Nicotine-induced dendritic remodeling in the insular cortex

The insular cortex has emerged as a novel target for nicotine addiction research. One unresolved question about the insular cortex is whether its neurons exhibit nicotine-induced dendritic remodeling similar to other brain regions implicated in nicotine addiction. To test this question, Long–Evans rats were administered nicotine via osmotic pump for two weeks. Thirty-seven days following the end of nicotine dosing, rats were sacrificed for Golgi-Cox staining and pyramidal neurons from the rostral agranular insular cortex were digitally reconstructed in three dimensions. Results from morphometric analyses revealed an increased complexity of dendrites in the insular cortex following nicotine. Increases were found for both total dendrite length and number of bifurcations. Sholl analyses revealed these changes depended on the distance from the soma, with the most prominent changes distributed at distal points along the dendritic tree. A follow-up comparison of length and bifurcation measurements from Sholl analyses suggested that new dendritic branches, rather than growth of existing dendrites, most likely contributed to overall changes in complexity. No change in dendrite morphology was found for apical dendrites. Together, these results show the insular cortex is a target for neuroplasticity following nicotine exposure.     

Renal sympathetic nerve activity is increased in monosodium glutamate induced hyperadipose rats

Repeated exposure to cocaine can induce persistent alterations in the brain's reward system, including increases in the number of dendrites and spine density on medium-sized spiny neurons (MSNs) in the nucleus accumbens (NAc). The structural remodeling of dendrites and spines in the NAc is thought to play a critical role in cocaine addiction. MSNs in the NAc can be classified by expression of either D1 or D2 dopamine receptors, which are localized to the direct and indirect pathway, respectively. It is unknown whether the dendritic changes induced by repeated cocaine treatment occur in MSNs of the direct or indirect pathway. Because the traditional Golgi-Cox impregnation of neurons precludes identifying particular subpopulations of MSNs, we performed dendritic morphology analysis after biocytin-labeling and Golgi-Cox impregnation. We found that the biocytin staining MSNs showed higher dendritic spine density and higher number of dendrites than that in Golgi impregnation group. In addition, we found that the increasing spine density induced by repeated cocaine treatment in female mice was higher than that in male mice. Next we used biocytin staining and dynorphin/D2 receptor colocalization to determine which cell type(s) displayed dendritic changes after repeated cocaine treatment. We found that cocaine-induced changes in dendritic parameters occurred in MSNs of both the direct (D1-expressing) and indirect (D2-expressing) pathways.     

D1-like and D2 dopamine receptor antagonists administered into the shell subregion of the rat nucleus accumbens decrease cocaine, but not food, reinforcement

Cocaine self-administration experiments were designed to assess the respective roles of D1-like and D2-like dopamine receptors in the ventral forebrain in cocaine reinforcement. D1-like or D2-like dopamine receptor antagonists were microinjected into the nucleus accumbens core, nucleus accumbens shell, neostriatum or lateral septum prior to sessions in which cocaine was self-administered under a progressive ratio schedule by rats. The results indicated that administration of a D1/5 (SCH-23390) or a D2/D3/D4 (eticlopride), but not a D3 (U99194A) or D4 (L-750,667), dopamine receptor antagonist into the core and shell of the nucleus accumbens decreased the reinforcing efficacy of cocaine. However, in control experiments intra-accumbal core administration of SCH-23390 or eticlopride decreased food self-administration, whereas administration of these drugs into the accumbens shell had no effect on food reinforcement. Neither SCH-23390 nor eticlopride influenced cocaine reinforcement when administered into the neostriatum or lateral septum. Collectively, these results indicate that D1-like and D2 dopamine receptors in the nucleus accumbens shell selectively modulate the reinforcing efficacy of cocaine, whereas D1-like and D2 dopamine receptors in the accumbens core have a more general influence on reinforced behaviors.     

Dopamine D1 and N-methyl-d-aspartate receptors and extracellular signal-regulated kinase mediate neuronal morphological changes induced by repeated cocaine administration

The development of drug addiction involves persistent cellular and molecular changes in the CNS. The brain dopamine and glutamate systems play key roles in mediating drug-induced neuroadaptation. Changes in dendritic morphology in medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and caudate putamen (CPu) accompany drug-induced enduring behavioral and molecular changes. We have investigated the potential involvement of dopamine D1 and D2 receptors, the N-methyl-d-aspartate (NMDA) receptor, and the extracellular signal-regulated kinase (ERK) in dendritic morphological changes induced by repeated cocaine administration. We show that either a genetic mutation or pharmacological blockade of dopamine D1 receptors attenuated cocaine-induced changes in both dendritic branching and spine density of MSNs in the shell of the NAc and CPu. In contrast, antagonism of dopamine D2 receptors had no obvious effect on changes in dendritic branching but had a partial effect on changes in spine density of MSNs in these brain regions following repeated cocaine injections. Pharmacological inhibition of either NMDA receptors or ERK attenuated cocaine-induced changes in both dendritic branching and spine density of MSNs in the shell of the NAc and CPu. These results suggest that dopamine D1 and NMDA receptors and ERK contribute significantly to neuronal morphological changes induced by repeated exposure to cocaine.     

Periadolescent nicotine administration produces enduring changes in dendritic morphology of medium spiny neurons from nucleus accumbens

The objective of the current study was to examine how periadolescent nicotine exposure affects dendritic morphology of medium spiny neurons from the nucleus accumbens shell. Male Long-Evans hooded rats were chronically administered nicotine or saline for a period extending from postnatal day 22 (p22) to p69. Nicotine and saline administration was via subcutaneously implanted osmotic pumps. At p144, 75 days after conclusion of nicotine administration, brains were processed for Golgi-Cox staining. Medium spiny neurons from the nucleus accumbens shell were digitally reconstructed. It was found that neurons from nicotine-treated animals possessed significantly longer dendrites and a greater number of dendritic segments than control animals. A branch order analysis indicated that differences in dendritic length and segment number were most pronounced in third and fourth order segments. A subsequent behavioral experiment suggests that the observed anatomical changes are associated with enduring psychomotor differences. These findings indicate that periadolescent exposure to nicotine can result in long-lasting structural changes in the nucleus accumbens shell and are consistent with behavioral data suggesting that adolescent nicotine exposure may result in vulnerability to nicotine addiction in adulthood.     

The roles of accumbal dopamine D1 and D2 receptors in maternal memory in rats

Female rats show enhanced maternal responsiveness toward their young if they have had maternal experiences before. This kind of maternal experience-based memory is critically dependent on the mesolimbic dopamine (DA) system, especially the nucleus accumbens (NA) shell. However, the relative contributions of the two main DA receptor systems (D1 and D2) within the shell have not been delineated. This study investigates the roles of dopamine D1 and D2 receptors in maternal memory by infusing a selective D1 antagonist, SCH-23390; a selective D2 antagonist, sulpiride; or a combination D-1/D-2 antagonist, cis-Z-flupenthixol, into the NA shell of postpartum female rats. Sulpiride-infused rats showed a significantly longer latency to exhibit full maternal behavior following a 10-day pup isolation period in comparison to the controls that received a vehicle. Cis-Z-flupenthixol disrupted maternal memory to a greater extent, as rats receiving this showed the longest latencies to express maternal behavior. SCH-23390 infusions had only marginal effects. These findings suggest that both the D1 and the D2 receptor subtypes play a role in the consolidation of maternal memory and they might do so by mediating the motivational salience of pup stimulation.     

Differential modulation of nucleus accumbens synapses

The nucleus accumbens (NAcc) is a brain region involved in functions ranging from motivation and reward to feeding and drug addiction. The NAcc is typically divided into two major subdivisions, the shell and the core. The primary output neurons of both of these areas are medium spiny neurons (MSNs), which are quiescent at rest and depend on the relative input of excitatory and inhibitory synapses to determine when they fire action potentials. These synaptic inputs are, in turn, regulated by a number of neurochemical signaling agents that can ultimately influence information processing in the NAcc. The present study characterized the ability of three major signaling pathways to modulate synaptic transmission in NAcc MSNs and compared this modulation across different synapses within the NAcc. The opioid [Met](5)enkephalin (ME) inhibited excitatory postsynaptic currents (EPSCs) in shell MSNs, an effect mediated primarily by micro-opioid receptors. Forskolin, an activator of adenylyl cyclase, potentiated shell EPSCs. An analysis of miniature EPSCs indicated a primarily presynaptic site of action, although a smaller postsynaptic effect may have also contributed to the potentiation. Adenosine and an adenosine A(1)-receptor agonist inhibited shell EPSCs, although no significant tonic inhibition by endogenous adenosine was detected. The effects of these signaling agents were then compared across four different synapses in the NAcc: glutamatergic EPSCs and GABAergic inhibitory postsynaptic currents (IPSCs) in both the core and shell subregions. ME inhibited all four of these synapses but produced a significantly greater inhibition of shell IPSCs than the other synapses. Forskolin produced an increase in transmission at each of the synapses tested. However, analysis of miniature IPSCs in the shell showed no sign of a postsynaptic contribution to this potentiation, in contrast to the shell miniature EPSCs. Tonic inhibition of synaptic currents by endogenous adenosine, which was not observed in shell EPSCs, was clearly present at the other three synapses tested. These results indicate that neuromodulation can vary between the different subregions of the NAcc and between the different synapses within each subregion. This may reflect differences in neuronal interconnections and functional roles between subregions and may contribute to the effects of drugs acting on these systems.     

Dopamine activity in the lateral anterior hypothalamus modulates AAS-induced aggression through D2 but not D5 receptors

Treatment with anabolic-androgenic steroids (AAS) throughout adolescence facilitates offensive aggression in Syrian hamsters. In the anterior hypothalamus (AH), the dopaminergic neural system undergoes alterations after repeated exposure to AAS, producing elevated aggression. Previously, systemic administration of selective dopamine receptor antagonists has been shown to reduce aggression in various species and animal models. However, these reductions in aggression occur with concomitant alterations in general arousal and mobility. Therefore, to control for these systemic effects, the current studies utilized microinjection techniques to determine the effects of local antagonism of D2 and D5 receptors in the AH on adolescent AAS-induced aggression. Male Syrian hamsters were treated with AAS throughout adolescence and tested for aggression after local infusion of the D2 antagonist eticlopride, or the D5 antagonist SCH-23390, into the AH. Treatment with eticlopride showed dose-dependent suppression of aggressive behavior in the absence of changes in mobility. Conversely, while injection of SCH-23390 suppressed aggressive behavior, these reductions were met with alterations in social interest and locomotor behavior. To elucidate a plausible mechanism for the observed D5 receptor mediation of AAS-induced aggression, brains of AAS and sesame oil-treated animals were processed for double-label immunofluorescence of GAD?? (a marker for GABA production) and D5 receptors in the lateral subdivision of the AH (LAH). Results indicate a sparse distribution of GAD?? neurons colocalized with D5 receptors in the LAH. Together, these results indicate that D5 receptors in the LAH modulate non-GABAergic pathways that indirectly influence aggression control, while D2 receptors have a direct influence on AAS-induced aggression.     

Subsets of neurotensin-immunoreactive neurons revealed following antagonism of the dopamine-mediated suppression of neurotensin immunoreactivity in the rat striatum.

The distribution of neurotensin-immunoreactive structures in the rat striatum was evaluated after blockade of dopamine neurotransmission by drugs that act presynaptically (6-hydroxydopamine, reserpine) and postsynaptically, preferentially at the D2 (eticlopride, haloperidol) and D1 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol, SCH-23390] receptor sites. Calbindin-D (mol. wt 28,000) immunoreactivity was used to delineate patch (striosome) and matrix in the caudate-putamen and core and shell in the nucleus accumbens. Antagonism at the D2 dopamine receptor and 6-hydroxydopamine lesions caused dense axonal immunoreactivity and moderate numbers of neurotensin-immunoreactive neurons to be distributed preferentially in the matrix of the caudate-putamen. D1 receptor antagonism was significantly less effective at eliciting neurotensin-immunoreactive neurons in the caudate-putamen. Reserpine or co-administration of the D1 and D2 receptor antagonists produced many neurotensin-immunoreactive neurons in both striatal compartments throughout the caudate-putamen and dense axonal neurotensin immunoreactivity in the medial patch compartment. To varying degrees, with SCH-23390 being least effective and reserpine most effective, all of the drug treatments elicited neurotensin immunoreactivity in neurons in the olfactory tubercle, rostral nucleus accumbens, accumbal shell and ventrolateral caudate-putamen, i.e. most of the ventral striatum.     

Calcium-dependent NMDA-induced dendritic injury and MAP2 loss in acute hippocampal slices

Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient N-methyl-d-aspartic acid (NMDA) exposure resulted in irreversible loss of synaptic function and loss of microtubule associated protein 2 (MAP2) from apical dendrites. The present study examined the initiation and progression of dendritic injury in mouse hippocampal slices following this excitotoxic stimulus. NMDA exposure (30 microM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20-90 min) involving most proximal dendrites. Over the same time course, immunoreactivity for the microtubule-associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the time course of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Deltaneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slices.     

Electrical and pharmacological manipulations of the nucleus accumbens core impair synaptic plasticity in the dentate gyrus of the rat

The interest on the physiology of the nucleus accumbens (NAcc) has grown in recent years given its relationship to addictive behaviours, and the possibility to treat them by interacting with NAcc function. We have shown that the prior stimulation of the core region blocks induction of long-term potentiation (LTP) at the dentate gyrus in anaesthetized rats, while the shell facilitated it. In the present study we have confirmed and expanded those results testing the effects of core and shell stimulation in freely moving rats, as well as the effect of blocking D1 receptors in the NAcc. Our results show that shell stimulation had no effect on baseline recordings of the field excitatory postsynaptic potential (fEPSP) or the population spike amplitude (PSA) for 24 h. Core stimulation did not modify baseline-fEPSP, but significantly depressed PSA up to 8 h. LTP maintenance was not modified; neither by core nor shell stimulation after its induction, but LTP induction was impaired (both in the fEPSP and PSA) by core stimulation 15 min before induction. Shell stimulation showed a slight facilitating effect. Previous, topical application of a dopaminergic-receptor antagonist (SCH23390) into the NAcc produced a significantly depressed baseline fEPSP and PSA, as well as LTP measured in both components of the evoked potentials. Our results confirm a dual role of stimulation of NAcc sub-regions on hippocampal baseline synaptic transmission, and LTP induction when activated before induction. In contrast, stimulation of the NAcc had no influence on an already ongoing dentate gyrus LTP. A role for dopaminergic innervation to the NAcc, modifying susceptibility for synaptic plasticity outside the NAcc is also suggested by our results.     

Genetic and dietary effects on dendrites in the rat hypothalamic ventromedial nucleus

Both genetic and environmental factors contribute to individual differences in body weight regulation. The present study examined a possible role for the dendritic arbor of hypothalamic ventromedial nucleus (VMH) neurons in a model of diet-induced obesity (DIO) in male rats. Rats were screened and selectively bred for being either susceptible, i.e., exhibiting DIO, or diet resistant (DR) when exposed to a 31% fat diet. A 2 × 2 experimental design was used, based on these two strains of rats and exposure to rat chow versus the 31% fat diet for seven weeks. Golgi-impregnated neurons were measured for soma size and dendrite parameters, including number, length, and direction. As previously observed, each VMH neuron had a single long primary dendrite. Genetic background and diet did not affect soma size or the number of dendrites of VMH neurons. However, genetic background exerted a main effect on the length of the long primary dendrites. In particular, the long primary dendrites were approximately 12.5% shorter on the VMH neurons in the DIO rats compared with DR rats regardless of diet. This effect was isolated to the long primary dendrites extending in the dorsolateral direction, with these long primary dendrites 19% shorter for the DIO group compared with the DR group. This finding implicates the connectivity of the long primary dendrites on VMH neurons in the control of energy balance. The functional significance of these shortened dendrites and their afferents warrants further study.     

Postweaning social isolation enhances morphological changes in the neonatal ventral hippocampal lesion rat model of psychosis

Neonatal ventral hippocampal (nVH) lesions in rats have been widely used as a neurodevelopmental model that mimics schizophrenia-like behaviors. Recently, we reported that nVH-lesions result in significant decreases in both length of dendrites and dendritic density of spines of pyramidal neurons of the prefrontal cortex (PFC) and in the density of dendritic spines of medium spiny neurons of the nucleus accumbens (NAcc). Moreover, postweaning social isolation induces major decreases in dendritic spiny density of PFC neurons. We investigated here the comparative dendritic morphology of PFC pyramidal neurons and NAcc medium spiny neurons in nVH rats, following social isolation after weaning (8 weeks). Morphological characteristics of dendrites were measured using the Golgi-Cox procedure followed by a Sholl analysis. Social isolation (SI) by itself induced decreases in dendritic length and dendritic spine density of the NAcc. In socially isolated nVH-lesion rats decrease in dendritic length in PFC and NAcc neurons were exacerbated whereas an increase in spine density of medium spiny neurons was observed in the NAcc. These results indicate that nVH-lesions alter dendritic morphology of NAcc and PFC neurons. These anatomical modifications in both structures may be relevant to behaviors observed in schizophrenia.     

Behavioral sensitization to apomorphine in pigeons (Columba livia): blockade by the D1 dopamine antagonist SCH-23390

Repeated administration of apomorphine leads to a context-dependent pecking response sensitization. Previously sensitized pigeons (Columba livia) challenged with saline in the same context show a conditioned response (CR). The authors studied the effects of intrastriatal injections of the dopamine (D(1)) antagonist SCH-23390 on both the sensitized response and the CR. When coadministered with apomorphine, SCH-23390 inhibited the initial response to apomorphine, prevented the development of sensitization, and impaired the maintenance of an already developed sensitization. However, SCH-23390 had no effect on the retrieval of a previously established CR. It is concluded that the activation of D(1) receptors in the caudal avian striatum is necessary for the acquisition and maintenance of the sensitization, but not for the expression, of the CR.     

Blockade of nicotinic acetylcholine or dopamine D1-like receptors in the central nucleus of the amygdala or the bed nucleus of the stria terminalis does not precipitate nicotine withdrawal in nicotine-dependent rats

Approximately 70% of tobacco smokers wish to quit, but attempts are often unsuccessful partly due to the aversive nicotine withdrawal syndrome. We investigated the possible involvement of nicotinic and dopaminergic signalling in the central nucleus of the amygdala (CeA) and dorsolateral bed nucleus of the stria terminalis (dlBNST) in the anhedonic depression-like effect of precipitated nicotine withdrawal in rats. Nicotine-dependent rats exhibit elevations in intracranial self-stimulation (ICSS) thresholds compared to control rats after cessation of chronic nicotine administration (spontaneous withdrawal) or systemic or intra-ventral tegmental area (VTA), but not intra-nucleus accumbens (NAcc), administration of nicotinic acetylcholine receptor (nAchR) antagonists while exposed to nicotine (precipitated withdrawal). We examined whether intracerebral administration of the nAChR antagonist dihydro-beta-erythroidine (DHbetaE; 0.6-20 microg total bilateral dose) or the dopamine D1-like receptor antagonist SCH 23390 (2-16 microg total bilateral dose) into the CeA and dlBNST results in withdrawal-like threshold elevations in nicotine-treated rats. Nicotinic acetylcholine and D1-like receptor blockade in the CeA or the dlBNST did not induce differential threshold elevations in nicotine- and saline-treated rats. Further, the highest SCH 23390 dose (16 microg bilateral dose) injected into the dlBNST, but not the CeA, elevated thresholds similarly in both saline- and nicotine-treated rats, suggesting that dopaminergic signalling in the dlBNST may regulate brain reward function under baseline conditions. These results suggest that nACh and D1-like signalling in the CeA and the dlBNST does not develop neuroadaptations with the development of nicotine dependence that may be involved in the depression-like aspects of nicotine withdrawal.     

Direct effect of nicotine on mesolimbic dopamine release in rat nucleus accumbens shell

Nicotine stimulates dopamine (DA) cell firing via a local action at somatodendritic sites in the ventral tegmental area (VTA), increasing DA release in the nucleus accumbens (NAcc). Additionally, nicotine may also modulate DA release via a direct effect in the NAcc. This study examined the contribution of the latter mechanism on NAcc DA release by applying nicotine systemically, as well as locally in the VTA and NAcc shell region in rats. Furthermore, the effect of i.v. nicotine on cell firing rate of dopaminergic neurons in the VTA was measured. Systemic administration of nicotine (0.32mg/kg s.c.) increased extracellular DA levels in the NAcc to ～1.5 fold of baseline, while DA levels in the VTA remained unaffected. A similar DA increase was observed after local NAcc infusion of nicotine (1μM and 10μM). However, 10-1000-fold higher nicotine concentrations were required in the VTA to produce a comparable 150% increase in extracellular DA levels in the ipsilateral NAcc. Additionally, electrophysiological experiments showed that the dopaminergic firing rate in the VTA showed a trend towards an increase after a nicotine dose of 0.1mg/kg i.v. Taken together these data indicate that the effects of nicotine on DA release at the level of the NAcc might be more important for the rewarding effects than originally proposed.     

Motor-skill learning in a novel running-wheel task is dependent on D1 dopamine receptors in the striatum

Evidence indicates that dopamine receptors regulate processes of procedural learning in the sensorimotor striatum. Our previous studies revealed that the indirect dopamine receptor agonist cocaine alters motor-skill learning-associated gene regulation in the sensorimotor striatum. Cocaine-induced gene regulation in the striatum is principally mediated by D1 dopamine receptors. We investigated the effects of cocaine and striatal D1 receptor antagonism on motor-skill learning. Rats were trained on a running wheel (40-60 min, 2-5 days) to learn a new motor skill, that is, the ability to control the movement of the wheel. Immediately before each training session, the animals received an injection of vehicle or cocaine (25 mg/kg, i.p.), and/or the D1 receptor antagonist SCH-23390 (0, 3, 10 microg/kg, i.p., or 0, 0.3, 1 microg, intrastriatal via chronically implanted cannula). The animal's ability to control/balance the moving wheel (wheel skill) was tested before and repeatedly after the training. Normal wheel-skill memory lasted for at least 4 weeks. Cocaine administered before the training tended to attenuate skill learning. Systemic administration of SCH-23390 alone also impaired skill learning. However, cocaine given in conjunction with the lower SCH-23390 dose (3 microg/kg) reversed the inhibition of skill learning produced by the D1 receptor antagonist, enabling intact skill performance during the whole post-training period. In contrast, when cocaine was administered with the higher SCH-23390 dose (10 microg/kg), skill performance was normalized 1-6 days after the training, but these rats lost their improved wheel skill by day 18 after the training. Similar effects were produced by SCH-23390 (0.3-1 microg) infused into the striatum. Our results indicate that cocaine interferes with normal motor-skill learning, which seems to be dependent on optimal D1 receptor signaling. Furthermore, our findings demonstrate that D1 receptors in the striatum are critical for consolidation of long-term skill memory.     

Spinal lamina I neurons that express neurokinin 1 receptors: morphological analysis

The morphology of neurons in lamina I of the dorsal horn of the lumbar spinal cord which express neurokinin 1 receptors in the rat has been investigated. On the basis of soma and dendritic measurements, these neurons form two populations. One group consists of large neurons that stain intensely for the neurokinin 1 receptor with the immunochemical methods employed. They have a large soma, typically giving rise to between three and five thick principal dendrites. The dendritic tree, however, is relatively sparse, with the principal dendrites giving rise to small numbers of second- and third-order branches. All these dendrites are almost spine free. The dendritic tree spreads extensively in the rostrocaudal (approximately 550microm) and mediolateral (approximately 30microm) orientations, with few ventrally directed branches. These cells give rise to a single axon from their soma or a principal dendrite that generates a few local branches and also ramifies sparsely in deeper laminae (II-IV). The details of axonal morphology were established from intracellularly labelled material. Ultrastructural analysis of the synaptic input to these neurons reveals that they receive synapses with both clear round, flattened and dense-core vesicles; however, they do not form components of glomerular synapses.The second neuron type stains less intensely and typically has a small fusiform soma, giving rise to dendrites at its rostral and caudal poles. The dendritic tree is long in the rostrocaudal orientation (approximately 350microm), but restricted mediolaterally (approximately 40microm). The primary dendrites of these neurons bifurcate and soon give rise to third-order branches that are spiny. No pattern of organization could be detected for the distribution of either neuron type. These observations are discussed in the light of other recent studies indicating a central role for lamina I neurons expressing neurokinin 1 in the perception of severe pain.     

Enkephalin-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal innervation of the ventrolateral dendritic bundle in the cat sacral spinal cord: An ultrastructural study

The distribution and synaptic arrangement of thyrotropin-releasing hormone-, substance P- and enkephalin-immunoreactive axonal boutons have been studied in the ventrolateral nucleus (Onuf's nucleus) of the upper sacral spinal cord segments in the cat. For this purpose, the peroxidase-antiperoxidase immunohistochemical technique was used. Immunoreactive axonal boutons were traced in complete series of sections in order to reveal synaptic contacts with the bundled dendrites of the ventrolateral nucleus. As judged from the cross-sectional diameter of the postsynaptic dendrites, the distribution of immunoreactive boutons was non-random. Enkephalin-immunoreactive axonal boutons, presumed to be mostly of segmental origin, displayed a rather restricted distribution to mainly (> 80%) medium-to-large dendrites. Thyrotropin-releasing hormone-immunoreactive boutons, that derive from supraspinal levels, were also found to impinge on medium-to-large dendrites (> 80%), indicating a proximal location within the dendritic trees. The skewness toward large postsynaptic dendrites was even more marked for thyrotropin-releasing hormone- than for enkephalin-immunoreactive boutons. Substance P-immunoreactive boutons, that are of either supraspinal or spinal origin, showed a more even distribution throughout the dendritic trees, including both thin distal branches and thick proximal dendrites. In view of the well-known fact that virtually all thyrotropin-releasing hormone-immunoreactive boutons in the ventral horn cocontain substance P (and serotonin) it was assumed that substance P-immunoreactive boutons in synaptic contact with the finest-calibre dendrites as well as most of those with a very proximal juxtasomatic location on the dendritic trees were of segmental origin, while those impinging on medium-to-large dendrites could be of either spinal or supraspinal origin. Fine-calibre dendrites (< 1 μm) represent about 25% of the dendritic branches in the ventrolateral nucleus, but receive, with the exception of substance P (8%), very little (< 3%) peptidergic or GABAergic (Ramírez-León and Ulfhake, 1993) input, although the degree of dendritic membrane covering by bouton profiles in the ventrolateral nucleus does not seem to vary much with the calibre of the postsynaptic dendrite (Ramírez-León and Ulfhake, 1993). Both substance P- and enkephalin-immunoreactive axonal boutons established synaptic contact with more than one dendrite. Furthermore, one and the same bouton could be found in contact with two dendrites that were coupled to each other by a dendro-dendritic contact of desmosomal or puncta adherentia type. This synaptic arrangement was, however, not seen among thyrotropin-releasing hormone-immunoreactive boutons, indicating that these axonal boutons act on a single postsynaptic element, while inputs intrinsic to the spinal cord can show a divergence also at the terminal level.