Genetically encoded fluorescent sensors of membrane potential.

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.

Chemosensitive serotonergic neurons are closely associated with large medullary arteries.

We have previously shown that serotonergic neurons of the medulla are strongly stimulated by an increase in CO(2), suggesting that they are central respiratory chemoreceptors. Here we used confocal imaging and electron microscopy to show that neurons immunoreactive for tryptophan hydroxylase (TpOH) are tightly apposed to large arteries in the rat medulla. We used patch-clamp recordings from brain slices to confirm that neurons with this anatomical specialization are chemosensitive. Serotonergic neurons are ideally situated for sensing arterial blood CO(2), and may help maintain pH homeostasis via wide-ranging effects on brain function. The results reported here support a recent proposal that sudden infant death syndrome (SIDS) results from a developmental abnormality of medullary serotonergic neurons.

Severe deficiencies in dopamine signaling in presymptomatic Huntington's disease mice.

In Huntington's disease (HD), mutation of huntingtin causes selective neurodegeneration of dopaminoceptive striatal medium spiny neurons. Transgenic HD model mice that express a portion of the disease-causing form of human huntingtin develop a behavioral phenotype that suggests dysfunction of dopaminergic neurotransmission. Here we show that presymtomatic mice have severe deficiencies in dopamine signaling in the striatum. These include selective reductions in total levels of dopamine- and cAMP-regulated phosphoprotein, M(r) 32 kDA (DARPP-32) and other dopamine-regulated phosphoprotein markers of medium spiny neurons. HD mice also show defects in dopamine-regulated ion channels and in the D(1) dopamine/DARPP-32 signaling cascade. These presymptomatic defects may contribute to HD pathology.

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

Molecular evolution of the synapsin gene family.

Synapsins, a family of synaptic vesicle proteins, play a crucial role in the regulation of neurotransmission and synaptogenesis. They have been identified in a variety of invertebrate and vertebrate species, including human, rat (Rattus norvegicus), cow (Bos taurus), longfin squid (Loligo pealei), and fruit fly (Drosophila melanogaster). Here, synapsins were cloned from three additional species: frog (Xenopus laevis), lamprey (Lampetra fluviatilis), and nematode (Caenorhabditis elegans). Synapsin protein sequences from all these species were then used to explore the molecular phylogeny of these important neuronal phosphoproteins. The ancestral condition of a single synapsin gene probably gave rise to the vertebrate synapsin gene family comprised of at least three synapsin genes (I, II, and III) in higher vertebrates. Synapsins possess multiple domains, which have evolved at different rates throughout evolution. In invertebrate synapsins, the most conserved domains are C and E. During the evolution of vertebrates, at least two gene duplication events are hypothesized to have given rise to the synapsin gene family. This was accompanied by the emergence of an additional conserved domain, termed A. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:360-377, 1999.

Regulation of synaptotagmin I phosphorylation by multiple protein kinases.

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.

Synapsins as regulators of neurotransmitter release.

One of the crucial issues in understanding neuronal transmission is to define the role(s) of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins and are the most abundant proteins on synaptic vesicles. Synapsins are able to interact in vitro with lipid and protein components of synaptic vesicles and with various cytoskeletal proteins, including actin. These and other studies have led to a model in which synapsins, by tethering synaptic vesicles to each other and to an actin-based cytoskeletal meshwork, maintain a reserve pool of vesicles in the vicinity of the active zone. Perturbation of synapsin function in a variety of preparations led to a selective disruption of this reserve pool and to an increase in synaptic depression, suggesting that the synapsin-dependent cluster of vesicles is required to sustain release of neurotransmitter in response to high levels of neuronal activity. In a recent study performed at the squid giant synapse, perturbation of synapsin function resulted in a selective disruption of the reserve pool of vesicles and in addition, led to an inhibition and slowing of the kinetics of neurotransmitter release, indicating a second role for synapsins downstream from vesicle docking. These data suggest that synapsins are involved in two distinct reactions which are crucial for exocytosis in presynaptic nerve terminals. This review describes our current understanding of the molecular mechanisms by which synapsins modulate synaptic transmission, while the increasingly well-documented role of the synapsins in synapse formation and stabilization lies beyond the scope of this review.

Galanin-5-hydroxytryptamine interactions: electrophysiological, immunohistochemical and in situ hybridization studies on rat dorsal raphe neurons with a note on galanin R1 and R2 receptors.

Galaninergic mechanisms related to 5-hydroxytryptamine neurons in the dorsal raphe nucleus of the rat were analysed using electrophysiology, immunohistochemistry and in situ hybridization. Galanin caused a dose-dependent hyperpolarization accompanied by a decrease in membrane resistance in most 5-hydroxytryptamine-sensitive dorsal raphe neurons. The galanin-induced outward current reversed at about - 105 mV and shifted to a more positive potential with increasing extracellular potassium concentrations. The 5-hydroxytryptamine-induced outward current was enhanced and prolonged by preincubation with a low concentration of galanin (1-10 nM). The immunohistochemical analysis showed (i) generally low levels of galanin in the 5-hydroxytryptamine cell bodies, (ii) moderate numbers of galanin-positive nerve endings around the 5-hydroxytryptamine cell bodies, (iii) presence of galanin-like immunoreactivity in 5-hydroxytryptamine-positive dendrites and (iv) galanin-positive, 5-hydroxytryptamine-negative boutons making synaptic contact with 5-hydroxytryptamine-positive dendrites. The in situ hybridization results suggest that the galanin receptor present in the galanin/5-hydroxytryptamine neurons is not of the recently cloned galanin-R1 type. Taken together these results indicate that galanin exerts an inhibitory effect via an increase in K+ conductance in 5-hydroxytryptamine neurons by acting on a postsynaptic receptor. In addition, galanin at low, possibly physiological concentrations enhances the inhibitory effect of 5-hydroxytryptamine at the cell soma level. We propose that galanin primarily is released from adjacent galanin boutons lacking 5-hydroxytryptamine and also from soma and dendrites of galanin/5-hydroxytryptamine dorsal raphe neurons. Galanin may thus be involved in the manifold functions hitherto ascribed to ascending 5-hydroxytryptamine neurons, for example in mood regulation.

Electrophysiologic effects of galanin on neurons of the central nervous system.

The neuropeptide galanin is found in a large number of neurons and nerve terminals throughout the nervous system. In nerve terminals, galanin is contained in large dense-core vesicles and is released upon electrical stimulation. A variety of electrophysiologic studies have examined the effects of galanin application onto neurons of the central nervous system. Overall, galanin appears to have inhibitory effects in the central nervous system, causing in most cases a potassium-mediated hyperpolarization accompanied by a decrease in input resistance. Other actions include a reduction in presynaptic excitatory inputs and an interaction with other applied neurotransmitters. These effects are robust and long lasting in most cases. Differences in the responses mediated by the various receptor subtypes have not been explored electrophysiologically. More complete analysis awaits the availability of more potent and specific receptor anatagonists.

Sustained neurotransmitter release: new molecular clues.

Chemical synapses convey impulses at high frequency by exocytosis of synaptic vesicles. To avoid failure of synaptic transmission, rapid replenishment of synaptic vesicles must occur. Recent molecular perturbation studies have confirmed that the recycling of synaptic vesicles involves clathrin-mediated endocytosis. The rate of exocytosis would thus be limited by the capacity of the synaptic clathrin machinery unless vesicles could be drawn from existing pools. The mobilization of vesicles from the pool clustered at the release sites appears to provide a mechanism by which the rate of exocytosis can intermittently exceed the rate of recycling. Perturbation of synapsins causes disruption of vesicle clusters and impairment of synaptic transmission at high but not at low frequencies. Both clathrin-mediated recycling and mobilization of vesicles from the reserve pool are thus important in the replenishment of synaptic vesicles. The efficacy of each mechanism appears to differ between synapses which operate with different patterns of activity.

The distribution and significance of CNS adrenoceptors examined with in situ hybridization.

Several of the established alpha 1-, alpha 2- and beta-adrenoceptors have now been isolated and cloned. The in situ hybridization method has been used to map the distribution of many of these adrenoceptors within cells of the CNS. These studies add complementary and new information to our knowledge of adrenoceptor localization provided previously by radioligand-mediated autoradiography. Neuronal cell groups containing one or more mRNAs for seven adrenoceptor subtypes throughout the rat CNS have been mapped. In the present review Anthony Nicholas, Tomas Hökfelt and Vincent Pieribone will examine these localizations and discuss the additional information these maps supply, as well as some implications for understanding central noradrenaline and adrenaline systems.

Distinct pools of synaptic vesicles in neurotransmitter release.

Nerve terminals are unique among cellular secretory systems in that they can sustain vesicular release at a high rate. Although little is known about the mechanisms that account for the distinctive features of neurotransmitter release, it can be assumed that neuron-specific proteins are involved. One such protein family, the synapsins, are believed to regulate neurotransmitter release through phosphorylation-dependent interactions with synaptic vesicles and cytoskeletal elements. Here we show that clusters of vesicles at synaptic release sites are composed of two pools, a distal pool containing synapsin and a proximal pool devoid of synapsin and located adjacent to the presynaptic membrane. Presynaptic injection of synapsin antibodies resulted in the loss of the distal pool, without any apparent effect on the proximal pool. Depletion of this distal pool was associated with a marked depression of neurotransmitter release evoked by high-frequency (18-20 Hz) but not by low-frequency (0.2 Hz) stimulation. Thus the availability of the synapsin-associated pool of vesicles seems to be required to sustain release of neurotransmitter in response to high-frequency bursts of impulses.

Synaptic vesicle depletion in reticulospinal axons is reduced by 5-hydroxytryptamine: direct evidence for presynaptic modulation of glutamatergic transmission.

5-hydroxytryptamine (5-HT; serotonin) is known to depress glutamatergic synaptic transmission in the spinal cord of vertebrates. To test directly whether 5-HT inhibits synaptic glutamate release, we examined its effect on the ultrastructure of synaptic vesicle clusters in giant reticulospinal axons in a lower vertebrate (lamprey; Lampetra fluviatilis). The size of these axons makes it possible to selectively expose only a part of the presynaptic element to 5-HT, while another part of the same axon is maintained in control solution. Action potential stimulation at 20 Hz for 20 min caused a marked reduction in the number of synaptic vesicles in active zones maintained in control solution, while in the part exposed to 5-HT (20 microM) the number of synaptic vesicles per active zone was approximately 3-fold higher. In contrast, 5-HT had no effect on the number of vesicles in resting axons. To examine whether 5-HT acts by reducing presynaptic Ca2+ influx, intra-axonal recordings of Ba2+ potentials were performed. No reduction of the axonal Ba2+ potential could be detected after application of 20 or 200 microM 5-HT. The present results show that 5-HT reduces the rate of synaptic exocytosis in reticulospinal axons. The effect appears to be mediated by a mechanism distinct from the presynaptic Ca2+ channels.

Galanin induces a hyperpolarization of norepinephrine-containing locus coeruleus neurons in the brainstem slice.

Galanin applied in the bath or by micropipette directly on to locus coeruleus neurons in an in vitro slice preparation caused a hyperpolarization accompanied by a small decrease in membrane resistance. Immunohistochemical staining of intracellularly filled neurons indicated that the effect of galanin was exerted on norepinephrine neurons of the locus coeruleus. The galanin effect was variable in amplitude and duration and often showed desensitization, with subsequent applications producing a smaller response. When cells were exposed to tetrodotoxin or tetrodotoxin/low calcium media, the galanin response was still present. Under voltage clamp galanin application caused a net outward current that did not reverse in normal potassium concentrations; however, by increasing extracellular potassium concentrations the net outward current was reversed and the reversal potential shifted to a less negative potential. The response to galanin was identical when either KCl or KAc was used as the intracellular electrode solution. Tetraethylammonium chloride significantly reduced or abolished the response to galanin in most cells, although in a few cells the galanin response was not affected. Glibenclamide, a blocker of ATP-sensitive potassium channels, did not affect the galanin hyperpolarization. In addition, diazoxide had no effect on the membrane properties of locus coeruleus neurons. These results demonstrate that galanin exerts its inhibitory effect in the locus coeruleus via an increase in K+ conductance; however, not via the pancreatic type of ATP-sensitive K+ channels. Cryostat sections of the locus coeruleus incubated in 125I-labeled galanin revealed binding sites in the locus coeruleus at all levels. Sections of the locus coeruleus processed for ultrastructural immunocytochemistry showed galanin immunoreactivity in many neuronal somata and dendritic processes within the nucleus, confirming earlier evidence for the coexistence of galanin and noradrenaline in locus coeruleus neurons. Galanin-immunoreactive soma and dendrites in the locus coeruleus less frequently received galanin-immunoreactive synapses of axonal origin. These findings suggest that endogenous galanin in the locus coeruleus is mainly released from noradrenaline galanin somata and/or dendrites to act on autoreceptors or on receptors on adjacent neurons.

The reticulospinal glutamate synapse in lamprey: plasticity and presynaptic variability.

1. The glutamatergic synapses formed between the unbranched giant reticulospinal axons onto spinal neurons in lamprey offer a central vertebrate synapse in which the presynaptic element can be impaled with one or several microelectrodes, which may be used for recording as well as microinjection of different substances. To provide a basis for the use of this synapse in studies of release mechanisms, we have examined the use-dependent modulation of the synaptic response under conditions of conventional cell body stimulation, and during direct stimulation of the presynaptic axon. 2. To examine the stability of the mixed electrotonic and chemical reticulospinal excitatory postsynaptic potential (EPSP) over time, action potentials were evoked at a rate of 1 Hz for 800-1000 trials. In three out of seven synapses the chemical component remained at a similar amplitude, while in four cases a progressive decrease (up to 35%) occurred. The electrotonic component remained at a similar amplitude in all cases. 3. During paired pulse stimulation of the reticulospinal cell body (pulse interval 65 ms) the chemical EPSP component showed a net facilitation in all cases tested [from 0.64 +/- 0.35 to 0.89 +/- 0.48 (SD) mV, n = 13], while the peak amplitude of the electrotonic component was unchanged (1.37 +/- 0.68 and 1.36 +/- 0.66 mV, respectively). Recording of the axonal action potential during paired pulse stimulation showed that the width of the first and second action potential did not differ [1/2 width (2.48 +/- 0.39 ms and 2.48 +/- 0.42 ms, respectively; n = 8)]. 4. The degree of facilitation varied markedly between different synapses, ranging from an increase of a few percent to a two-fold increase (24 +/- 16% mean change of total EPSP amplitude, corresponding to 44 +/- 26% mean change of chemical EPSP amplitude). This type of variability was also observed in synapses made from the same unbranched reticulospinal axon onto different postsynaptic cells. 5. When paired pulse stimulation was applied to the reticulospinal axon in the very vicinity of the synaptic area (0.1-1 mm) a net depression of the chemical component occurred in 11 out of 19 cases, and in the remaining cases the level of net facilitation was lower as compared with cell body stimulation (range between +17 and -23% change of total EPSP amplitude; mean -5%; n = 19). 6. To test if the change of the EPSP plasticity during local stimulation correlated with an increased transmitter release, two microelectrodes were placed in the same reticulospinal axon at different distances from the synaptic area.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of alpha 1 adrenoceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes.

The distribution of neurons in the rat CNS that synthesize mRNA for the alpha 1A/D and alpha 1B adrenoceptors was revealed by the in situ hybridization method. Forty-eight-mer DNA probes were synthesized to two different and unique regions of both the alpha 1A/D and alpha 1B mRNAs. Tissue sections from all levels of the CNS and some peripheral ganglia were incubated in a hybridization cocktail containing one of these four probes. The two mRNAs were expressed in a discrete and often complementary manner to each other, and identical hybridization patterns were seen for the probes directed against the same mRNA. The alpha 1A/D probes hybridized heavily with neurons in the internal granular and internal plexiform layers of the olfactory bulb, in layers II-V of most areas of the cerebral cortex, and in the lateral aspect of the lateral amygdaloid nucleus, with pyramidal neurons of CA1-CA4 regions, hilar and granular neurons of the dentate gyrus, and neurons in the reticular thalamic nucleus, cranial and spinal motor nuclei, and the inferior olivary nucleus. Light labeling was seen in a variety of other regions in the brain and spinal cord. The alpha 1B probes hybridized heavily with neurons in the mid layers of cerebral cortex and with virtually all neurons in the thalamus, except the reticular and habenular nuclei. In addition, labeling was seen in the lateral and central amygdaloid nuclei, in brainstem and spinal motor nuclei, over most neurons of the dorsal and medullary raphe nuclei and neurons of the intermediolateral cell column in the spinal cord. Light labeling was seen in the septal nucleus, the horizontal limb of the diagonal band, the paraventricular and lateral hypothalamic nuclei, the pontine and medullary reticular formation, and in most laminae in the spinal cord. The patterns of labeling obtained with the alpha 1B probes resemble the labeling seen in previous autoradiographic ligand binding studies utilizing "general" alpha 1 ligands, while the labeling patterns seen with the alpha 1A/D probes do not correspond to any published alpha 1 receptor distribution pattern, indicating that this mRNA likely encodes for a novel adrenoceptor. The present findings further expand the heterogeneity of adrenoceptor mRNAs presented in two accompanying studies (Nicholas et al., 1993a,b). This differential distribution of adrenoceptors subtypes provides a framework for the functional diversity to the apparently widespread, diffuse, and rather homogeneous noradrenergic innervation of the CNS.

Neuropeptide role of both peptide YY and neuropeptide Y in vertebrates suggested by abundant expression of their mRNAs in a cyclostome brain.

The evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY-related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60-70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY- and PYY-expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved.

Immunohistochemical analysis of the relation between 5-hydroxytryptamine- and neuropeptide-immunoreactive elements in the spinal cord of an amphibian (Xenopus laevis).

In mammals, a large proportion of the bulbospinal 5-hydroxytryptamine (5-HT) neurons also contain neuropeptides, such as substance P (SP) and galanin (GAL). To examine whether a similar coexistence occurs in an amphibian, an immunofluorescence double-labelling technique was employed on sections of the Xenopus laevis spinal cord. Antisera raised against SP, GAL, enkephalin (ENK), corticotropin-releasing factor (CRF), calcitonin gene-related peptide (CGRP), and cholecystokinin (CCK) produced a labelling of fibers at all rostrocaudal levels of the spinal cord, with the highest fiber densities for SP and ENK and intermediate densities for GAL, CCK, and CGRP, while CRF-immunoreactive fibers were barely detectable in intact animals. 5-HT-immunoreactive fibers were widely distributed in the spinal cord, and they often occurred in the vicinity of different types of peptide-immunoreactive fibers. However, no coexistence between 5-HT and the different peptide immunoreactivities could be detected, although SP and GAL immunoreactivities were sometimes found to be colocalized in the same fiber. Similar negative results were obtained when 5-HT+SP- and 5-HT+GAL-labelled sections were examined in single focal planes with a confocal microscope. After a spinal transection, (survival period 6 weeks to 4 months), almost all 5-HT-immunoreactive fibers below the lesion were lost, and a build-up of immunoreactive material occurred in fibers just rostral to the cut. In contrast, no significant loss of peptide-immunoreactive fibers occurred, although some swollen SP-, GAL-, ENK-, CRF-, and CCK-immunoreactive fibers were present rostral to the cut. The distribution of swollen peptide-immunoreactive fibers did not overlap with that of the swollen 5-HT-immunoreactive fibers. Although negative immunohistochemical data must be interpreted with caution, in conjunction with previous studies (Brodin et al. [1988] J. Comp. Neurol. 271:1-18; Sakamoto and Atsumi [1991] Cell Tissue Res. 264:221-230), the present results indicate that bulbospinal 5-HT neurons in nonmammalian vertebrates cocontain neuropeptides to a lesser extent than in mammals.