Neurons of the Paraventricular Hypothalamic Nucleus Under Normal and Modified Illumination Conditions: Immunohistochemical and Morphometric Parallels

Using an immunohistochemical technique and densitometric analysis in experiments on rats, we examined the expression of an early-response protein, c-Fos, in the medial parvicellular subnucleus of the hypothalamic paraventricular nucleus (mpcPVN). During 7 days, the animals were kept under conditions of (i) normal photoperiod (group LD), (ii) continuous illumination (group LL), and (iii) continuous darkness (light deprivation, group DD). It was found that both normal photoperiodicity and artificial modifications of the latter result in significant changes of c-Fos immunopositivity in the nuclei of mpcPVN neurons (cells that synthesize corticotropin-releasing factor). Changes in the indices of concentration and amount of c-Fos in mpcPVN neurons were, from some aspects, similar to those observed under analogous experimental conditions in neurons of a leading pacemaker of the circadian periodicity, the suprachiasmatic nuclei (SChNs) of the hypothalamus [10], but, at the same time, demonstrated noticeable specificity from other aspects. In all experimental groups, the concentration and amount of c-Fos in mpcPVN neurons were greater during daytime than at night. Dramatic increases in the above parameters observed at daytime in rats subjected to light deprivation were the most significant effect of modifications of the photoperiod. Both continuous illumination and light deprivation resulted in some increase of the number (density) of c-Fos-immunopositive units in the mpcPVN. The nuclei of neurons of this structure demonstrated the phenomenon found earlier in SChN neuron; geometrical dimensions of the cell nuclei of these structures depended noticeably on both illumination conditions and periodicity. Probable mechanisms of the dependence of c-Fos expression in the hypothalamic nuclei on the photoperiod and its modifications, interdependence of such changes observed in the circadian pacemaker (SChN) and an important component of the system controlling the stress reactions (mpcPVN), and the relation of the above phenomena to the level of melatonin produced by the epiphysis cerebri (main neuroendocrine intermediator organizing the above-mentioned periodicity) are discussed.     

Expression of <Emphasis Type="Italic">c-fos</Emphasis> as an index of functional interaction of the frontal cortex and limbic cerebral structures in the course of food-procuring stereotyped movements in rats

We estimated expression of the c-fos gene (a marker of increase in neuronal activity) and manifestations of the histochemical reaction to NADPH-diaphorase (a marker of NO-generating neurons) in the medial prefrontal cortex (MPFC) and forebrain limbic structures of rats in the norm, in the state of starvation, and during realization of long-lasting (60 min) periodic (several times per minute) food-procuring movements of the forelimb. The starvation state or realization of motivated stereotyped forelimb movements were related to significant bilateral increases (P < 0.05) in the levels of c-fos expression in the anterior olfactory (AOP) and cortical (ACo) nuclei and the central (Ce) and basolateral (BLA) nuclei of the amygdala, and also in the pyriform (Pyr), prelimbic (PrL), and inferior limbic (IL) cortices. The described findings demonstrate that the high Fos immunoreactivity in the MPFC and amygdalar structures is related to the motivation state in animals and reflects the active involvement of limbic cerebral structures in the formation of motor programs and also in the stabilization and realization of operant reflexes. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 256–259, May–June, 2008.     

Suppression of activation of c-fos protein in the striatum by systemic administration of nitroglycerin in a rat model of Parkinson’s disease

We found sustained activation of the proto-oncogenec-fos in neurons of the medial and dorsal neostriatum and suppression of this activation in NO-synthase (NOS) -containing cells within the islands of Calleja after unilateral lesions of the dopaminergic mesostriatal system induced by 6-hydroxydopamine (6-OHDA). Systemic administration of nitroglycerin (NTG) resulted in a decreased expression of c-Fos protein in the dorsal part of the denervated neostriatum: respective numbers of Fos-positive neuronal nuclei were 420.71 ± 21.32 in control and 141.38 ± 11.48 after NTG injection (the mean numbers in 50-μm-thick slices). However, other brain structures, NTG evoked sustained expression of c-Fos protein in the groups of NOS-containing neurons and sources of catecholaminergic projections involved In the control of cardiovascular reactions. We hypothesize that systemic administration of an NO donor and a potent vasodilator, NTG, partially normalized an excessively Increased glutamate level in the denervated neostriatum manifested in c-Fos protein expression.     

Coupling of c-fos expression in the spinal cord and amygdala induced by dorsal neck muscles fatigue

c-fos gene expression in the cervical spinal cord and amygdala was examined in anaesthetized rats following muscle fatigue caused by intermittent high-rate (100 s⁻¹) electrical stimulation of the dorsal neck muscles (m. trapezius and m. splenius). Fatigue-related increases in c-fos expression were observed on the stimulated muscle side in the cervical C2–C4 (layers 1, 3–5, 7 and 10) spinal segments, bilaterally in the lumbar L4–L6 (layer 1) segments and in contralateral central (Ce), medial (Me), and basomedial (BM) amygdaloid nuclei. A scarce number of staining cells were found within lateral and basolateral nuclei. The rostro-caudal extent of c-fos expression in the spinal cord supports functional coupling of the cervical and lumbar regions during the neck muscle fatigue development. The distinct c-fos expression in the Ce and Me amygdaloid nuclei suggests that they may contribute to mediating the neck muscle fatigue-related nociception, autonomic and behavioural responses.     

Peripheral ChE Inhibition Modulates Brain Monoamines Levels and <Emphasis Type="Italic">c-fos</Emphasis> Oncogene in Mice Subjected to a Stress Situation

The present study examined, in mice, whether regional patterns of brain monoamines concentrations (DA, 5-HT and their metabolites) and expression of c-Fos protein, that may represent a prolonged functional change in neurons, could be changed after a combined exposure to stress and the peripheral cholinesterase reversible inhibitor pyridostigmine (PYR). Animals were subjected every day to a random combination of mild unescapable electric footshocks and immobilization over a 12-day period, resulting in a significant increase of glucocorticoids levels and an activation of c-fos in hippocampus, thalamus and piriform cortex. This stress protocol induced a significant increase of 5-HT levels in striatum, hippocampus and ponto mesencephalic area (PMA) but failed to induce any DA activation. When PYR (0.2 mg/kg s.c. inducing 19–35% inhibition of the plasmatic ChE activity) was administered twice a day during the last 5 days of the stress session, 5-HIAA levels and expression of c-fos oncogene were significantly increased in the most of the brain areas studied. DA levels were also enhanced in striatum/hippocampus as a result of a possible activation of mesolimbic and nigrostriatal dopamine systems. Taken together, these results suggest that a combined exposure to certain stress conditions and PYR leads, in mice, to functional changes in neurons and may affect centrally controlled functions. The mechanisms underlying these modifications and their behavioral implications remain to be further investigated.     

Fatigue of the dorsal neck muscles initiates c-fos expression in the rat spinal cord and hypothalamus

Fatiguing stimulation of the dorsal neck muscles in rats is shown to induce significant up-regulation of the c-fos expression in the ipsilateral cervical cord, contralateral hypothalamic nuclei, periaqueductal gray, and lateral parabrachial nucleus. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 354–357, July–August, 2006.     

Topography of Fos-Immunoreactive and NADPH-d-Reactive Neurons in the Limbic Structures of the Basal Forebrain and in the Hypothalamus during Realization of Motivated Operant Movements in Rats

We estimated in rats the expression of early gene c-fos (marker of neuronal activation) and NADPH-diaphorase activity (NO-synthase marker) in the limbic structures of the basal forebrain and in the hypothalamus. Estimations were performed in the norm, in the state of starvation, and after realization of long-lasting (repeated 4 to 12 times per minute for 30 min) motivated stereotyped food-procuring forelimb movements. In food-deprived animals, a significantly greater (Р < 0.05), as compared with the control, number of Fos-immunoreactive (Fos-ir) and NADPH-diaphorase-reactive (NADPH-dr) neurons was observed in limbic structures, namely in the medial septum (MS), nuclei of the vertical and horizontal branches of the diagonal fascia (VDB and НDB), magnocellular preoptic nucleus (MCPO), complex of the substantia innominata−basal nucleus of Meynert of the pallidum, SI-GP(B), as well as in the laterodorsal tegmental nucleus (LDTg), medial part of the pallidum (MGP), paraventricular and lateral nuclei of the hypothalamus (Pa and LH), and islands of Calleja (ICj and ICjM). In the limbic structures and pontine nuclei of rats of the experimental group (that performed operant movements), greater mean densities of labeled neurons were found in the succession LDTg < SI < MCPO < GP(B) < MS < VDB < HDB. The maximum mean density of Fos-ir neurons (13.8 ± 0.9 labeled nuclei within 200 × 200 μm² area) was found in the HDB. In the hypothalamic nuclei of starving rats, c-fos expression was two times higher than that in the control. After realization of operant movements, the intensity of expression in the LH was somewhat smaller, while in the Ра it was higher. The maximum density of NADPH-dr neurons was observed in the Pa (303.4 ± 18.7 cells), in the ICj and ICjM (287 ± 11.6 and 260 ± 8.7 neurons, respectively), and in the MGP (93 ± 6.7 labeled cells). When analyzing the distribution of labeled neurons in experimental rats, we found high densities of double-labeled cells (Fos + NADPH-d positivity) in the Pa, MGP, ICj, and ICjM. Such specificity of changes in the c-fos expression and NADPH-d reactivity in the hypothalamus correlates, perhaps, with the formation of motivation signals related to a delay in food accessibility and supply of food. Modifications of neuronal activity in limbic structures reflect involvement of the latter in the formation of motor programs for food-procuring movements and their realization. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 32–40, January–February, 2009.     

Changes in the levels of activity of spinal neurons after long-lasting vibrational stimulation of the shin muscles in rats

Vibrational stimulation of the tendon of the mm.gastrocnemius+soleus (100 sec^–1) in rats anesthetized with chloral hydrate (400 mg/kg) resulted in the appearance of considerable Fos immunoreactivity in the lumbar spinal cord (L1-L6), as compared with that in intact animals. Total densities of Fos-immunopositive (Fos-ip) neurons in each of the examined segments were higher than 40 units per 40-μm-thick slice; the respective index reached the maximum at the L4 level (78.9 ± 2.3 cells). Most Fos-ip neurons were localized in laminae 4 to 7 of the gray matter, both ipsi- and contralaterally with respect to the side of stimulation (28.5 ± 0.6 and 28.4 ± 0.6, respectively). Single Fos-ip motoneurons were found bilaterally in the ventral horn motor nuclei. Thus, activation of muscle spindle receptors induced by vibrational stimulation applied to the Achilles tendon induces noticeable bilateral c-fos expression in spinal neuronal networks related to transmission of proprioceptive muscle-born impulsation.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

Hormones, subjective night and season of the year

Production and release of many mammalian hormones exhibit circadian rhythms controlled by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Under conditions when the circadian pacemaker free-runs with a period close to, but not equal to 24 h, subjective day and night may not be identical with the environmental day and night. The present study was aimed to define the phase and state of the circadian pacemaker when the circadian system is experiencing subjective night and to ascertain whether and how such a defined subjective night depends on the photoperiod. The results indicate that the subjective night may be defined as the time interval when i) light stimuli can reset the circadian system, ii) pineal melatonin production and photic induction of the c-Fos gene in the ventrolateral SCN are high, and iii) the spontaneous c-Fos protein production in the dorsomedial SCN is low. Such a defined subjective night and, logically, the whole circadian pacemaking system depend on the photoperiod and hence on the season of the year which the animals are experiencing.     

Neurodegenerative changes in the hippocampus within the early period of experimental diabetes mellitus

We studied the dynamics of modifications of the structure and architectonics in different zones of the pyramidal layer of the rat hippocampus within the early periods (3, 7, and 14 days) after induction of diabetes mellitus by streptozotocin. Using confocal immunofluorescence microscopy, we found neurons containing a specific protein, NeuN; a fluorescence dye, Hoechst 33258, allowed us to visualize the cell nuclei. The density of localization of neurons in the CA2 area decreased significantly on the 3rd day of development of diabetes. In the CA1 and CA3 areas, a significant decrease in this index was observed beginning from the 7th day. Within this time interval, we observed neurons with clear condensation of chromatin in the nuclei of these cells. The obtained data indicate that formation of appreciable neurodegenerative changes in the hippocampus occurs within the initial stages of development of experimental diabetes mellitus; this phenomenon can be a factor in the development of diabetic encephalopathy. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 30–37, January–February, 2008.     

Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

Glutamate antibodies repress expression of <Emphasis Type="Italic">Dffb</Emphasis> gene in brain of rats in experimental Alzheimer’s disease

The rat model of Alzheimer’s disease including injection of neurotoxic fragment of β-amyloid protein Aβ_25–35 into giant-cell nuclei basalis of Meynert was used for experiments. We have investigated the influence of glutamate antibodies administered intranasally in a dose of 300 μg/kg after 1 h of the mentioned alteration on the level of expression of Dffb mRNA. Dffb gene codes caspase-dependent DNase, which participates in the internucleosomal fragmentation of genome DNA during apoptosis. On the third day after the injection of Aβ_25–35, we obtained a significant decrease in Dffb gene expression in the prefrontal cortex (37% decrease) and hippocampus (62% decrease) in the group of experimental animals compared to the control group. In the hypothalamus, there were no such differences. Seemingly, the repressing action of glutamate antibodies on the mRNA expression of the Dffb gene reflects the stabilization of processes that take place in the brain cells during experimental Alzheimer’s disease; meanwhile, the intensity of the apoptotic death of neurons and glial cells decreases.     

Effects of NMDA-Receptor Antagonist Treatment on <Emphasis Type="Italic">c-fos</Emphasis> Expression in Rat Brain Areas Implicated in Schizophrenia

1. The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists produce behavioral responses that closely resemble both positive and negative symptoms of schizophrenia. These drugs also induce excitatory and neurotoxic effects in limbic cortical areas.2. We have here mapped the brain areas which show increased activity in response to noncompetitive NMDA-receptor antagonist administration concentrating especially to those brain areas that have been suggested to be relevant in the pathophysiology of schizophrenia.3. Rats were treated intraperitoneally with a NMDA-receptor antagonist MK801 and activation of brain areas was detected by monitoring the expression of c-fos mRNA by using in situ hybridization.4. MK801 induced c-fos mRNA expression of in the retrosplenial, entorhinal, and prefrontal cortices. Lower c-fos expression was observed in the layer IV of the parietal and frontal cortex. In the thalamus, c-fos mRNA expression was detected in the midline nuclei and in the reticular nucleus but not in the dorsomedial nucleus. In addition, c-fos mRNA was expressed in the anterior olfactory nucleus, the ventral tegmental area, and in cerebellar granule neurons.5. NMDA-receptor antagonist ketamine increased dopamine release in the parietal cortex, in the region where NMDA-receptor antagonist increased c-fos mRNA expression.6. Thus, the psychotropic NMDA-receptor antagonist induced c-fos mRNA expression in most, but not all, brain areas implicated in the pathophysiology of schizophrenia. The high spatial resolution of in situ hybridization may help to define regions of interest for human imaging studies.     

Fos Immunoreactivity and NADPH-d Reactivity in the Brain Cortex of Rats Realizing Motivated Stereotyped Movements by the Forelimb

A comparative study of mmunoreactivity with respect to c-Fos protein in the motor (zones М1 and М2), medial prefrontal (PrL and IL), and cingular (Cg1 and Cg2) cortices allowed us to find significant differences between the intensities of expression of gene c-fos in these cortical regions in control rats (group 1) and animals trained to perform catching of food globules by the forelimb (i.e., realizing an operant food-procuring reflex, group 2). The density of distribution of Fos-immunoreactive (Fos-ir) neurons in rats of group 2 in motor and limbic cortical zones at +2.2 to +0.2 levels rostrally from the bregma were significantly lower than in control rats (Р < 0.05). In animals of group 2, we also found significantly greater numbers of Fos-ir neurons in the contralateral (with respect to the active extremity) zones of the cortex at all examined levels. These changes are probably related to functional changes in the cortex resulting from learning of motor habits in the course of training sessions for stabilization of the operant reflex. Histochemical estimation of the NADPH-diaphorase (NADPH-d) activity in the motor and limbic cortex showed that, in rats of both groups, the maximum number of labeled interneurons per slice in the М1 zone were observed in layers II/III, V, and VI (5.6 ± 0.4, 6.4 ± 0.5, and 14.0 ± 0.8, respectively, within 200 × 200 μm₂ areas). In the limbic cortex, NADPH-d-reactive (NADPH-d-r) interneurons were also met in layers II/III, V, and VI. Cortical NADPHd-r neurons with the Fos-ir nuclei were not found. The presence of spatial associations of the somata or processes of NADPH-d-r neurons with intraparenchimal arterioles and microvessels was a typical feature of the distribution of NADPH-d-reactivity in the М1 and М2 zones, as well as in Cg1, Cg2, PrL, and IL. The following succession of the density of neurovascular associations was observed: Cg1 > Cg2 > М1 > М2 > > PrL. As is supposed, NADPH-d-r neurons (i.e., cells generating NO) are involved in the control of regional blood flow in the studied cortical regions. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 348–358, July–August, 2008.     

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn¹³-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn¹³-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day.