Dimitar E. Itzev

Orexinergic innervation of the extended amygdala and basal ganglia in the rat

The orexinergic system interacts with several functional states of emotions, stress, hunger, wakefulness and behavioral arousal through four pathways originating in the lateral hypothalamus (LH). Hundreds of orexinergic efferents have been described by tracing studies and direct immunohistochemistry of orexin in the forebrain, olfactory regions, hippocampus, amygdala, septum, basal ganglia, thalamus, hypothalamus, brain stem and spinal cord. Most of these tracing studies investigated the whole orexinergic projection to all regions of the intracranial part of the CNS. To identify the orexinergic efferents at the subnuclear level of resolution, we focussed on the orexinergic target in the amygdala, which is substantially involved in the LH output and contributes mostly to the functional outcome of the orexinergic system and the basal ganglia. Immunohistochemical identification of axonal orexin A and orexin B in male adult rats has been performed on serial sections. In the extended amygdala many new orexinergic targets were found in the anterior amygdaloid area (dense), anterior cortical nucleus (moderate), amygdalostriatal transition region (moderate), basolateral regions (moderate), basomedial nucleus (moderate), several bed nucleus of the stria terminals regions (few to dense), central amygdaloid subdivisions (dense), posteromedial cortical nucleus (moderate) and medial amygdaloid subnuclei (dense). Furthermore, the entopeduncular nucleus has been newly identified as another target for orexinergic fibers with a high density. These results suggest that subdivisions and subnuclei of the extended amygdala are specific targets of the orexinergic system.

Efferent Projections of the Anterior and Posterodorsal Regions of the Medial Nucleus of the Amygdala in the Mouse

The efferent projections of the anterior and posterodorsal part of the medial nucleus (MePD) in the mouse were studied by means of anterograde axonal tracing using biotinylated dextran amine. The MePD axons ran mainly via the stria terminalis and to a lesser extent via the ventral amygdalofugal pathway. The projections to the forebrain were broadly distributed and varied from very strong to scant. The most significant connections were destined to the bed nucleus of the stria terminalis in which all parts of the medial division were innervated by MePD neurons. Moderate projections reached the limbic striatum (nucleus accumbens), olfactory tubercle and the lateral septal nucleus. The substantia innominata was also innervated by the MePD, and especially the projection to its ventral portion was substantial. The profuse innervation of the medial preoptic nucleus and medial preoptic area indicated significant involvement of the MePD in sexual behavior. Many hypothalamic nuclei were innervated but to a different extent. The very strong innervation of the ventral premammillary nucleus further indicated the involvement of the MePD in the neuronal circuitry for sexual behavior. Substantial projections also reached the anterior hypothalamus and tuber cinereum, while the connections to the lateral hypothalamus were widespread but showed moderate density. MePD strongly innervated the ventrolateral part of the ventromedial hypothalamic nucleus and moderately its remaining parts. The neurosecretory hypothalamic nuclei and the arcuate nucleus contained only a few MePD terminals. The thalamic innervation was very scant and reached the lateral habenular nucleus and the nuclei of the midline. The mesencephalic connections were moderate to sparse and projected to the mesolimbic dopaminergic groups in the ventral tegmental area, the pars lateralis and the dorsal tier of the substantia nigra pars compacta, the periaqueductal gray and the dorsal raphe nucleus. The present results principally resembled data known in other rodent species; however, the efferents of the MePD often differed in extent and/or topical distribution.

Brain stem afferent connections of the amygdala in the rat with special references to a projection from the parabigeminal nucleus: a fluorescent retrograde tracing study

A recently revealed important function of the amygdala (Am) is that it acts as the brain’s “lighthouse”, which constantly monitors the environment for stimuli which signal a threat to the organism. The data from patients with extensive lesions of the striate cortex indicate that “unseen” fearful and fear-conditioned faces elicit increased Am responses. Thus, also extrageniculostriate pathways are involved. A multisynaptic pathway from the retina to the Am via the superior colliculus (SC) and the pulvinar was recently suggested. We here present data based on retrograde neuronal labeling following injection of the fluorescent tracer Fluoro-Gold in the rat Am that the parabigeminal nucleus (Pbg) emits a substantial, bilateral projection to the Am. This small cholinergic nucleus (Ch8 group) in the midbrain tegmentum is a subcortical relay visual center that is reciprocally connected with the SC. We suggest the existence of a second extrageniculo-striate multisynaptic connection to Am: retina–SC–Pbg–Am, that might be very effective since all tracts listed above are bilateral. In addition, we present hodological details on other brainstem afferent connections of the Am, some of which are only recently described, and some others that still remain equivocal. Following selective injections of Fluoro-Gold in the Am, retrogradely labeled neurons were observed in parasubthalamic nucleus, peripeduncular nucleus, periaqueductal gray, dopaminergic nuclear complex (substantia nigra pars lateralis and pars compacta, paranigral, parabrachial pigmented and interfascicular nuclei, rostral and caudal linear nuclei, retrorubral area), deep mesencephalic nucleus, serotoninergic structures (dorsal, median and pontine raphe nuclei), laterodorsal and pedunculopontine tegmental nuclei (Ch6 and Ch5 groups), parabrachial nuclear complex, locus coeruleus, nucleus incertus, ventrolateral pontine tegmentum (A5 group), dorsomedial medulla (nucleus of the solitary tract, A2 group), ventrolateral medulla (A1/C1 group), and pars caudalis of the spinal trigeminal nucleus. A bilateral labeling of the upper cervical spinal cord was also observed.

Diurnal rhythms of spontaneous recurrent seizures and behavioral alterations of Wistar and spontaneously hypertensive rats in the kainate model of epilepsy

Attention deficit hyperactivity disorder (ADHD) can coexist with epilepsy. Spontaneously hypertensive rats (SHRs) are considered to model ADHD with overactivity, impulsiveness, deficient sustained attention, and alterations in circadian autonomic profiles. The present study explored spontaneous recurrent seizures (SRSs) and behavioral diurnal activity rhythms in normotensive Wistar rats and SHRs in the kainate model of epilepsy. Rats were video monitored (24 h/3 months) to detect SRSs. SHRs manifested a lower seizure frequency during the light phase in the 8th and 10th weeks and a lower frequency of SRSs during the night phase accompanied by attenuated responses in hyperexcitability tests. Both epileptic strains were hyperactive, with lower anxiety levels, and their diurnal rhythms were abolished. Epileptic Wistar rats and SHRs exhibited less exploration during the dark phase. This study suggests that SHRs may be useful in modeling some aspects (particularly hypertension-related diurnal rhythm disturbance) of behavior associated with epilepsy.

Changes in NADPH-diaphorase neurons of the rat laterodorsal and pedunculopontine tegmental nuclei in aging

The aim of the present study was to compare the morphological pattern and the quantitative parameters of nitric oxide (NO)-containing neurons in the laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats. NADPH-diaphorase (NADPH-d) histochemical reaction, as a marker of the cholinergic neurons in the two mesopontine nuclei, and computer-assisted image analysis were used. The relationships between the neurons stained for NADPH-d and choline acetyltransferase (ChAT) were examined using a double-labelling procedure. The results demonstrated only occasional ChAT positive somata that did not exhibit NADPH-d staining. The volume of the LTD and PPN and the number of NADPH-d neurons remained unaltered with advancing age. However, ANOVA demonstrated a significant effect of age and level on the cross-sectional areas, maximum diameters and staining intensity of NADPH-d somata in the LTD and PPN. The three parameters were reduced in 26-month-old rats compared to 3-month-old rats. The changes in the morphological appearance of NADPH-d somata and processes as well as the quantitative analysis pointed to age-related neuronal atrophy. It was accompanied by hypertrophy of some neighbouring neurons, suggesting a compensatory mechanism which would counteract the degenerative changes. The age-dependent alterations in the LTD and PPN were rather similar.

Muscarinic modulation of nitrergic neurotransmission in guinea-pig gastric fundus.

Abstract  Muscarinic receptor activation by (4‐Hydroxy‐2‐butynyl)‐1‐trimethylammonium‐m‐chlorocarbanilate chloride (McN‐A‐343) was investigated both on NADPH‐d staining and on electrically induced responses in guinea‐pig gastric fundus. McN‐A‐343 (10 μmol L−1) significantly increased the optical density of NADPH‐d positive neurones, while blockade of nitric oxide synthase with Nω‐nitro‐L‐arginine (L‐NA) decreased it, suggesting facilitation of nitric oxide (NO) production. Electrical field stimulation (EFS; 2 Hz, 0.2 ms, supramaximal current intensity, 10 s train duration) elicited on‐contraction followed by off‐relaxation in the circular muscle strips. McN‐A‐343 (10 μmol L−1) transformed the EFS‐evoked response from on‐contraction into on‐relaxation, which was neurogenic, tetrodotoxin‐sensitive and hexamethonium‐resistant. L‐NA partly reduced the EFS‐evoked relaxation, revealing two components: a nitrergic and a non‐nitrergic one. The effect of McN‐A‐343 on the amplitude of the EFS‐evoked relaxation was not changed by the M3 receptor antagonist para‐fluoro‐hexahydro‐sila‐difenidol hydrochloride, but was significantly enhanced by M1 receptor blockade with telenzepine. In the presence of telenzepine, the L‐NA‐dependent nitrergic component of the EFS‐induced relaxation predominates. We suggest that cholinergic receptor activation has a dual effect on nitrergic neurotransmission: (i) stimulation of NOS by muscarinic receptor(s) different from M1 and M3 subtype, (ii) prejunctional inhibition of NO‐mediated relaxation via M1 receptors. In addition, M1 receptors may facilitate the non‐nitrergic relaxation.

Aging and the dendritic morphology of the rat laterodorsal and pedunculopontine tegmental nuclei

The morphological appearance and quantitative parameters characterizing the dendrites of NADPH-diaphorase-stained neurons in the laterodorsal (LDT) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats were studied. All dendritic segments were classified according to the number of terminal and link segments they drain and the vertex analysis was used to quantify the dendritic tree and to determine its configuration. Morphological aberrations of the dendrites as local swelling, nodulation, thinning, shrinkage, folding and even the appearance of stumps were observed with advancing age. The quantitative analysis demonstrated a significant reduction (one-way ANOVA) of the total dendritic length, mean terminal path length, maximal segment length, total segment number and number of terminal segments at the rostral two thirds of the LDT and in the PPN. The mean vertex path length and the mean segment length significantly decreased only at the rostral level of the LDT. Plotting of the segment length against equivalent orders showed a decrease in all generations of the dendritic segments. The vertex ratios remained constant and indicated that the configuration of the dendritic tree remained unchanged during aging. The alterations in the dendrites mainly developed after 12 months of age. The age-related changes in the morphology and quantitative parameters of the dendrites in the rostral two thirds of the LDT and PPN were rather similar, which could be explained by the common anatomical, neurochemical and electrophysiological features. Thus, the present results suggest a mild, but continuous regression of the dendritic tree of the rat LDT and PPN in normal aging.

Neurons in the dorsal column nuclei of the rat emit a moderate projection to the ipsilateral ventrobasal thalamus.

The dorsal column nuclei (DCN; gracile and cuneate nuclei) give rise to the medial lemniscus, the fibre system that provides an organised somatosensory input to the thalamus. Unlike the spinothalamic and trigeminothalamic tracts that project, also to the ipsilateral thalamus, the medial lemniscus system is believed to be entirely crossed. We demonstrate that DCN emit a small number of axons that reach the ipsilateral thalamus. As retrograde fluorescent neuronal tracer Fluoro-gold was stereotaxically injected in the ventrobasal thalamus of nine young adult Wistar rats. The injection foci were voluminous and encroached upon adjacent nuclei, but the periphery of the injection halo never spilled over to the contralateral thalamus. All sections of the contralateral gracile and cuneate nuclei and the midline nucleus of Bischoff contained abundant retrogradely labelled neurons. The comparison with the Nissl-stained parallel sections suggests that approximately 70–80% of the DCN neurons project to the contralateral thalamus. Counting of retrogradely labelled neurons in two cases revealed 4,809 and 4,222 neurons in the contralateral and 265 and 214 in the ipsilateral DCN, respectively. Thus, although less prominent than the ipsilateral spinothalamic tract, the lemniscal system also emits an ipsilateral projection that accounts for about 5% of the neuronal population in DCN that innervates the ventrobasal thalamus.

Amygdalotrigeminal projection in the rat: An anterograde tracing study.

Previous neurophysiological studies have demonstrated that the amygdala has a direct influence upon trigeminal motoneuron activity. The existence of a direct amygdalotrigeminal pathway in rats was proved by anterograde tracing with the neuroanatomical tracer, biotinylated dextran amine (BDA). After ipsilateral BDA application to the central nucleus of the amygdala (AmCe), widespread ipsilateral projections emerging from its medial subnucleus were traced to the trigeminal brainstem nuclear complex, including the principal sensory (Pr5) and mesencephalic trigeminal nucleus (Me5), and their premotoneurons and interneurons, located in the supratrigeminal, intertrigeminal and peritrigeminal nuclei. Sparse BDA-labeled axons and their terminals were also distributed in the contralateral Pr5, interpolar and caudal subnuclei of the spinal trigeminal nucleus. The central lateral amygdaloid nucleus gives rise to a light ipsilateral projection to the pontine part of the Me5. The present data indicate that AmCe sends massive efferents to the trigeminal nuclei in the brainstem, wherein its medial subnucleus sends the major input to them. The medial amygdaloid nucleus sparsely innervates Me5 neurons, specifically those located in its mesencephalic portion, while basomedial and basolateral efferents do not target the trigeminal nuclear complex. These results suggest that the amygdaloid input may modulate the activity of trigeminal sensory and motor neurons and, thus, the amygdala is possibly involved in the control of masticatory behavior.

Glutamate stimulation of acetylcholine release from myenteric plexus is mediated by endogenous nitric oxide.

Glutamate was found to be an excitatory neurotransmitter in the enteric nervous system. Although several lines of evidence indicate a role of glutamate in the regulation of gut motility and secretion the physiological significance of glutamatergic transmission is not clear yet. We studied the effect of glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining in longitudinal muscle strips with attached myenteric plexus of guinea pig ileum. L-glutamate (100 microM) significantly enhanced both the evoked [3H]acetylcholine release and the optical density of nicotinamide adenine dinucleotide phosphate-diaphorase positive neurones, i.e. the intensity of staining. The non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (3 microM) abolished the stimulatory effect of L-glutamate on acetylcholine efflux. Similarly, the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (100 microM) significantly reduced the effect of L-glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining. Our data suggest that endogenous nitric oxide seems to mediate the stimulatory effect of glutamate on acetylcholine release from guinea pig myenteric neurons.