the ventral olfactory bulb (two-fold increase, p<0.036). In contrast, no significant TMT-induced c-fos induction could be observed in the dorsal olfactory bulb or in the amygdala. Our results display robust fear responses of GAD67-GFP knock-in mice exposed to TMT and suggest that the ventral olfactory bulb and the BNST are strongly activated during the elicitation of fear through predator odor in these transgenic mice.

Topographical connections of the substantia nigra pars reticulata to higher-order thalamic nuclei in the rat.

The substantia nigra pars reticulata (SNR) is the ventral subdivision of the substantia nigra and contains mostly GABAergic neurons. The present study explores whether the SNR relates to all dorsal thalamic nuclei equally or just to a particular group of nuclei, such as first or higher-order nuclei. Injections of biotinylated dextran amine (BDA) were made into the SNR of 10 male adult rats. The distribution of anterogradely labelled axon terminals in the thalamic nuclei was documented. The projections of the SNR to the thalamic nuclei were exclusively to some motor higher-order, but not to first-order thalamic relays. There were bilateral projections to the ventromedial (VM), parafascicular (PF), centromedian (CM) and paracentral (PC) nuclei and unilateral projections to the centrolateral (CL), mediodorsal (MD) and thalamic reticular nucleus (Rt). Labelled axon terminals in the thalamic nuclei ranged from numerous to sparse in VM, PF, CM, CL, PC, MD and Rt. Further, injections into the SNR along its rostral-caudal axis showed specific topographical connections with the thalamic nuclei. The rostral SNR injections showed labelled axon terminals of VM, PF, CL, PC, CM, MD and Rt. Caudal SNR injections showed labelling of VM, PF, PC, CM and MD. All injections showed labelled axons and terminals in the zona incerta. The nigrothalamic GABAergic neurons can be regarded as an important system for the regulation of motor activities. The SNR is in a position to influence large areas of the neocortex by modulating some of the motor higher-order thalamic nuclei directly or indirectly via Rt.