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354
G.
THE
BERNOCCHI
JOURNAL OF
ET EXPERIMENTAL
AL.
ZOOLOGY 280:354–367 (1998)
Bioactive Peptides and Serotonin
Immunocytochemistry in the Cerebral Ganglia
of Hibernating Helix aspersa
G. BERNOCCHI,* C. VIGNOLA, E. SCHERINI, D. NECCHI, AND M.B. PISU
Dipartimento di Biologia Animale, Centro di Studio per l’Istochimica del
C.N.R., Universita´ degli Studi di Pavia, I-27100 Pavia, Italy
ABSTRACT
The role of some neuromodulators and neurotransmitters in the functioning of molluskan cerebral neurons and in their metabolic changes during hibernation has been considered.
The cerebral ganglion of mollusks is a center for the integration of different inputs from the sensory
areas of the head and for the generation of motor command impulses. During hibernation, animals
are deprived of many external sensory stimuli and do not have locomotion and feeding. Immunocytochemistry for bioactive peptides (BAPs), such as SP (Substance P), CCK8 (Cholecystokinin 8/
Gastrin), CGRP (Calcitonin-Gene-Related Peptide) and ET (Endothelin), and serotonin was performed on cerebral ganglia of active and hibernating Helix aspersa. The distribution of the
immunopositivity was analyzed in different cell-containing areas (procerebrum, mesocerebrum,
metacerebrum) and in the neuropiles. With all the antibodies raised against peptides, we observed
that only a few neurons, mainly of small and medium size, had immunopositivity during the period
of activity, the patterns of distribution being quite similar to those previously described in Helix or
other gastropods. Fibers and varicosities with BAP immunopositivity were found in the procerebral
and central neuropiles and sometimes around neurons. Serotonin-immunopositive neurons, including the giant neuron, were observed in the metacerebrum; numerous fibers and varicosities
immunopositive for serotonin were present in the neuropile areas. In hibernating snails, the number of fibers with BAP and serotonin immunopositivity decreased in several areas of the neuropiles.
Moreover, an increased number of neurons of the metacerebrum (two- to four-fold) and mesocerebrum
(8- to 28-fold) had BAP-like immunopositivity, and the intensity of the immunoreaction for serotonin
of the metacerebral neurons was also higher than in the active snails. These results are discussed,
taking into account two hypotheses. The first hypothesis assumes that the increased immunocytochemical staining was really linked to accumulation of BAPs and serotonin. The second hypothesis
considers that the antibodies for BAPs recognized a preprotein, the synthesis of BAPs being completed during the active period only. Both the hypotheses account for the co-occurrence and colocalization of two or ore peptides and serotonin and stress that the hibernation condition is of
interest for studies on the actual function of single neurons in the cerebral ganglia. Finally, the data
are consistent with the changes recently found in other markers of the morphological and functional
activity of neurons, demonstrating that the neuromodulation and the neurotransmission are slowed
during hibernation. J. Exp. Zool. 280:354–367, 1998. © 1998 Wiley-Liss, Inc.
A wide spectrum of biological problems can be
investigated using hibernation as a model. In fact,
studies of hibernating animals are of particular
interest in learning about the physiology and metabolism of organisms (Hunter, ’64; Machin, ’75;
Heller, ’79; Lyman et al., ’82), because they contribute to the understanding of some important
aspects of the structure and function of cells
(Kolaeva et al., ’80; Bernocchi et al., ’86; Barni et
al., ’87; Giacometti et al., ’89; Anderson et al., ’90;
Barni and Bernocchi, ’91; Fenoglio et al., ’92;
Malatesta et al., ’94, ’95; March and Reisman, ’95).
Our research was focused on studying the
© 1998 WILEY-LISS, INC.
mechanism by which neurons change their metabolism when there is suppression or reduction
of some physiological activities of the organism
(Hunter, ’64; Machin, ’75; Heller, ’79; Lyman et
al., ’82). This may be considered a primitive form
of cellular adaptability, similar to that used in a
variety of situations associated with neuronal
plasticity.
*Correspondence to: Prof. Graziella Bernocchi, Dipartimento di
Biologia Animale, Piazza Botta 10, I-27100 Pavia, Italy. E-mail:
BERN@IPV36.UNIPV.IT
Received 20 February 1997; Accepted 24 October 1997
HIBERNATION AND NEURONAL ACTIVITY
A large number of biactive peptides (BAPs) has
been demonstrated in specific cell populations of
both vertebrates and invertebrates. In particular,
in the nervous system, the amino acid sequence
of specific peptides is often similar in different
phyla, suggesting that this class of molecules is
widely distributed and highly conserved (for a review, see Fasolo and Clairambault, ’91).
BAPs have a pervasive role in governing essentially all physiological activities, and there are
probably only a few biological processes in which
they are not involved. In particular, in invertebrates, the role of BAPs in the regulation of a variety of activities has been extensively investigated
in mollusks. They are involved in feeding (CCK8/
Gastrin, FMRF-amide, see Engelhardt et al., ’82;
Vigna et al., ’84; Elekes and Naessel, ’90; Sonetti
et al., ’90; Cottrel, ’92; Cottrel et al., ’92), pain
perception, blood flow regulation, and vasodilation
(CGRP, ET, SP, see Osborne et al., ’82; Osborne
’84; Breimer et al., ’88; Giaid et al., ’91). In addition, recent studies (Elekes et al., ’93, ’94) on the
distribution of enkephalins and leucokinin in the
central nervous system of Helix point to an involvement of opioid substances in several behavioral phenomena (Gutierrez and Asai, ’91; Marchang et al.,
’91; Elekes et al., ’93, ’94). Peptides of the VIP (vasoactive intestinal peptide) family are present in
the nervous system of Helix, where they are involved in several functions such as vasodilation,
water and electrolyte secretion, regulation of metabolism, modulation of circadian rhythm, promotion of glycogenolysis, etc. (Kaufmann et al., ’95).
Recent reports have demonstrated that the number of VIP-immunoreactive cells is lower in active snails collected in summer in comparison with
hibernating animals (Kaufmann et al. ’95).
In addition to BAPs, the biogenic amine serotonin, which occurs in relatively high concentrations
in the central nervous system (CNS) of mollusks
(Osborne et al., ’82; Osborne, ’84; Hernadi et al.,
’89; Elekes, ’91; Vehovszky et al., ’93), can affect
and modulate a variety of behavioral and physiological processes, such as feeding, withdrawal and
escape reaction, blood circulation, and some forms
of learning. Some data indicate that, in Helix, the
number of serotonin-immunoreactive neurons is
lower during hibernation (Hernadi et al., ’89).
In order to have a histological and histochemical picture of CNS physiology during hibernation,
we have carried out a comparative analysis of
changes in immunopositivity for BAPs, whose distribution is at present unknown in hibernating
Helix aspersa. The BAPs we have analyzed are
355
involved in many functions accomplished by the
cerebral ganglion of H. aspersa, which is a center
of integration of several stimuli (Kandel, ’76;
Balaban, ’93) that are at least partially suppressed
during hibernation. The BAP-immunoreactivity
changes were compared with the immunocytochemical pattern of serotonin, one of the first neurotransmitters studied by fluorescence histochemistry
(Cardot, ’71; Kerkut and Walker, ’75). A preliminary
report of some of these findings has already appeared in abstract form (Bernocchi et al., ’93).
MATERIALS AND METHODS
Adult terrestral snails (Helix aspersa), collected
in the garden of the Section of Ecology (Department of Genetics and Microbiology, University of
Pavia) were used. The active animals were collected in September and remained active in the
laboratory for 10 days before killing. The hibernating snails were collected in November with the
shell closed by the epyphragm and kept in this
condition at 4°C in a cage until February. A total
of 54 animals of similar size was used.
Fixation and sectioning
The cerebral ganglia were fixed in Bouin solution (12 for both active and hibernating animals)
for 12 h at room temperature or in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 (12
for both active and hibernating animals), for 12 h
at 4°C. After being washed in 70% ethanol for 3
days (Bouin) or in phosphate buffer for 12 h
(paraformaldehyde), the ganglia were dehydrated
and embedded in paraffin. Eight-µm thick horizontal or transverse sections were collected on silane-coated slides.
Three other ganglia fixed in Bouin for each
experimental group were cut serially (series of
4 slides) for counting immunoreactive neurons
(see below).
Immunocytochemistry
Immunocytochemical reactions were carried out
simultaneously for active and hibernating animals. For every antibody and fixative solution, two
slides from at least three animals for both the active and hibernation periods were used.
On Bouin- and paraformaldehyde-fixed specimens, the following rabbit polyclonal antibodies
were used: 1:500 anti-Substance P (SP, Sera Lab,
Sussex, U.K.), 1:500 anti-Cholecystokinin/Gastrin
(CCK8/Gastrin, Incstar, MN), 1:500 anti-CalcitoninGene-related Peptide (CGRP, Peninsula Lab, CA),
1:500 anti-Endothelin (ET, Peninsula Lab). Seroto-
356
G. BERNOCCHI ET AL.
nin immunocytochemistry (1:500 antiserotonin,
Incstar) was performed on paraformaldehyde-fixed
specimens only. For all antibodies, the incubation
time was 18 h. The peroxidase-antiperoxidase (PAP)
method of Sternberger et al. (’70) was used to reveal the sites of antigen-antibody reaction. Briefly,
the sections were incubated in 3% H2O2 in 10%
methanol for 7 min; 5% normal goat serum for 45
min; primary antibodies; 1:50 goat-anti-rabbit IgG
for 90 min; 1:200 rabbit PAP for 60 min; and 0.05%
3,3´diaminobenzidine tetrahydrochloride in 0.05 M
Tris/HCl buffer, pH 7.6, with 0.01% H2O2 for 20 min.
All washings and incubations were in PBS and at
room temperature (20°C). For negative controls,
some sections were incubated in normal rabbit serum instead of the primary antibodies at the same
dilution; for positive staining, control tissues and
cells that contain the antigen were used.
Counting neurons
The number of immunoreactive for BAPs and
immunonegative neurons of the metacerebrum
and mesocerebrum was estimated on serial sections of Bouin-fixed ganglia. To avoid multiple
counting of a neuron, its cell body was traced out
on consecutive sections. The number of immunoreactive neurons was also expressed as percentage
of the total neuronal population. The differences
Fig. 1. Immunoreaction for CGRP in paraformaldehydefixed ganglia. (a) Active snails: the neurons (n) are immunonegative; in the central neuropile there are varicosities and
strongly labeled fibers. (b) Hibernating snails: several small-
between the active period and hibernation were
significant by a Mann-Whitney test (P < 0.001).
RESULTS
All the antibodies recognized epitopes in the neuronal perikarya and/or in the fibers of neuropile
and interneuronal area (trophospongium) of the
land snail CNS. The staining pattern was consistent in the different animals of the same period
(activity or hibernation). No immunohistochemical
labeling was observed in the cerebral areas and
neuropiles in negative control slides.
In the following paragraphs we shall describe
analytically the patterns of the metacerebrum and
mesocerebrum (Figs. 1–7) and the central neuropile. In the procerebrum, with all the antibodies,
no positivity was detectable in the cytoplasm of the
small neurons during both the active period and
the period of hibernation. During the active period,
around the neurons and in the neuropile, there were
fibers and varicosities immunopositive for some of
the antibodies, e.g., serotonin (Fig. 5a), SP, CGRP,
ET. This BAP and serotonin immunoreactivity was
less detectable during the hibernation (Fig. 5b).
Influence of fixatives
Paraformaldehyde permitted a better detection
of the immunopositivity to some BAPs of the fibers in the neuropiles and around neurons. For in-
and medium-sized neurons of the metacerebrum are strongly
immunopositive, but some areas (stars) of the central neuropile do not show immunopositive fibers or varicosities. a: ×90;
b: ×100.
HIBERNATION AND NEURONAL ACTIVITY
357
Fig. 2. Immunoreaction for CGRP in Bouin-fixed ganglia.
(a) Active snails: immunopositivity is detectable in some small
and large neurons and the giant neuron (GN, arrow) of the
metacerebrum. (b) Hibernating snails: most neurons of the
metacerebrum, including the GN (arrow) are strongly immunostained. a, b: ×100.
Fig. 3. Immunoreaction for CCK8/gastrin in Bouin-fixed
ganglia. (a, b) Active snails: the fibers around neurons are
immunopositive (arrows). On the contrary, the neurons are
negative. (c, d) Hibernating snails: in the meso (Ms) and
metacerebrum (Mt), part of the cytoplasm of several neurons
is immunopositive; the fibers around neurons maintain
immunopositivity (arrows). a, c: ×90; b, d: ×360.
358
G. BERNOCCHI ET AL.
Fig. 4. Immunoreaction for SP in Bouin-fixed sections. (a)
Active snails: immunopositive fibers (arrows) are present
around neurons that appear negative. (b) Hibernating snails:
a part of the cytoplasm of some mesocerebral neurons is immunoreactive (stars). a, b: ×360.
stance, after immunoreaction for CGRP (Fig. 1a),
CCK8 (not shown) and SP (not shown) strong positivity was present in the varicosities and fibers of
the central neuropile of active snails. This positivity was not observed in several areas of the neuropile during hibernation (Fig. 1b) and was decreased
after immunoreaction for SP around neurons.
In general, as analytically described below,
Bouin solution allowed a better visualization of
the BAP immunocytochemical reaction in the neuronal cytoplasms (see Figs. 2–6).
of the metacerebrum and mesocerebrum were
negative in the active period (Fig. 4a) but very
reactive in the period of hibernation (Fig. 4b). In
some of the large neurons, the positivity was located in a discrete area of the cytoplasm. The GN
was weakly positive (not shown).
Immunocytochemistry for CGRP
In comparison with the active period (Fig. 2a),
a greater number of neurons were immunopositive
in both metacerebrum and mesocerebrum during
hibernation (Fig. 2b). The metacerebral giant neuron (GN) was entirely stained.
Immunocytochemistry for CCK8/gastrin
A few weakly immunopositive neurons and
immunopositive fibers around neurons were observed in the active snails (Fig. 3a,b). During
hibernation, the fibers maintained some immunopositivity (Fig. 3c,d). Several large neurons of the metacerebrum and mesocerebrum
(Fig. 3c,d), including GN (Fig. 6d), were reactive and had the immunopositivity often confined to a discrete area of the cytoplasm.
Immunocytochemistry for SP
Only a few of the small neurons were strongly
positive in the active animals. The large neurons
Immunocytochemistry for ET
In active animals (Fig. 5a,b), the immunopositivity
was in the fibers around the neurons and in the
cytoplasm of some neurons (Fig. 5b). Hibernation
determined a decrease in the immunoreactive fibers
and an increase in the number of immunopositive
neuronal cytoplasms (including the GN, Fig. 5c,d;
6c) in both the metacerebrum and mesocerebrum.
The immunopositivity in neurons was mainly located in a distinct area of the cytoplasm (Fig. 5c,d).
Coexistence/colocalization of ET and CGRP
in the mesocerebrum
Adjacent sections of hibernating snail ganglia
showed that in both the left and right mesocerebra,
ET-immunopositive neurons (Fig. 6a) were present.
The positivity was stronger in the peripheral area of
the left mesocerebrum. Almost all the neurons of the
Fig. 5. Immunoreaction for ET in Bouin-fixed cerebral
ganglia. (a, b) Active snails: immunopositive fibers or varicosities are found around neurons, of which only a few are
labeled (arrows). (c, d) Hibernating snails: most of the neurons, including the GN (arrow), are immunoreactive. Fibers
around neurons are less immunopositive. a, c: ×140; b, d: ×360.
HIBERNATION AND NEURONAL ACTIVITY
Figure 5.
359
360
G. BERNOCCHI ET AL.
Fig. 6. Immunoreaction for ET, CGRP and CCK8/gastrin
in Bouin-fixed ganglia of hibernating snails. (a) Immunoreaction for ET in the mesocerebrum: in the right (R) mesocerebrum, all the neurons are immunostained; in the left
(L) mesocerebrum, the peripheral neurons are more strongly
labeled than those of the central area. (b) Immunoreaction
for CGRP in the mesocerebrum: in the right mesocerebrum,
a group of central-lateral neurons are immunostained, while
in the left, almost all the neurons are strongly positive.
(c) Immunoreaction for ET in the GN: the immunoreaction
(arrow) is confined to a segregated cytoplasmic area. (d) Immunoreaction for CCK8/gastrin in the GN: the same cytoplasmic area (arrow) as in c is weakly positive. a, b: ×90; c,
d: ×580.
HIBERNATION AND NEURONAL ACTIVITY
Fig. 7. Immunoreaction for serotonin in paraformaldehyde-fixed cerebral ganglia. (a, b, c) Active snails: in the neuropile of the procerebrum (Pnp), there are immunopositive
varicosities with granular appearance and fine fibers (a); the
GN is immunopositive (b), and a large number of labeled fibers and varicosities (arrows) are present in the central neu-
361
ropile (c). (d, e, f) Hibernating snails: in the procerebrum,
lesser granular varicosities and fine fibers are evident (d);
increased immunoreactivity is shown in the GN (e). In the
neuropile surrounding the GN (e) and in the central neuropile (f), a reduction in the positive varicosities can be observed.
a, b, c, d, e, f: ×360.
362
G. BERNOCCHI ET AL.
left mesocerebrum were also positive for CGRP (Fig.
6b). In the right mesocerebrum, on the contrary, only
the central area was immunoreactive (Fig. 6b).
The GN was immunopositive for ET (Fig. 6c)
and CCK8 (Fig. 6d). For both the immunoreactions, the distribution of immunoprecipitates was
confined to the same distinct cytoplasmic area.
Immunocytochemistry for serotonin
In the active snails, the immunopositivity was
observed in the GN (Fig. 7b) and in some neurons of the metacerebrum; numerous positive varicosities and fibers were also present in the central
neuropile (Fig. 7c).
A marked increase in the intensity of the reaction was found in the same neurons, including the
GN (Fig. 7e), of the hibernating animals, whereas
immunopositive fibers of the neuropile adjacent the
GN were decreased in number (Fig. 7e,f).
Number and percentages of BAPimmunoreactive neurons
For all BAPs, the number (Table 1) and the percentages (Fig. 8) of immunoreactive neurons during the active period were very low, mainly in the
mesocerebrum (from 4 ± 0.9 to a maximum of 12
± 2.5 that corresponded to about 2.5% and 7% of
the neuronal population). In hibernating snails,
the percentages were 2- to 4-fold in the metacerebrum and 28-fold in the mesocerebrum. The highest percentages during hibernation were in the
mesocerebrum (64–79%).
DISCUSSION
Critical aspects
Though we used antibodies that are raised
against vertebrate antigens, they recognized epiTABLE 1. Changes in the number of BAP-like
immunoreactive neurons of the metacerebrum and
mesocerebrum during active period and hibernation*
Active
period
CCK8
SP
CGRP
ET
metacerebrum
mesocerebrum
metacerebrum
mesocerebrum
metacerebrum
mesocerebrum
metacerebrum
mesocerebrum
26
10
46
12
40
4
39
4
±
±
±
±
±
±
±
±
5
2
6
3
7
1
3
1
Hibernation
103
85
100
114
136
112
150
92
±
±
±
±
±
±
±
±
5
8
11
6
10
7
14
3
*Values are given as mean ± S.D.; n = 3 per each period; MannWhitney test significant level P < 0.001. CCK8, cholecystokinin; SP,
substance P; CGRP, calcitonin-gene-related peptide; ET, endothelium.
Fig. 8. Percentages of BAP-like immunoreactive neurons
in the metacerebrum (MT) and mesocerebrum (MS) during
active period (Ac) and hibernation (Hi). CCK8: Cholecystokinin; SP: Substance P; CGRP: Calcitonin-gene-related peptide;
ET: Endothelin.
topes in the Helix aspersa CNS. Indeed, the data
obtained with vertebrate antibodies to reveal invertebrate antigens must be interpreted with some
caution. In particular, the results may be affected
by cross-reaction with unrelated molecules, or immunocytochemical reaction may have detected
parts of a molecule that can be either identical to
or shorter or longer than the original antigen because we used polyclonal antibodies that contain
several immunoglobulins against different epitopes
of the same immunogenic antigen. As a consequence, we describe an “antigen-like immunoreactivity.” Nonetheless, as reported below, most of the
BAPs considered here and serotonin have been
chemically identified in Helix (for a review, see
Elekes et al., ’94) or other gastropods (Vigna et al.,
’84; Sonetti et al., ’90; Giaid et al., ’91).
It is known that the type of fixation can influence
immunoreactivity, in particular of peptidergic cells
(Schot et al., ’84; Andriès et al., ’91). We emphasize
that with the two fixatives we used immunolabeling
for BAPs was often detected in different places. For
instance, after immunoreaction to CGRP, CCK8 and
SP, fibers and varicosities were better visualized in
HIBERNATION AND NEURONAL ACTIVITY
formaldehyde-fixed specimens, whereas neurons
showed immunopositivity mainly after fixation with
Bouin solution. Only for immunoreactions to CCK8
and ET were fibers and neurons labeled from
samples fixed in formaldehyde and those fixed in
Bouin solution.
Occurrence and function of BAPs and
serotonin in gastropods
Several BAPs have been mapped in the nervous system of gastropods and in particular in
Helix in order to increase the knowledge about
putative peptidergic pathways. Some BAPs, such
as CCK8/gastrin (Osborne, ’84; Vigna et al., ’84;
Gesser and Larsson, ’85; Sonetti et al., ’90),
FMRF-amide (Cottrel et al., ’92), myomodulin
(Santama et al., ’94), and buccalin (Santama et
al., ’94) are implicated in control of feeding behavior. SP, which has been described in some molluskan neurons (Osborne, ’84; Sonetti et al., ’90),
is involved in sensory pathways concerned with
pain and touch. In mammals, calcitonin and
CGRP are involved in fundamental physiological
functions such as pain perception, appetite and
blood flow regulation, and vasodilation (Breimer
et al., ’88), and in mollusks, CGRP has been found
in slugs (Sasayama et al., ’91). First recognized
as a potent vasoconstrictor, ET has been immunocytochemically demonstrated in neurons controlling the vascular system and water retention
in Aplysia (Giaid et al., ’91). Opioid peptides are
involved in a wide spectrum of physiological functions, such as thermoregulation, locomotion, pain
resistance, and immunomodulation (Kavaliers
and Hirst, 1984). In particular, neurons containing enkephalins have been recently mapped in
ganglia of Helix (Marchand et al., ’91; Elekes et
al., ’93). Finally, the distribution of peptides of
the VIP-family, which are involved in many functions, has been described in the CNS of Helix
(Kaufmann et al., ’95).
With all the antibodies against the peptides we
have considered, individual neurons and fibers
were positive in the cerebral ganglia of H. aspersa
during the active period; the patterns of distribution were quite similar to those previously described. In gastropods and in land snails of the
genus Helix (Osborne et al., ’82; Osborne, ’84;
Cottrel et al., ’92; Elekes and Naessel, ’90; Gutierrez and Asai, ’91; Marchand et al., ’91; Elekes et
al., ’93, ’94; Kaufmann et al., ’95); several peptides
have been localized in the circumesophageal ganglia, and we have found also fibers immunopositive
for antibodies against SP, CCK8, CGRP, and ET
363
in the neuropile or around the neurons of the meta
and mesocerebrum. Some neuron somata were also
stained in these areas. It must be recalled that
there are small, medium, and large neurons in the
metacerebrum, whereas the mesocerebrum comprises only large neurons (for a review, see Kerkut
and Walker, ’75). In the metacerebrum, the neurons that showed SP-, CCK8-, CGRP- and ET-like
immunoreactivity were mainly small in size, and
in the mesocerebrum, the immunopositive neurons
occurred sporadically.
In invertebrates, including mollusks, serotonin
acts as neurotransmitter, modulator, and neurohormone (Hernadi et al., ’89; Hernadi and Elekes,
’93). In gastropods in particular, serotonin can affect and modulate a variety of behavioral and
physiological processes, such as feeding (Weiss et
al., ’82; Kupfermann and Weiss, ’82; Kemenes and
Srozsa, ’87), withdrawal and escape reaction
(Kupfermann and Weiss, ’82), blood circulation
(Liebeswar et al., ’75), and some forms of learning (Kandel, ’76; Balaban, ’93). In the Helix central
nervous system, it has been found by immunocytochemistry that both the neuropile and the cell
body layer are richly innervated by serotonergic
fibers (Hernadi et al., ’89; Elekes, ’91). Accordingly,
during the active period, we have found neurons
with serotonin immunoreaction in the Helix
metacerebrum, which sends numerous projections
to the central neuropile, the body wall, and different internal organs. The metacerebral giant
neuron, which innervates buccal ganglia, also had
serotonin immunoreactivity, as previously reported (Osborne et al., ’82; Osborne, ’84).
BAP immunocytochemistry in hibernating
pulmonate snails
The most striking result of the present research
was the observation that, in the hibernating Helix
aspersa, a high number of neurons had immunopositive cytoplasm for all BAPs. The immunoreactivity of fibers decreased for some BAPs in several
areas of the neuropile and for SP and ET around
neurons. In the mesocerebrum, a high number of
neurons (64% to 78%) were immunopositive for
CGRP, ET, CCK8, SP, and in the metacerebrum
(30% to 57%) several small- and medium-sized neurons were also immunopositive. In the metacerebrum, the GN was strongly immunopositive for
CGRP and SP, and a part of the cytoplasm was also
intensely immunoreactive for CCK8 and ET.
The high number of immunopositive neurons and
the increase in the intensity of staining in the immunoreactive cells in hibernating terrestrial snails
364
G. BERNOCCHI ET AL.
suggest an accumulation of reactive epitopes and
molecules in the cytoplasm. This probably gives rise
to the co-occurrence of two or more peptides in the
same cells, which were negative during activity, and
to the co-localization of differently immunopositive
neurons. Two hypotheses may be put forward as
explanations for these points. The first one assumes
that the immunopositivity is really linked to BAP
presence. Actually, the segregation of immunoprecipitates—for instance, for CCK8 and ET—in discrete areas of the cytoplasm of neurons that are
entirely positive for CGRP and SP (e.g., the GN) is
consistent with a storage of BAPs (two or more) in
hibernating snail neurons. Therefore, hibernation
may be a tool for studies of the functional role of
different cerebral neurons. In this context, the
small nervous systems not only give lessons for the
study of functional organization of cotransmission
systems (Marder et al., ’95), but the seasonal cycle
of some invertebrates also offers an unique opportunity to unravel the true function of brain areas.
Though this was not the aim of our study, we cannot exclude the possibility of the co-occurrence of
two or more peptides, which suggests that a neuron can be involved in different functions. Co-occurrence of neurotransmitters and neuropeptides
has been widely documented (Osborne, ’84; Fasolo
and Clairambault, ’91; Marder et al., ’95). Moreover, the co-localization of neurotransmitters and
neuropeptides in the cerebral ganglia of Helix is
evident in the paper by Hernadi and Elekes (’93).
However, co-reactivity does not necessarily
mean co-occurrence of two or more peptides. In
fact, during the biosynthesis of neuropeptides, a
preprotein is incorporated into dense-core vesicles
and subsequently processed to give several BAPs
and then transported to the cell terminals. Thus,
the preprotein is a precursor for several bioactive
peptides with distinct biological properties. In this
way, all the peptidergic neurons can putatively
manufacture different mixtures of peptides, each
with a markedly different spectrum of biological
activity. The recent findings of Kaufmann et al.
(’95) show that hibernating snails contained the
highest number of VIP and mainly of prepro-VIP
immunoreactive neurons. In this light, the positivity of the neurons for two or more antibodies
against bioactive peptides also may be ascribed
to the presence of a preprotein molecule, synthesized in the cell cytoplasm and blocked in this localization. In this case, all the neurons may have
the capability and possess the preteolytic enzymes
to manufacture several peptides. The hypothesis
will be tested by further research.
According to this interpretation, during hibernation the neurons are ready to elaborate and release
more than one bioactive peptide. The eventual diversification of neurons occurs only during the period of activity. This places under discussion the
multipotential role of neurons and the compartmentalization of ganglia in invertebrates, as tentatively
analyzed by Hernadi and Elekes (’93).
During hibernation the large neurons of the
mesocerebrum, which has a role in mating and
avoidance behavior (for a review, see Chase and
Li, ’94; Li and Chase, ’95), showed immunoreactivity for ET, CGRP, SP, and CCK8. This indicates
that all these BAPs are involved in these activities or that the mesocerebral neurons have more
numerous roles than expected. The same consideration may be made for GN, which controls the
feeding behavior (Chase and Tidd, ’91; Chase and
Tolloczko, ’92) and is immunopositive not only to
CCK8, but also to CGRP and ET.
Finally, it is particularly interesting that there
are also differences in the immunoreactivity of the
paired mesocerebral lobes during hibernation; this
supports a different involvement of the right and
left mesocerebrum in the hormonal and/or neuronal
activities (for a review, see Chase and Li, ’94).
Serotonin immunocytochemistry in
hibernating pulmonate snails
Another interesting result concerns the changes,
not in only number but also in intensity in immunoreactivity for serotoninergic neurons of the
metacerebrum. In particular, the metacerebral GN
was described as a typical serotonin-containing
neuron (Osborne et al., ’82; Osborne, ’84; Hernadi
et al., ’89). This neuron increased its immunoreactivity during hibernation, suggesting an accumulation of serotonin in the neuronal cytoplasm,
as observed for peptides. It has been shown, however, that 3H-serotonin injected into serotoninergic
somata accumulates in both synaptic vesicles and
large lysosomes (Cottrel and Osborne, ’70; Schkolnik and Schwartz, ’80). Consequently, it has been
suggested that serotoninergic synaptic vesicles
that are not released are ultimately transported
back to the soma, where they are degraded by lysosomes. Electron immunocytochemistry will
clarify whether the increase in the immunopositivity of the soma of the GN is due to the storage of the neurotransmitter before the release or
before being degraded by lysosomes. It must be
stressed that the immunoreactivity of serotonin
was decreased in the nearby neuropile.
HIBERNATION AND NEURONAL ACTIVITY
Functional changes in the CNS
during hibernation
The physiology and metabolism of the organism change during hibernation in both vertebrates
and invertebrates (Hunter, ’64; Machin, ’75; Heller,
’79; Lyman et al., ’82), to different extents, according to the modality of hibernation and, probably,
to the systematic position.
Studies carried out mainly on organs, tissues, and
cells from vertebrate species (Kolaeva et al., ’80;
Barni et al., ’87; Bernocchi et al., ’86; Giacometti et
al., ’89; Anderson et al., ;90; Barni and Bernocchi,
’91; Fenoglio et al., ’92; Malatesta et al., ’94, ’95;
March and Reisman, ’95) have explained some control mechanisms of adaptation of the animals to different environmental conditions and have provided
interesting contributions to the knowledge of the
structure and function of quiescent cells. In the vertebrate CNS, information has been acquired concerning some structural and functional changes of
neurons during natural hibernation, mainly regarding the condensation state of chromatin (Bernocchi
et al., ’86) and nucleolar and nuclear organization
(Giacometti et al., ’89; Malatesta et al., ’94, ’95).
These studies showed a lower activity of the nucleus
both in amphibians and in mammals.
There are few data about seasonal variation in
serotonin, dopamine and enkephalin contents in
gastropod ganglia (Cardot, ’71; Hiripi and Salanki,
’73; Hernadi et al., ’89, ’93; Gutierrez and Asai,
’91). The biochemical analysis demonstrated that
levels of serotonin and dopamine were higher in
the spring than in winter (Hernadi et al., ’89;
Juhos et al., ’93).
Furthermore, there are few studies about the histochemistry of circumesophageal ganglia during the
seasonal cycle (Gesser and Larsson, ’85; Hernadi
et al., ’89; Kaufmann et al., ’95). In particular,
Hernadi et al. (’93) observed a notable amount of
dopamine in the CNS of Helix, accompanied by a
low number of dopamine-immunopositive neurons
in summer, but the highest number of dopamineimmunopositive neurons was present in winter,
when the dopamine content was lower. The different distribution and intensity of immunopositivity
we have found in the neuropile and in neuronal
somata could explain this discrepancy.
The immunocytochemistry of some markers of
systems that regulate neuronal morphology and
activity showed clear changes in the cerebral ganglion of H. aspersa during hibernation in comparison with the active period (Vignola et al., ’95).
In hibernating snails, there was an increased immunoreactivity for calmodulin, in agreement with
365
an increase of the intracellular Ca2+ concentration. Calmodulin is the major intracellular Ca2+
recipient in most eukaryotic cells and is thus an
important mediator for many basic cellular
processes, including the synthesis and release of
neurotransmitters. This suggests that, during hibernation, neurons decrease or suppress the Ca2+dependent mechanisms. The lowered Ca2+-ATPase
activity (Vignola et al., ’95), which also plays a
role in regulating the intracellular ionized calcium
concentration, supports this interpretation.
Finally, the immunocytochemical expression of
some cytoskeletal components also changed during hibernation (Vignola et al., ’95). There was an
increase in the high-molecular-weight phosphorylated neurofilament-like immunoreactivity in the
cytoplasm of some clusters of neurons and, in contrast, a decrease in the immunopositivity for alpha-tubulin and microtubule-associated proteins
2 in the neuropile, which suggests that the transport of vesicles in the axons may be slowed during hibernation (Vignola et al., ’95). This is
consistent with the modification in the pattern of
neurotransmission and neuromodulation shown in
the present investigation.
ACKNOWLEDGMENTS
The research was supported by Italian M.U.R.S.T.
(40% and 60%) and C.N.R. grants to G. Bernocchi.
We are grateful to Mrs. P. Veneroni and Mr. S.
Veneroni for their excellent technical assistance.
Special thanks are also due to Mrs. T. Bosini for
typing the manuscript.
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