Bertram Peretz

Habituation and Dishabituation in the Absence of a Central Nervous System

Habituation and dishabituation have been observed in a semi-intact Aplysia preparation in which the central nervous system is removed. The amplitude of withdrawal responses in the gill decreases in proportion to the rate of water drops applied (one drop per 0.5 minute to one drop per 2.5 minutes at 15�C). The effects of habituation last for at least 2 hours. A dishabituated response is elicited by stopping the water drops or electrically stimulating the preparation. Furthermore, the gill contains nerve cell bodies, and habituation and dishabituation appear to be properties of these peripheral neurons.

Age-dependent CNS control of the habituating gill withdrawal reflex and of correlated activity in identified neurons inAplysia

SummaryThe parieto-visceral ganglion (PVG) control of the gill withdrawal reflex to tactile stimulation of the gill is absent in young animals, weighing 25 g or less. In older animals, weighing 160 g or more, the PVG control causes suppression of the reflex amplitude and a faster rate of habituation.1)In youngAplysia the PVG did not suppress the reflex amplitude nor did it accelerate the rate of habituation (Fig. 2). The gill neural plexus, through which presumably, PVG control of the reflex is mediated, is as competent in young as in older animals to mediate habituation (Fig. 2).2)PVG control was not evoked by increasing the intensity of the tactile stimulus 12 fold, from 0.15 g to 1.8 g, to the gill; in older animals the extent of control was dependent on stimulus intensity (Fig. 3).3)In young animals the 0.15 g stimulus evoked spiking in L7, a gill motor neuron, and it evoked only an EPSP in L7 in olderAplysia (Figs. 4, 5). The decrement of evoked activity in L7 from both groups of animals was correlated with habituation of the reflex (Fig. 8).4)Induced spiking of L7, in young and olderAplysia, resulted in dishabituation of the gill reflex (Fig. 8). These results and those in (3) show that the pathways between the gill and the PVG are functional in young animals.5)No differences were found in membrane properties of L7 from young and older animals; L7s were only differentiated by cell size (Table 1, Figs. 6, 7).6)In contrast R2, which is not involved in the reflex, was responsive to the weaker gill stimuli only in young animals (Figs. 4, 5). R2s greater responsiveness is attributed to small cell size which accounts for greater input resistance (Table 1, Fig. 7).7)The absence of control of the gill withdrawal reflex and the greater synaptic efficacy in L7 to gill stimulation, we suggest, results from incompletely developed inhibitory mechanisms in the PVG of young animals (Fig. 9).

Central influence on peripherally mediated habituation of anAplysia gill withdrawal response

SummaryThe parieto-visceral ganglion (PVG) modulates gill habituation, which in turn is mediated by a peripheral neural plexus. 1.A central influence accelerates the rate of habituation and depresses the responsiveness to waterdrops applied to a gill lobe (Figs, 2, 3). The branchial nerve is the major pathway for this influence (Figs. 4, 5). The ctenidio-genital nerve, on the other hand, elicits facilitatory effects in response to gill stimulation (Fig. 4).2.Complete recovery, with and without the PVG connected to the gill, occurs in 3 hours, indicating that the gill plexus is responsible for the recovery (Fig. 4).3.PVG influence increases with repeated stimulation, and it is suggested that the influence evokes inhibition in the gill neural plexus (Figs. 3C, 4).4.Waterdrops evoked spike and PSP activity in L7; EPSPs decremented to repeated application of the stimulus (Fig. 6). L7 cannot be responsible for the observed central effects, however, because they were not suppressed when the cell was hyperpolarized.5.The PVG is a more efficient analyzer of repeated stimulation to the gill than is the neural plexus. A model is presented for the interaction between the CNS and the gill neural plexus during habituation (Fig. 7).

Survival and aging of a small laboratory population of a marine mollusc, Aplysia californica

In an investigation of the postmetamorphic survival of a population of 112 Aplysia californica, five animals died before 100 days of age and five after 200 days. The number of survivors among the 102 animals which died between 100 and 220 days declined approximately linearly with age. The median age at death was 155 days. The animals studied were those that died of natural causes within a laboratory population that was established to provide Aplysia for sacrifice in an experimental program. Actuarial separation of the former group from the latter was justified by theoretical consideration. Age-specific mortality rates were calculated from the survival data. Statistical fluctuation arising from the small size of the population was reduced by grouping the data in bins of unequal age duration. The durations were specified such that each bin contained approximately the same number of data points. An algorithm for choosing the number of data bins was based on the requirement that the precision with which the age of a group is determined should equal the precision with which the number of deaths in the groups is known. The Gompertz and power laws of mortality were fitted to the age-specific mortality-rate data with equally good results. The positive values of slope associated with the mortality-rate functions as well as the linear shape of the curve of survival provide actuarial evidence that Aplysia age. Since Aplysia grow linearly without approaching a limiting size, the existence of senescence indicates especially clearly the falsity of Bidders hypothesis that aging is a by-product of the cessation of growth.

Excitability and branching of neuroendocrine cells during reproductive senescence

In the mollusc Lymnaea stagnalis, neuroendocrine caudodorsal cells (CDCs) were studied physiologically and morphologically from egg layers (EL) (aged 154-400), and animals 4 weeks (CEL-4) (342-455 days), and 8 weeks (CEL-8) (477-660 days) after production of their last egg mass. After recording chemical transmission, electrical coupling and stimulation induced afterdischarges (ADs), CDCs then were filled with Lucifer Yellow. Based on the axonal branching revealed by Lucifer Yellow, CDCs were classified as extensively, moderately, or minimally branched. In EL-CDCs, induction of AD activity, which normally (9) precedes egg-laying, only was initiated in the resting state. CEL-4 CDCs exhibited ADs whereas CEL-8 CDCs did not. CEL-8 CDCs exhibited significantly reduced chemical and electrical transmission, and CEL-4 CDCs did not differ from resting state EL-CDCs. CDC branching was significantly reduced with both increasing age and declining egg-laying. Minimally branched CDCs most frequently failed to exhibit an AD and exhibited reduced electrical coupling. We conclude that both physiology and morphology of CDCs are related to age and reproductive state.

Effect of age on acetylcholinesterase and other hemolymph proteins in Aplysia

SummaryHemolymph was examined in young (ca. 86 days old), mature (ca. 163 days old), and old (ca. 294 days old) Aplysia for age-related changes in constituent proteins. In young, mature, and old animals protein concentrations were 1.6±0.27, 1.41±0.53, and 1.45±0.43 mg·ml-1, respectively. The copper-containing respiratory protein, hemocyanin, measured by determining the copper concentration, was found to increase significantly from young (0.98±0.51 μg·ml-1) to mature (2.02±0.95 μg·ml-1) Aplysia, with little change between mature and old (1.92±0.43 μg·ml-1) animals. These findings were consistent with the results obtained when hemocyanin was directly measured by spectrophotometric absorption at 340 nm. Acetylcholinesterase (AChE) was present in the hemolymph of Aplysia. Its activity was highest in mature animals (3121±1627 units·mg-1) and least in old animals (1463±599 units·mg-1). Young animals had intermediate levels (2080±762 units·mg-1). SDS-PAGE revealed a distinct pattern of protein bands for hemolymph from each age group; hemolymph from the young group contained six prominent protein bands with molecular weights (MW) from 13 to 300 kDa. Hemolymph of mature and old animals exhibited four and three prominent protein bands, respectively, with MW between 45 and 300 kDa. A prominent band at 97 kDa was present in samples from the mature group, but was faint in samples from the old group and absent in samples from the young group. Under non-denaturing conditions the hemolymph protein band patterns for each group differed from the others, thereby demonstrating that the age-dependent differences in the protein profiles are intrinsic to hemolymph in vivo. Isoelectric focusing of the hemolymph samples revealed that the proteins were all acidic (pI ca. 3.0–6.5). The hemolymph from the young differed from the other two groups in having an additional acidic protein (pI ca. 4.0). A possible link between age-related changes in hemolymph proteins and age-related changes in the nervous system is proposed.

Motor neuronal function in old Aplysia is improved by long-term stimulation of the siphon/gill reflex.

As Aplysia age, motor neuronal (L7) elicited gill-pinnule contractions are significantly decreased, as is transmission at pinnule junctions. To determine whether this reduced function of L7 with increased age could be altered, the siphon/gill reflex, which involves the L7-pinnule pathway, was stimulated regularly in unrestrained old animals. Aplysia, more than 240 days old, were assigned randomly into trained and untrained groups. For more than 3 weeks, a 1-s, 25-g water jet stimulus was administered to the siphon of the trained animals 10 times per day at 20-min intervals. The duration of siphon withdrawal increased significantly during training. In semi-intact preparations, pinnule contractions and junctional transmissions were then measured during 3-s depolarized pulses to L7 (frequency range = 1-44 per 3-s interval). The trained animals had (a) significantly higher pinnule contractions, (b) a significantly greater increase in pinnule contractions elicited by increasing L7 spike rates, (c) significantly higher double-spike facilitation, and (d) significantly higher facilitation per spike across the four spike trains below or at pinnule contraction threshold. Long-term stimulation of the siphon/gill reflex in old Aplysia did not produce the same functional efficiency observed in mature Aplysia; nevertheless, the significant training-induced increases in both pinnule contraction and junction transmission indicate that a considerable level of plasticity still exists in an aging nervous system. Whether this long-term training of Aplysia retards the same processes responsible for the age-related decline in neuromuscular transmission or produces a compensatory change in other neuronal processes is discussed.

Effect of acetylcholinesterase inhibition on behavior is age-dependent in freely moving Aplysia

The significance of age-dependent changes in acetylcholinesterase (AChE) activity level is poorly understood. Reported here is one approach to understand AChEs function as it relates to age: to investigate how inhibition of AChE affects behavior in freely moving Aplysia of two age groups, mature and old. The siphon/gill withdrawal reflex (S/GWR) and gill pumping movement (GPM) were examined to assay the effects of AChE inhibition by BW284c51, a specific and reversible AChE inhibitor. In mature Aplysia AChE inhibition by 2 microM and 5 microM of BW284c51 resulted in a significantly shortened S/GWR duration, and in suppression of habituation and dishabituation. In old animals, AChE inhibition by 2 microM of BW284c51 did not affect S/GWR and only dishabituation was suppressed by inhibition by 5 microM of BW284c51. AChE inhibition reduced the GPM rate significantly only in mature animals. AChE inhibition did not alter the decrement in GPM rate which is regularly observed in both age groups during repetitive exposure to acidified sea water. Thus both S/GWR and GPM were affected by AChE inhibition, and a significant age effect on the two behaviors was observed. Comparisons of the results of AChE inhibition which would elevate acetylcholine (ACh) levels with those of carbachol administration revealed that AChE inhibition affects both cholinergic and non-cholinergic mechanisms underlying the two behaviors.

Increased age affects properties characterizing behavioral plasticity in freely behaving Aplysia

In the marine mollusc Aplysia, in vitro studies showed that the gill withdrawal reflex (GWR) and its neuronal substrates were altered by age. In contrast, age minimally affected the gill respiratory pumping movements (GPM) and its neuronal substrates. Based on the respective properties of the GWR- and GPM-pathways in vitro, we proposed that the more pronounced the effect of age, the greater the expression of plasticity in a pathway. This conclusion may hold for in vitro preparations, but it remained to be demonstrated in intact animals. Based on this conclusion, the GWR should exhibit greater plasticity than the GPM in intact animals. Using freely behaving Aplysia, we tested for plasticity of the GWR and the GPM in three age groups (young, mature, and old). The tests for behavioral plasticity were: Graded responses to varying stimulus strength, response decrement (or habituation) to repetitive stimulation, enhanced response to dishabituating stimuli, and the effect of the GWR stimulus on the GPM and the GPM stimulus on the GWR. The GWR in mature animals exhibited all four properties, but in old animals, graded responses and habituation were significantly altered and in young animals habituation and dishabituation were absent. The GPM exhibited fewer of the properties than the GWR, only graded responses and response decrement, both of which were generally the same in the three groups. We found that behavioral plasticity and age-induced plasticity are related in freely behaving animals and are consistent with in vitro findings. The effect of age on properties characterizing plasticity at both the behavioral and pathway levels is discussed.

Chronic stimulation increases acetylcholinesterase activity in old Aplysia

In the marine mollusc Aplysia, a reduced level of activity of circulating AChE (acetylcholinesterase) signals the onset of aging [28], as it does in mammals [23,25]. In old Aplysia, coincident with the reduced AChE activity is impaired neuron function [17], which chronically applied sensory stimulation (CSS) improves [35]. As a first step to establish the link between the CSS and improved neuronal function, we investigated if CSS alters the level of AChE activity in old Aplysia. Before and after 4 weeks of CSS of the siphon-gill withdrawal reflex (S/GWR), we measured circulating and neural levels of AChE and behaviors involving the gill in freely moving mature and old Aplysia. Only in old animals did the CSS produce increased AChE activity levels in both the CNS and serum, and the increased levels were correlates of a change in the S/GWR, the behavior elicited by the CSS. This result shows that aging animals are able to up regulate enzymatic activity in response to specific sensory input. It also suggests that age influences how the level of AChE activity responds to persistent changes in sensory input. Parallels exist between the results here and those in higher vertebrates and are discussed.