Single CNS neurons link both central motor and cardiosympathetic systems: a double-virus tracing study.

Two anatomical experiments were performed to test the hypothesis that single CNS neurons link the central areas that regulate the somatomotor and sympathetic systems. First, the retrograde neuronal tracer cholera toxin beta-subunit was injected into the lateral parafascicular thalamic nucleus, a region that projects to both the motor cortex and striatum. Several days later, a second injection of the retrograde transneuronal tracer, pseudorabies virus (PRV), was made in the same rats in the stellate ganglion, which provides the main sympathetic supply to the heart. Using immunohistochemical methods, we demonstrate that the cholinergic neurons of the pedunculopontine tegmental nucleus (PPN) are connected to both systems. The second experiment used two isogenic strains of Bartha PRV as double transneuronal tracers. One virus contained the unique gene for green fluorescent protein (GFP) and the other had the unique gene for beta-galactosidase (beta-gal). GFP-PRV was injected in the stellate ganglion and beta-gal-PRV was injected into the primary motor cortex. Double-labeled neurons were found in the lateral hypothalamic area (50% contained orexin) and PPN (approximately 95% were cholinergic). Other double-labeled neurons were identified in the deep temporal lobe (viz., amygdalohippocampal zone and lateral entorhinal cortex), posterior hypothalamus, ventral tuberomammillary nucleus, locus coeruleus, laterodorsal tegmental nucleus, periaqueductal gray matter, dorsal raphe nucleus, and nucleus tractus solitarius. These results suggest these putative command neurons integrate the somatomotor and cardiosympathetic functions and may affect different behaviors (viz., arousal, sleep, and/or locomotion).

Suprachiasmatic nucleus projection to the medial prefrontal cortex: a viral transneuronal tracing study.

The viral transneuronal labeling method was used to examine whether the suprachiasmatic nucleus (SCN) is linked by multisynaptic connections to the medial prefrontal cortex of the rat. In separate experiments, pseudorabies virus (PRV) was injected into one of the three different cytoarchitectonic regions that comprise the medial prefrontal cortex: infralimbic (Brodmann area 25), prelimbic (Brodmann area 32), and cingulate (Brodmann area 24) cortical areas. After 4-days survival, extensive SCN transneuronal labeling was found following infralimbic cortex (ILC) injections, but almost none occurred when the PRV injections were centered in the prelimbic or cingulate areas. In the ILC cases, transneuronal labeling was localized mainly in the dorsomedial SCN, although a moderate number of labeled neurons were found in the ventrolateral SCN. About 13% of the infected neurons were vasopressin immunoreactive and 4% were vasoactive intestinal polypeptide-positive. Another set of experiments was performed in which the paraventricular thalamic nucleus (PVT) was destroyed 2 weeks prior to making PRV injections into the ILC. Almost no SCN transneuronal labeling occurred in these animals, suggesting that the SCN projection to the ILC is dependent on a relay in the PVT. We propose that the SCN sends timing signals, via its relay in the PVT, to the ILC. This pathway may modulate higher-level brain functions, such as attention, mood, or working memory. Assuming that a homologous circuit exists in humans, we speculate that neurochemical changes affecting this pathway may account for some of the symptoms associated with clinical depression and attention-deficit/hyperactivity disorder.

CNS inputs to the suprachiasmatic nucleus of the rat.

The neural circuits that modulate the suprachiasmatic nucleus (SCN) of the rat were studied with the retrograde transneuronal tracer--pseudorabies virus. First-order afferents were also identified using cholera toxin beta subunit. Olfactory processing regions (viz., main olfactory bulb, anterior olfactory nucleus, taenia tecta, endopiriform nucleus, medial amygdaloid nucleus, piriform cortex, and posteriomedial cortical amygdaloid nucleus) were virally labeled. The subfornical organ directly innervates SCN; two other circumventricular organs: organum vasculosum of the lamina terminalis and area postrema provide multisynaptic inputs. Direct limbic afferents arise from lateral septum, bed nucleus of the stria terminalis, amygdalohippocampal zone, and ventral subiculum; multineuronal connections come from the basolateral and basomedial amygdaloid nuclei, ventral hippocampus, amygdalopiriform area, as well as lateral entorhinal, perirhinal, and ectorhinal cortices. Most preoptic regions project directly to SCN. Multisynaptic inputs come from the lateral preoptic region. Hypothalamic inputs originate from the anterior, arcuate, dorsal, dorsomedial, lateral, paraventricular, posterior, periventricular posterior, retrochiasmatic, subparaventricular, ventromedial and tuberomammillary nuclei. Paraventricular thalamic nucleus, intergeniculate leaflet and zona incerta directly innervate SCN. Polyneuronal inputs arise from the subparafascicular parvicellular thalamic nucleus. Brainstem afferents originate from the pretectum, superior colliculus, periaqueductal gray matter, parabrachial nucleus, pedunculopontine nucleus, raphe system, locus coeruleus, nucleus incertus and reticular formation. Nucleus tractus solitarius, C3 catecholamine region, rostral ventrolateral medulla and spinal trigeminal nucleus provide indirect inputs. We propose that the SCN receives feedback primarily from interoceptive systems such as the circumventricular, autonomic, and neuroendocrine systems that are important in the central regulation of glucose metabolism (e.g., insulin and glucocorticoids).

Quantitative response characteristics of thermoreceptive and nociceptive lamina I spinothalamic neurons in the cat.

The physiological characteristics of antidromically identified lamina I spinothalamic (STT) neurons in the lumbosacral spinal cord were examined using quantitative thermal and mechanical stimuli in barbiturate-anesthetized cats. Cells belonging to the three main recognized classes were included based on categorization with natural cutaneous stimulation of the hindpaw: nociceptive-specific (NS), polymodal nociceptive (HPC), or thermoreceptive-specific (COOL) cells. The mean central conduction latencies of these classes differed significantly; NS = 130.8 +/- 55.5 (SD) ms (n = 100), HPC = 72.1 +/- 28.0 ms (n = 128), and COOL = 58.6 +/- 25.3 ms (n = 136), which correspond to conduction velocities of 2.5, 4.6, and 5.6 m/s. Based on recordings made prior to any noxious stimulation, the mean spontaneous discharge rates of these classes also differed: NS = 0.5 +/- 0.7 imp/s (n = 47), HPC = 0.9 +/- 0.7 imp/s (n = 59), and COOL = 3.3 +/- 2.6 imp/s (n = 107). Standard, quantitative, thermal stimulus sequences applied with a Peltier thermode were used to characterize the stimulus-response functions of 76 COOL cells, 47 HPC cells, and 37 NS cells. The COOL cells showed a very linear output from 34 degrees C down to approximately 15 degrees C and a maintained plateau thereafter. The HPC cells showed a fairly linear but accelerating response to cold below a median threshold of approximately 24 degrees C and down to 9 degrees C (measured at the skin-thermode interface with a thermode temperature of 2 degrees C). The HPC cells and the NS cells both showed rapidly increasing, sigmoidal response functions to noxious heat with a fairly linear response between 45 and 53 degrees C, but they had significantly different thresholds; half of the HPC cells were activated at ~45.5 degrees C and half of the NS cells at approximately 43 degrees C. The 20 HPC lamina I STT cells and 10 NS cells tested with quantitative pinch stimuli showed fairly linear responses above a threshold of approximately 130 g/mm(2) for HPC cells and a threshold of approximately 100 g/mm(2) for NS cells. All of these response functions compare well (across species) with the available data on the characteristics of thermoreceptive and nociceptive primary afferent fibers and the appropriate psychophysics in humans. Together these results support the concept that these classes of lamina I STT cells provide discrete sensory channels for the sensations of temperature and pain.

Superior colliculus projections to midline and intralaminar thalamic nuclei of the rat.

The superior colliculus (SC) projections to the midline and intralaminar thalamic nuclei were examined in the rat. The retrograde tracer cholera toxin beta (CTb) was injected into one of the midline thalamic nuclei-paraventricular, intermediodorsal, rhomboid, reuniens, submedius, mediodorsal, paratenial, anteroventral, caudal ventromedial, or parvicellular part of the ventral posteriomedial nucleus-or into one of the intralaminar thalamic nuclei-medial parafascicular, lateral parafascicular, central medial, paracentral, oval paracentral, or central lateral nucleus. After 10-14 days, the brains from these animals were processed histochemically, and the retrogradely labeled neurons in the SC were mapped. The lateral sector of the intermediate gray and white layers of the SC send axonal projections to the medial and lateral parafascicular, central lateral, paracentral, central medial, rhomboid, reuniens, and submedius nuclei. The medial sector of the intermediate and deep SC layers project to the parafascicular and central lateral thalamic nuclei. The paraventricular thalamic nucleus is innervated almost exclusively by the medial sectors of the deep SC layers. The superficial gray and optic layers of the SC do not project to any of these thalamic areas. The discussion focuses on the role these SC-thalamic inputs may have on forebrain circuits controlling orienting and defense (i.e., fight-or-flight) reactions.

Parabrachial nucleus projections to midline and intralaminar thalamic nuclei of the rat.

The projections from the parabrachial nucleus to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin-beta (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus (the lateral parafascicular, medial parafascicular, oval paracentral, central lateral, paracentral, and central medial nuclei), as well as the midline thalamic nuclei (the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, parvicellular part of the ventral posterior, and caudal ventral medial nuclei). The retrograde cell body labeling pattern within the parabrachial subnuclei was then analyzed. The paracentral thalamic nucleus received an input only from the internal lateral parabrachial subnucleus. However, this subnucleus also projected to all the other intralaminar thalamic nuclei, except for the central lateral thalamic nucleus, which received no parabrachial afferent inputs. The external lateral parabrachial subnucleus projected to the lateral parafascicular, reuniens, central medial, parvicellular part of the ventral posterior, and caudal ventromedial thalamic nuclei. Following CTb injections in the paraventricular thalamic nucleus, retrogradely labeled cells were found in the central lateral, dorsal lateral, and external lateral parabrachial subnuclei. The medial and ventral lateral parabrachial subnuclei projected to the oval paracentral, parafascicular, and rhomboid thalamic nuclei. Finally, the waist area of the parabrachial nucleus was densely labeled after CTb injections in the parvicellular part of the ventral posterior thalamic nucleus. Nociceptive, visceral, and gustatory signals may reach specific cortical and other forebrain sites via this parabrachial-thalamic pathway.

Periaqueductal gray matter projections to midline and intralaminar thalamic nuclei of the rat.

The periaqueductal gray matter (PAG) projections to the intralaminar and midline thalamic nuclei were examined in rats. Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected in discrete regions of the PAG, and axonal labeling was examined in the thalamus. PHA-L was also placed into the dorsal raphe nuclei or nucleus of Darkschewitsch and interstitial nucleus of Cajal as controls. In a separate group of rats, the retrograde tracer cholera toxin beta-subunit (CTb) was injected into one of the intralaminar thalamic nuclei-lateral parafascicular, medial parafascicular, central lateral (CL), paracentral (PC), or central medial nucleus-or one of the midline thalamic nuclei-paraventricular (PVT), intermediodorsal (IMD), mediodorsal, paratenial, rhomboid (Rh), reuniens (Re), or caudal ventral medial (VMc) nucleus. The distribution of CTb labeled neurons in the PAG was then mapped. All PAG regions (the four columns of the caudal two-thirds of the PAG plus rostral PAG) and the precommissural nucleus projected to the rostral PVT, IMD, and CL. The ventrolateral, lateral, and rostral PAG provided additional inputs to most of the other intralaminar and midline thalamic nuclei. PAG inputs to the VMc originated from the rostral and ventrolateral PAG areas. In addition, the lateral and rostral PAG projected to the zona incerta. No evidence was found for a PAG input to the ventroposterior lateral parvicellular, ventroposterior medial parvicellular, caudal PC, oval paracentral, and reticular thalamic nuclei. PAG --> thalamic circuits may modulate autonomic-, nociceptive-, and behavior-related forebrain circuits associated with defense and emotional responses.

Periaqueductal gray matter projection to the parabrachial nucleus in rat.

The efferent projections from the periaqueductal gray matter (PAG) to the parabrachial nucleus (PB) were studied in the rat following microinjections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into restricted regions of the PAG. The dorsomedial and dorsolateral PAG columns project almost exclusively to the superior lateral PB subnucleus, whereas the lateral and ventrolateral PAG columns project to five lateral PB sites: dorsal lateral subnucleus, medial and lateral crescent areas (which flank the dorsal lateral PB subnucleus), central lateral subnucleus (rostral portion), and superior lateral subnucleus. The PAG region lying near the cerebral aqueduct projects to five lateral PB sites: external lateral subnucleus (inner subdivision), medial and lateral crescent areas, central lateral subnucleus (rostral portion), and dorsal lateral subnucleus. The internal lateral PB subnucleus, which projects exclusively to the intralaminar thalamic nuclei, and the Kölliker-Fuse nucleus were not innervated by the PAG. The PAG selectively innervates individual PB subnuclei that may be part of the spino-parachio-forebrain pathway. All PAG columns, including the aqueductal region, project to the superior lateral PB subnucleus, a presumed nociceptive relay site that receives inputs from multiple spinal cord regions (laminae I, V, and VIII) and projects to the ventromedial and retrochiasmatic hypothalamic areas-two regions that have been implicated in complex goal-directed behavior (e.g., food intake and reproductive function). Earlier studies demonstrated that the dorsal lateral and external lateral PB subnuclei (inner division) receive overlapping inputs from the superficial dorsal horn (laminae I and II) and the nucleus tractus solitarius, and both PB subnuclei send projections to limbic forebrain areas (e.g., hypothalamus, preoptic region, amygdala). Because the PAG projects to both of these PB subnuclei, this projection system possibly functions as a behavioral state-dependent filter system that modulates ascending nociceptive and/or visceral information as it is relayed through the PB to forebrain sites.

Fine structural organization of spinothalamic and trigeminothalamic lamina I terminations in the nucleus submedius of the cat.

We examined lamina I trigemino- and spinothalamic tract (TSTT) terminals labeled with Phaseolus vulgaris leucoagglutinin in the nucleus submedius (Sm), a nociceptive relay in the cat's thalamus. Volume-rendered (three-dimensional) reconstructions of ten lamina I TSTT terminals identified with light and electron microscopy were built from serial ultrathin sections by computer, which enabled the overall structures of the terminal complexes to be characterized in detail. Two fundamentally different terminations were observed: compact clusters of numerous boutons, which predominate in the dense focus of a lamina I terminal field in the Sm, and boutons-of-passage, which are present throughout the terminal field and predominate in its periphery. Reconstructions of cluster terminations reveal that all boutons of each cluster make synaptic contact with protrusions and branch points on a single dendrite and involve presynaptic dendrites (PSDs) in triadic arrangements, providing a basis for the secure relay of sensory information. In contrast, reconstructions show that boutons-of-passage are generally characterized by simple contacts with PSDs, indicating an ascending inhibitory lamina I influence. These different synaptic arrangements are consistent with physiological evidence indicating that the morphologically distinct nociceptive-specific and thermoreceptive-(cold)-specific lamina I TSTT neurons terminate differently within the Sm. Thus, a suitable structural substrate exists in the cat's Sm for the inhibitory effect of cold on nociception, a behavioral and physiological phenomenon of fundamental significance. We conclude that the Sm is more than a simple relay for nociception, and that it may be an integrative comparator of ascending modality-selective information that arrives from neurons in lamina I.