A long ascending pathway of enkephalin-like immunoreactive spinoreticular neurons in the rat

In the present study, we examined whether some long ascending spinal cord neurons contain enkephalin by combining the retrograde transport of the fluorescent dye True Blue with enkephalin immunocytochemistry. Evidence is presented for the existence of enkephalin in a subpopulation of spinoreticular neurons in the rat located in the central canal region and adjacent gray matter.     

Effects of static tilt on cervical spinoreticular tract neurons.

The activity of axons located mainly in the ventral part of the lateral funiculi of the cervical segments C2-C3 were recorded in precollicular decerebrate cats and their responses to lateral tilt of the whole animal studied. Units were identified according to their antidromic and/or transynaptic responses to stimulation of the ipsilateral lateral reticular nucleus (NRL) as well as their responses to ipsilateral and/or contralateral forelimb nerve stimulation. In most respects, those units which could be antidromically identified as cervical ascending tract axons showed properties similar to those described for neurons of the spinoreticular pathway, the bVFRT. Among the 106 recorded units, 42 responded to 15 degrees tilts in the median plane. Steady changes in unit discharge frequency were evoked by tilting, which lasted as long as the position of the cat was maintained. The response of the units to tilting consisted of increased in discharge rate during tilt in one direction, while tilt in the opposite direction resulted in a decrease in discharge rate. The magnitude of the responses was clearly related to the degree of tilt. All the units affected by tilt, except one, received inputs from both ipsilateral and contralateral forelimb nerves and some of them also from high-threshold neck muscle afferents. Furthermore, 12 of these units could be orthodromically excited at short latency from the ipsilateral brain stem, suggesting a possible monosynaptic input perhaps transmitted via the lateral vestibulospinal tract. The responses of the cervical ascending spinal tract units to tilt did not appear to depend on peripheral proprioceptive or cutaneous feedback since a number of precautions were taken to eliminate such influences, while control experiments in animals submitted to partial or complete bilateral VIIth nerve section showed the number of neurons responsive to tilt to be profoundly reduced or eliminated. It appeared also that the average spontaneous discharge rate of the ascending units was much lower following bilateral VIIth nerve section than that obtained from the corresponding units recorded in preparations with the VIIth nerves intact. These findings suggested that the responses of the cervical ascending neurons to tilt as well as their spontaneous background activity depended on influences arising from the macular labyrinthine receptors. The observation that the responses of these ascending neurons to peripheral nerve stimulation are modified by tilt further suggested that the macular system may interact with the somatosensory system originating from the forelimb and the neck musculature, thus being able to produce the fine adjustments that cerebellar and brain stem structures exert in the control of posture and movements.     

Spontaneous spike activity of spinoreticular tract neurons during sleep and wakefulness

Sleep mentation studies infer that pain sensation in humans may be reduced during active REM sleep. However, to provide a mechanistic explanation for this phenomenon, few, if any neurophysiological studies have been performed at the lumbar level from neurons comprising classical pain pathways during sleep and wakefulness. The spinoreticular tract is one such classical pathway that has been implicated in the rostral transmission of nociceptive information. The present study was performed to determine if the activity of spinoreticular tract (SRT) neurons is dependent upon behavioral state. Accordingly, extracellular recording techniques were used to monitor the activity of identified SRT neurons in unanesthetized chronic cats during sleep and wakefulness. The ongoing spike activity of SRT neurons was found to be relatively uniform when the states of quiet wakefulness and quiet sleep were compared. However, during active sleep, the majority of the SRT neurons sampled underwent a sustained reduction in spike activity. Marked facilitation of SRT cell activity occurred in a few instances. These data provide the first unitary evidence supporting earlier evoked potential, psychophysical and clinical studies that ascending sensory information in a classical pain pathway is regulated in a state-dependent fashion.     

Intracellular records of the effects of primary afferent input in lumbar spinoreticular tract neurons in the cat

1. The afferent-evoked synaptic input to lumbar spinal cord (L5-S1) neurons that were activated antidromically from the medial pontomedullary reticular formation (nucleus reticularis gigantocelluaris and vicinity) was investigated with the use of intracellular recordings in pentobarbital sodium-anesthetized cats. 2. Spinoreticular tract (SRT) neurons (n = 33) were categorized into three types ("deep-inhibited," "deep-complex," and "intermediate") on the basis of their locations and of their responses to natural and electrical stimulation. 3. The deep-inhibited-type neurons, located in the medial part of the deeper laminae (approximately VI-VIII), comprised a large component of the sample (20/33). They had no demonstrable excitatory receptive field (RF). However, electrical stimulation of low-threshold cutaneous afferents of hindlimb nerves evoked inhibitory postsynaptic potentials (IPSPs) via an oligosynaptic linkage. High-threshold cutaneous and muscle afferents also evoked IPSPs. 4. In the deep-complex-type neurons (8/33), electrical stimulation of low-threshold cutaneous afferents evoked complex IPSP-excitatory postsynaptic potential (EPSP) sequences. With intense stimuli, long-latency C-fiber-like EPSPs were evoked. Two of these eight neurons were characterized as wide-dynamic-range (WDR) neurons with large, excitatory and inhibitory cutaneous RFs. 5. Intermediate-type neurons (5/33) were concentrated in the lateral spinal gray and relatively superficially (approximately lamina V). These neurons had convergent low- and high-threshold cutaneous inputs (WDR neurons). Electrical stimulation of low-threshold cutaneous afferent fibers from within the excitatory RF evoked mono- or disynaptic EPSPs followed by IPSPs. High-threshold muscle and cutaneous afferents also evoked EPSPs. 6. These results show that SRT neurons have a variety of response characteristics resulting from various degrees of spatial and temporal summation of primary afferent input. Neurons with widespread inhibitory responses but no excitatory drive from the periphery comprise a surprisingly large component of the SRT: the function of these cells is unknown. It is apparent that the spinoreticular projection has considerable functional heterogeneity.     

Activity of peptidergic neurons in the amygdala during sexual behavior in the male rat

The medial amygdaloid nucleus (AME) occupies a central position in the circuitry that organizes sexual behavior in the male rat. It receives a projection from olfactory structures that are activated by pheromonal cues indicating receptivity in the female and projects in turn to limbic and hypothalamic structures that are thought to organize aspects of coitus. Electrical stimulation of the AME elicits a behavioral state that is indistinguishable by several measures from the post-ejaculatory interval. We used chronic single-unit recording techniques to determine the behavioral conditions in which the AME is normally active. We found that the cells indeed fired selectively during the presence of a receptive female, but that the discharge considerably anticipated copulation in time. We propose that sexual behavior in the male rat is a reaction chain of fixed action patterns, each one acting as a releaser for the next. The AME mediates an early event in the reaction chain, namely recognition of the receptive female, but electrical activation of the AME causes the reaction chain to proceed to its culminating behavior, the post-ejaculatory interval.     

Dye-coupled magnocellular peptidergic neurons of the rat paraventricular nucleus show homotypic immunoreactivity

Magnocellular neurons in rat hypothalamic slices are known to exhibit dye coupling: the transfer of the fluorescent dye, Lucifer Yellow, from an intracellularly-injected neuron to one or more nearby neurons. The question of the hormonal identity of coupled cells and the possibility of dye coupling as an artefact led us to determine the immunoreactivity of dye-coupled magnocellular neurons in the paraventricular nucleus of the rat hypothalamus using antisera to oxytocin- and vasopressin-associated neurophysins. In 23 pairs, one triplet, and one quadruplet, immunoreactivity to one or the other antiserum was always exclusive, and dye coupling was always homotypic, that is, coupled neurons in each instance were reactive to the same antiserum. The quadruplet, triplet and 17 pairs were immunoreactive to vasopressin-associated neurophysin, and oxytoxin-associated neurophysin immunoreactivity was observed in the remaining pairs. Immunoreactivity to each antiserum was found for somasomatic and non somasomatic modes of coupling and for coupled neurons in the three magnocellular areas of the nucleus. A relationship between mode of coupling and hormone content was not detected. The data support the hypothesis that coupling is a real, functionally significant mechanism for coordinating neuronal activity in this nucleus, particularly under conditions of high hormone demand. They do not support the idea that coupling is artefact. The possibility of a relationship between hormone content and mode of coupling, and the projection pathway(s) of the coupled neurons of each type require further study.     

Characterization of peptidergic efferents from the lateral parabrachial nucleus to identified neurons in the rat dorsal raphe nucleus

The peptidergic content of the lateral parabrachial nucleus (LPB) efferents to the dorsal raphe nucleus (DRN) was studied by combining visualization of the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin within fibers that were immunocytochemically stained for neurotensin (NT), calcitonin gene-related peptide (CGRP) or galanin (GAL). The identity of DRN target neurons was determined with simultaneous immunocytochemical labelling for serotonin, the major transmitter within the nucleus. Within the DRN, we estimated that about two-thirds of the anterogradely labelled fibers arising from the LPB also showed peptidergic immunoreactivity. NT was the most commonly observed neuropeptide in LPB neuronal efferents directed to the DRN, followed by CGRP and GAL. The peptidergic afferents in the DRN were oriented preferentially in the dorsoventral plane. Peptidergic fibers from the LPB possessed varicosities (diameters not exceeding 3 microns) and were apposed on serotoninergic neuronal somata. Some of the anterogradely labelled peptidergic fibers were not associated with cells showing immunoreactivity for serotonin. The present results suggest that NT-ergic, CGRP-ergic and GAL-ergic neurons within the LPB are in contact with serotoninergic and non-serotoninergic neurons within the DRN. Since the DRN is known to project to the LPB, it is likely that bi-directional interconnections between these nuclei exist. Such linkages may provide anatomical substrates for coordinated autonomic responses.     

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulbs

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat olfactory bulb was obtained with sheep antiserum to glutamate decarboxylase (GAD) and rabbit antiserum to tyrosine hydroxylase (TH). GAD-positive neurons include periglomerular cells, granule cells, superficial and deep short axon cells. TH-positive neurons represent periglomerular cells. Two-color immunocytochemistry shows that GABA and dopamine periglomerular cells are separate populations. The accessory olfactory bulb has rare dopamine cells and few superficial short axon cells. Radial gradients of GAD-immunostaining are evident in the main but not in the accessory olfactory bulb.     

A long ascending projection in the rat brain containing vasoactive intestinal polypeptide

The effects of knife cut lesions of the medial forebrain bundle on the distribution of forebrain vasoactive intestinal polypeptide (VIP)-like immunoreactivity (VIP-LI) in the rat has been studied with radioimmunoassay and immunocytochemistry. The extent of depletion of tyrosine hydroxylase activity from corpus striatum was used to monitor lesion efficiency. Medial forebrain bundle lesions produced substantial depletions of VIP-LI from nucleus accumbens, hypothalamus, amygdala and bed nucleus of the stria terminalis ipsilateral to the lesion. No changes were seen in frontal cortex, olfactory tubercle, striatum and hippocampus. Possible origins of this long ascending projection, including an extensive group of cell bodies containing VIP-LI in the mesencephalic central grey matter, are discussed.     

Neurons in the nucleus of the solitary tract with ascending projections to the subfornical organ in the rat

Thirty-one neurons in the region of the nucleus of the solitary tract (NTS) were antidromically activated by electrical stimulation of the subfornical organ (SFO) in male rats under urethane anesthesia. The activity of these identified neurons was tested for a response to activation of peripheral baroreceptors, achieved by rising arterial blood pressure with an intravenous administration of the alpha-agonist metaraminol. Of the neurons tested, 17 displayed an increase and 6 exhibited a reduction in neuronal firing that accompanied a 40- to 60-mmHg elevation in mean arterial pressure, while 8 were unresponsive. The results suggest that neurons projecting to the SFO in the region of the NTS may be important for carrying peripheral baroreceptor information to the SFO.     

Immunocytochemical characterization of afferents to estrogen receptor-containing neurons in the medial preoptic area of the rat.

Double-label immunocytochemistry has been employed to elucidate the chemical nature of the afferent neuronal projections to the estrogen receptor-containing neurons located in the medial preoptic area of the rat brain. To ensure a clear separation of the immunolabelled afferent profiles from the estrogen receptors, the former were visualized first and the diaminobenzidine reaction product was silver-gold intensified. Using a monoclonal antibody raised against purified human estrogen receptors, we observed an intense nuclear immunoreactivity in Vibratome, semithin and ultrathin sections. Neuropeptide-Y, serotonin-, phenylethanolamine N-methyltransferase- and adrenocorticotrophin-immunoreactive axons and varicosities were observed in close apposition to the estrogen receptor-positive cells. At the ultrastructural level, neuropeptide-Y-immunoreactive boutons were seen in synaptic contact with cells showing estrogen receptor immunoreactivity in their nucleus. These results indicate that neurons located in the medial preoptic area, one of the principal sites for the control of female reproductive function, may be influenced by both estrogen and neurotransmitters/neuropeptides via, respectively, nuclear receptors and synaptic contacts.     

The effects of lesions to ascending noradrenergic neurons on discrimination learning and performance in the rat

Three experiments examined the effect of central noradrenergic depletion on the acquisition and performance of a temporal discrimination in auditory or visual modalities. In Experiment 1, 6-hydroxydopamine-induced lesions of the dorsal noradrenergic bundle significantly retarded acquisition compared to a similarly lesioned ventral noradrenergic bundle-group, two sham-operated and one unoperated control group. In Experiment 2, the acquisition impairment produced in the dorsal noradrenergic bundle group was replicated, for both auditory and visual modalities, by lesions depleting hippocampal and neocortical noradrenaline by over 80%. In rats subsequently switched to discriminations involving the unfamiliar modality, the dorsal-bundle lesion also impaired acquisition several weeks after surgery. Experiment 3 showed significantly impaired performance in rats with dorsal bundle lesions when training prior to surgery had not resulted in better than chance performance. In rats previously trained to criterion, the dorsal bundle lesion transiently, but non-significantly, impaired performance. In rats performing better than chance, but not having reached criterion, there were no significant effects of the dorsal bundle lesion. Subsequent manipulations of deprivation and difficulty of discrimination in general failed to distinguish between the dorsal bundle lesion and sham-groups, suggesting that the acquisition impairment did not result from simple sensory or motivational effects. Reducing the interstimulus interval did impair the dorsal bundle group more than controls. However, there was no evidence of altered "distractibility" in the lesioned group when the alternative modality was introduced as a distractor. The results are discussed in terms of other acquisition deficits shown by rats with central noradrenaline depletion and their significance for determining the functions of the projections from the locus caeruleus via the dorsal noradrenergic bundle to the neocortex and hippocampus.     

Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus

Summary The structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic cells.     

Distribution and morphology of spinothalamic tract neurons in the rat

 Previous studies showed that spinothalamic tract (STT) neurons in the dorsal horn head have a distinct laminar organization that corresponds to the cytoarchitectonic divisions. However, groupings of STT neurons in the deeper part of the spinal gray matter have not yet been established, since, in these regions, the distribution of STT neurons with similar morphology does not follow the cytoarchitectonic subdivisions. To classify STT neurons in the deep part of the rat spinal gray matter, not only the distribution but also the size and shape of STT neurons were investigated in the present study by the method of retrograde axonal transport of wheat germ agglutinin–horseradish peroxidase. In addition to the confirmation of the previously described groups of STT neurons in the dorsal horn head, this study showed novel groupings of STT neurons in the deeper part of the spinal gray matter. First, the interomedio-ventral group of STT neurons, which spreads over laminae VII through IX, were classified into four subgroups in the cervical cord according to their side of projection and medio-lateral location: ipsilateral-medial, ipsilateral-lateral, contralateral-medial and contralateral-lateral. These subgroups were different not only in distribution of neurons but also in mean size of their cell bodies. On the ipsilateral side, neurons in the lateral subgroup were smaller than those in the medial subgroup, while on the contralateral side, the lateral subgroup had larger neurons than the medial subgroup. Second, STT neurons in laminae IV–VI were classified into the dorsal horn neck and base groups based on the orientation of their somata and their rostrocaudal distribution. Neurons in the dorsal horn neck group were more densely distributed and more horizontally aligned than those in the dorsal horn base group. The dorsal horn base group contained more STT neurons projecting to the medial thalamus than the dorsal horn neck group. Accepted: 20 June 1997     

Immunocytochemical identification of proteins involved in dopamine release from the somatodendritic compartment of nigral dopaminergic neurons

We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters.     

Immunocytochemical studies of GABAergic neurons in rat basal ganglia and their relations to other neuronal systems

GABAergic neurons were localized in the rat basal ganglia by glutamate decarboxylase (GAD) immunohistochemistry. In the striatum (caudato-putamen, accumbens nucleus) a medium density of GAD-positive terminals was observed; a small number of medium-to-large size neurons and the vast majority of medium-size neurons were GAD immunoreactive. In addition, opioid peptide-like immunoreactivity was colocalized in a subclass of GAD-positive medium-size striatal neurons. The pallido-nigral system (GP, VP, EP, SNR) displayed a high density of GAD-positive axon terminals which synapsed upon dendrites and nerve cell bodies. The majority of pallido-nigral neurons also were GAD-immunoreactive. In contrast, the substantia nigra pars compacta and the subthalamic nucleus contained only few GAD-immunoreactive neurons.     

Electrophysiology of ascending, possibly cholinergic neurons in the rat laterodorsal tegmental nucleus: comparison with monoamine neurons

In urethane-anesthetized rats single neuronal activity was recorded in the laterodorsal tegmental nucleus (LDT), which has a dense complement of cholinergic neurons, and in the dorsal raphe and locus coeruleus for comparison. Most LDT neurons responded antidromically to stimulation of either one or more of several rostral loci, and gave rise to broad spikes. They showed various properties very similar to those of monoaminergic neurons. Others generated brief spikes. The former may be cholinergic. The most frequent response of broad-spike LDT neurons to noxious stimulation was phasic excitation, which tended to become weak or disappear upon repetition.     

Immunocytochemical localization of cell adhesion molecule L1 in developing rat pyramidal tract

L1 is a representative of a family of carbohydrate neural cell adhesion molecules. The expression of L1 was studied during postnatal development of the rat pyramidal tract by immunohistology using polyclonal antibodies to L1 in spinal cord cervical intumescences. On postnatal day 1 (P1), L1 immunoreactivity was present in the entire dorsal funiculus, consisting of the ascending fasciculus gracilis and fasciculus cuneatus and the descending pyramidal tract. At that time the cervical pyramidal tract contains the first outgrowing corticospinal axons. At P4 both the fasciculus gracilis and the pyramidal tract are immunoreactive whereas the fasciculus cuneatus is negative. At P10 the pyramidal tract is intensely labelled whereas both ascending bundles are negatively stained. In the period between P4 and P10 the pyramidal tract is characterized by a massive outgrowth of corticospinal axons. During pyramidal tract myelination, between P10 and the end of the third postnatal week (P21), L1 immunoreactivity is progressively reduced. These observations suggest that L1 may play a prominent role in outgrowth, fasciculation and the onset of myelination of rat pyramidal tract axons. The differential L1 immunoreactivity of the pyramidal tract and the earlier developing ascending systems in rat dorsal funiculus indicate that this polyclonal antiserum is a useful differentiating marker for outgrowing fibre tracts.